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Abstract—Soil contaminated with iron and chromium was planted with Psoralea pinnata under greenhouse condition. The growth of the plants and phytoextraction of the metal contaminants from the soil were studied for a period of three months. The results showed that Psoralea pinnata was able remove both chromium and iron from the contaminated soil during the period of experimentation. The percentage reduction in chromium and iron concentrations in the experimental soil varied greatly at different concentration of both contaminants in the two soils used. It was observed however that at some points in the experiment involving mixed concentration of iron and chromium, there were preferences on accumulation of metals by Psoralea pinnata. Results show that chromium was initially most accumulated by Psoralea pinnata (up to 68%). As the concentration of contaminants increased, at high concentrations, iron was recorded to have been accumulated more in Psoralea pinnata (up to 55%). Index Terms—Chromium, iron, phytoextraction, Psoralea pinnata. I. INTRODUCTION Soils contamination by heavy metals and metalloids has become a serious environmental issue today. A number of metals including chromium, iron, arsenic, zinc, cadmium, mercury and copper are known to significantly compromise the quality of soil and cause adverse effects to human and health and the well being of other organisms that comes in contact with such soil. Heavy metals are extremely persistent in the environment because they are not biodegradable and may not be broken down by chemical oxidation  or through thermal processes, as a result their accumulation readily reaches to toxic levels . Some metals are essential for plant growth however, very high or low concentrations of some these heavy metals may be inhibitory to plant growth. Human activities such as metal smelting, electroplating and mining are sources through which heavy metals enter the environment. According to Kuhndt , about 100-350 tons of residues are generated during the extraction processes for every ton of copper produced. South Africa has about 70% of the world’s chrome reserve and is the world’s largest producer of ferrochrome (75%). South Africa has about 6000 abandoned mines most of which have potential to contaminate the environment . The contamination of soil Manuscript received November 8, 2013; revised February 17, 2014. R. O. Ochonogor is with the Department of Environmental Sciences, University of South Africa, P.O. Box 392, Pretoria 0003, South Africa. H. I. Atagana is with the Institute for Science and Technology Education, University of South Africa, P.O. Box 392, Pretoria 0003, South Africa (e-mail: [email protected]). with heavy metals in each of the sites is dependent on length of operation of mines. Rain and runoff waters help to increase the chance of extending metal contamination beyond the primary contaminated sites. Metals have the potential to accumulate in human body when contaminated plants are ingested and may produce unwanted side effects -. Methods used for remediation of heavy metal contaminated soil include soil flushing, solidification/stabilization, vitrification, thermal desorption and encapsulation . Other methods include burying of the contaminated soil or dilution of the contaminated soil with clean soil. These methods contribute to long-term risks such as leaching into groundwater and surrounding soil . Due to the expensive nature of the conventional remediation methods for heavy metal contamination , phytoremediation technologies are continuously being researched for possible solutions. The level of heavy and toxic metals (Pb, Cr, Hg, etc.) in the environment can be reduced from contaminated sites or media using a number of aquatic and terrestrial plants. Metals are taken up in solution by the root system of plants and transported to the stems and leaves without showing toxicity syndromes and this have been supported by many studies , . As a developing technology , phytoremedaition, particularly phytoextraction have been applied to metals contaminations containing (e.g. Ag, Cr, Fe, Cu, Hg, Mn, Mo Ni, Pb, Zn), metalloids (e.g. As, Se), radionuclides (e.g. 90Sr, 137Cs, 234U, 238U) and non-metals , . Phytoextraction employs plants to transport and accumulate high quantities of metals from soil into the harvestable parts of roots and above ground shoots , , and has emerged as a cost effective, environmentally friendly clean up alternative . The phytoextraction or hyperaccumulation of metals in various plant species have been extensively investigated and substantial progress has been made. The potential of duck weed was investigated by Zayed et al.  for the removal of Cd, Cr, and Cu from nutrient-added solution and the results indicated that duck weed is a good accumulator for Cd and Cu, but his result was unable to establish potential plant for abstracting Cr from the soil. Brooks,  investigated the uptake of Cr from soil by the use of some plants including Indian mustard (Brassica juncea). He indicated that there is no evidence of Cr hyperaccumulation by any vascular plants. Robinson et al.  investigated the potential of Berkheya Coddii to phytoextract Co from artificial metalliferous media. Although, Co was readily taken up by the plant, cobalt was toxic to the plant above a certain limit. Although, majority of phytoextraction investigations have focused on Cd, Pb and Zn , Fe contamination is a more prominent problem in many soils particularly where iron extraction is common and Phytoremediation of Heavy Metal Contaminated Soil by Psoralea Pinnata International Journal of Environmental Science and Development, Vol. 5, No. 5, October 2014 440 DOI: 10.7763/IJESD.2014.V5.524 where conversion of iron into various kinds of steel carried out . The hyperaccumulators that have been extensively studied include s Thlaspi spp., Arabidopsis spp., sedum alfredii spp., belong ing to the families Brassicaceae and Alyssum  . Psoralea pinnata belon gs to the family Fabaceae thriving well in both wetland and upland habitats. The use of Psoralea pinnata in phytoextraction has not been investigated. The aim of this study is to investigate the use of Psoralea pinnata in phytoextracting Fe and Cr from con taminated soil under green house conditions. II. M ATERIALS AND METHODS A. Plant Psoralea pinnata , seeds were collected from Silver Hills Seeds and Brook, Cape Town. The seeds were planted and watered in a green house for four weeks. Healthy plants with a height of about 11.50cm were selected for the phytoextraction experiments. B. Treatments TABLE I: C HARACTERISTICS OF TH E SOIL USED IN THE EXPERIMENTS Garden soil Commercial Potting Soil (PS)/ Soil B pH – H 2 O 7.41±0.25 6.43±0.49 CEC (meq/100g soil) 11.2 21.8 Org anic carbon ((% wt)) 12.12 0.87 N tot (% wt) 0.02 0.05 P tot (% wt) 4.4 9.1 K (ppm) 3.2±0.29 14.8±0.52 Sand (%) 63.9 8.9 Silt (% wt) 15.3 18.0 Gravel (% wt) ≤ 5 N/A Clay (% wt) 19.0 69.8 Ca (ppm) 61.5±0.39 82.8±0.53 Mg tot (ppm) 1.5±0.79 8.5±0.82 Mn (ppm) 9.7±0.89 75.6±0.64 Na(ppm) 147±0.03 44.0±0.61 Fe tot (ppm) 57.2±0.61 4.6±0.45 Cr tot (ppm) 78.0±0.27 10.2±0.31 The two soils types were separately mixed with compost in a ratio of 5:1 (w/w) (soil: compost) (see Table I ). Eight experiments were set up in triplicates in PVC pots (550 × 413mm) by contaminating each soil with a 1.5:1 ratio (v/v) of Cr ( KCrO 4) and Fe ( Fe(NO 3) 3.9H 2O) to mimic the composition of both metals in a typical ferrochrome. The combined total concentration of both metals in the treatments ranged from 40 to 320 mg kg -1 . The treatments for both soil types contained Cr and Fe in mg kg -1 as follows: T 40 = 24 Cr + 16 Fe, T 80 = 48 Cr + 32 Fe, T 120 = 72 Cr + 48 Fe , T 160 = 96 Cr + 64 Fe , T 200 = 120 Cr + 80 Fe , T 240 = 144 Cr + 96 Fe, T 280 = 168 Cr + 112 Fe, T 320 = 192 Cr + 128 Fe Two sets of control experiments were separately set up using the garden soil and commercial potting soil without metals. Four week old Psorelea pinnata plants from the nursery were transplanted into the contaminated soils and the controls and allowed to grow for 3 months in the green house. Moisture was kept at 60-70% field capacity. Leaching was avoided by adding only a little amount of the water at a time. Plant s were harvested after 3 months growth, washed, dried and homogenised before digesting 15g in a mixture of HNO 3 : HCl (1:3) and analyzing in Atomic Absorption Spectrophotometer (AAS) Ten grams of soil samples wer e digested in an acid mixture of HNO 3 : HCl (1:3). T he chromium and iron content of the samples were analyzed using Atomic Absorption Spectrophotometer (AAS) . III. RESULTS AND DISCUSSION The result of the analysis of the two soils are shown in Table 1. Most measured parameters including organic carbon varied considerably in both soils. The results of analysis for Cr and Fe in plant tissues from the experimental plants shows that the plant tissues accumulated between 12 and 27% Cr and 18 and 22% Fe of the amoun t of Cr and Fe present in the garden soil. The largest amounts (%) accumulated were in T 40 (27), T 80 (20) and T 120 (20.5) for Cr ( see Fig. 1 ) and T 40 (20.5), T 160 (21) and T 200 (22) for Fe ( see Fig. 2). Fig. 1. Amount of Cr accumulated in plant tissues (% of soil concentration) in garden soil. Values are means of 3 +/ – SE. Fig . 2. Amount of Fe accumulated in plant tissues (% of soil concentration) in garden soil. Values are means of 3 +/ – SE. Both metals were taken up well by the experimental plants. The difference in the concentration of Fe between T 40 and T 320 in the mixed contamination in the garden soil did not significantly affect the rate of accumulation of Fe in the plant International Journal of Environmental Science and Development, Vol. 5, No. 5, October 2014 441 tissues. However, the rates of accumulation of Cr was significantly affected by the increases in concentrat. Although all plants grew well in the soil, leaf yellowing was observed in some of the plants in T 240 – T 320 . Generally, there tended to be a decrease in the amount of both metals accumulated as concentration of metals increased . This is an indication of toxicicty at elevated concentrations, however, it could not determined which of the metals was responsible for the toxic effect or whether the effect was due to both metals. Fig . 3. Amount of Cr accumulated in plant tis sues (% of soil concentration) in commercial potting soil. Values are means of 3 +/ – SE. Fig . 4. A comparison of the amounts of Cr and Fe accumulated in plant tissues in garden soil. Values are means of 3 +/ – SE. Changes in metal concentration did not significantly affect the rate of accumulation of Cr in the commercial potting soil ( see Fig. 3). The difference in the responses to concentration of the metals could not be readilly explained. However, there were a number of differences in both chemical an d physical parameters of the two soils. The cation exchange capacity and (CEC) and the organic carborn are to parameters that could be responsible for the difference. From Treatments T40 to T160, Cr was the dominant metals accumulated by Psoralea pinnata i n preference to Fe. In the T reatment T200, there was no significant difference between the accumulation of Cr and Fe although the accumulation of Fe was slightly higher (see Fig. 4). The treatment with the most iron absorption in relation to chromium absor ption is T320. It was observed that with rising concentration of metals in the soil, Psoralea pinnata absorbed more of Fe than of Cr . The results show that plants in the control experiments with garden soil and commercial potting soil showed a very low amo unts of both metals. The total amounts of Fe accumulated in the garden soil 6.34% and 1.38% in the commercial potting soil. The total amounts of Cr accumulated in the garden soil was 3.48% and 3.11% in the commercial potting soil. These results are not une xpected, as the control experiments were not spiked with Fe and Cr and the concentrations of both metals in the soil were very low ( see Table I ). These result s support those of previous studies where it w as observed that there wa s competition between Cr a nd other metals for binding sites . Sharma and Pant, , showed that in maize plants, the effects of Cr on Fe concentration varied with plant organ s and also with Cr levels. They observed that Mn, Fe and Cu concentrations generally decreased w ith increase in Cr level s. In a study on Cr (III) –Fe interaction, Bonet et al.  reported that Cr enhanced growth of both Fe -control ed and Fe -deficient plants. However, Cr concentration s correlated neither with changes of Mn, P nor Fe tissue concentration s or Cr -in duced alterations of the Fe/Mn and P/Fe ratios. The reduction in the uptake of Fe could be mainly due to the chemical similarity of Fe and Cr ions in solution. Hence, the competitive binding to common carriers by Cr (VI) could have reduced the uptake of ma ny nutrients . IV. CONCLUSION From the results obtained in this study, Psorelea piñata has demonstrated that it can accumulate Fe and Cr in contaminated soils under green house conditions. It has also shown that it can tolerate high levels of metal contamination with minimal inhibition in growth processes. It would therefore be a useful plant to test further for hyperaccumulation of toxic heavy metals . R EFERENCES  A. N. Matthew, ―Phytoremediation of heavy metal contaminated soil ,‖ MSc dissertation, Graduate College, Oklahoma State Univ., Tulsa, Ok, 2005.  H. L. Bohn, B. L. McNeal, and G. A. O’Connor , Soil Chemistry , 2nd ed. New York : Wiley, 1985, pp. 50- 55.  M. Kuhndt, J. Von Geibler, V. Türk, S. Moll, K. O. Schallaböck, and S. Steger. (May 2012). Virtual dematerialisation: ebusiness and factor X. Wuppertal Institute Final Report. Digital Europe, March. [Online]. 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R. Brooks and B. H. Robinson, ―Aquatic phytoremediation by accumulator plants, ‖ in Plants that Hyperaccumulate Heavy Metals: Their Role in Phytoremediation, Microbiology, Archaeology, Mineral Exploration and Phytomining , CAB International, Oxon, UK , 1998, pp. 203- 226.  B. H. Robinson, M. Leblanc. and D. Petit, ―The potential of Thlaspi caerulescens for phytoremediation of contaminated soils, ‖ Plant Soil , vol. 203, no. 1 , pp. 47–56, 1998.  J. W. Huang, J. Chen, W. R. Berti et al., ―P hytoremediation of lead contaminated soil: role of synthetic chelates in lead phytoextraction, ‖ Environ. Sci. Technol ., vol. 31, no. 3, pp. 800-805, 1997.  M. N. V. Prasad and H. M. O. Freitas, ―Metal hyperaccumulation in plants —Biodiversity prospecting for phytoremediation technology, ‖ Electronic J. Biotechnol , vol. 6, pp. 285-321, 2003.  D. C. Sharma and R. C. Pant , ―Chromium uptake its effects on certain plant nutrients in maize (Zea mays L. CV) Ganga, ‖ Journal of Environmental Science and Health , Part A, vol. 29, pp. 941-948, 1994.  A. Bonet, C. Poschenrieder, and J. Barcelo. ―Chromium III ion interaction in Fe deficient and Fe sufficient bean plants. I. Growth and Nutrient content, ‖ J. Plant Nutr ., vol . 14, pp. 403–414, 1991.  A. K. Shanker , ―Physiological, biochemical and molecular aspects of chro-mium toxicity and tolerance in selected crops and tree species, ‖ PhD Thesis, Tamil Nadu Agricultural University, Coimbatore, India, 2003. Richie O. Ochonogor has BSc in biochemistry and is currently studying towards the MSc degree in environmental management at the University of South Africa. The title of his MSc dissertation, which is currently being examined, is Phytoextraction of chromium and iron from contaminated soil using Psoralea pinnata . Richie hopes to continue studying towards the PhD degree in environmental management on completion of his MSc. Harrison I. Atagana is a professor and the head of Institute for Science and Technology Education (ISTE) , University of South Africa . His field of interests is in bioremediation, exotoxicology, phytoremediation, wasterwater treatment, freshwater quality, pollution studies, and science education. He is a rated researcher of the South African National Research Foundation and has held three research grants of the fo undation. He has published extensively in peer reviewed international journals and has presented over twenty papers in at international and national conferences around the world. Professor Atagana currently has six masters and doctoral students in environm ental biotechnology. International Journal of Environmental Science and Development, Vol. 5, No. 5, October 2014 443
See d is c u ssio n s, s ta ts , a n d a u th or p ro fil e s f o r t h is p ub lic a tio n a t: http s:/ /w ww.r e se a rc h ga te .n et/ p ub lic a tio n /5 1416114 Mult i- sit e A naly sis R ev eals W id esp re ad Antib io tic R esis ta n ce i n t h e M arin e P ath ogen Vib rio v u ln if ic u s Artic le in Mic ro b ia l E co lo gy · J a n uary 2 009 DO I: 1 0.1 007/s 0 0248-0 08-9 413-8 · So u rc e : P ub M ed CIT A TIO NS 38 REA D S 48 9 a u th ors , i n clu d in g: Cra ig B ake r-A ustin Cen tr e f o r E n vir o n m en t, F is h erie s a n d A q u … 69 PU BLIC ATIO NS 1,6 85 CIT A TIO NS SEE P R O FIL E J V au n M ca rth ur Univ e rs it y o f G eo rg ia 91 PU BLIC ATIO NS 2,7 64 CIT A TIO NS SEE P R O FIL E An gela H L in d ell Univ e rs it y o f G eo rg ia 14 PU BLIC ATIO NS 436 CIT A TIO NS SEE P R O FIL E Ja m es D O liv e r Univ e rs it y o f N orth C aro lin a a t C harlo tte 210 PU BLIC ATIO NS 8,4 92 CIT A TIO NS SEE P R O FIL E All i n -t e xt r e fe re n ce s un d erlin ed i n b lu e a re l in ke d t o p ub lic a tio n s o n R ese a rc h G ate , le ttin g y o u a cce ss a n d r e a d t h em i m med ia te ly . Ava il a b le f r o m : J a m es D O liv e r Retr ie ve d o n : 1 5 A ugu st 2 016 ORIGINAL ARTICLE Multi-site Analysis Reveals Widespread Antibiotic Resistance in the Marine PathogenVibrio vulnificus Craig Baker-Austin &J. V. McArthur & Angela H. Lindell &Meredith S. Wright & R. Cary Tuckfield &Jan Gooch &Liza Warner & James Oliver &Ramunas Stepanauskas Received: 10 December 2007 / Accepted: 21 May 2008 # Springer Science + Business Media, LLC 2008 AbstractVibrio vulnificusis a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. There- fore, few studies have verified the effectiveness of commonly prescribed antibiotics inV. vulnificustreatment. Here we screened 151 coastal isolates and 10 primarysepticaemia isolates against 26 antimicrobial agents repre- senting diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed forV. vulnifi- cusinfections, such as doxycycline, tetracycline, amino- glycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implica- tions of our findings. Among environmental isolates, there were no consistent differences in the frequency of resis- tance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the impor- tance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resis- tance factors. Introduction Bacteria of the genusVibrioare commonly found in coastal and estuarine waters. Select strains ofV. cholerae,V. parahaemolyticus,V. vulnificusandV. mimicusare consid- ered serious human pathogens, .V. vulnificuscauses food-borne diseases and wound infections. It carries the highest fatality rate of any food-borne pathogen in the US, often exceeding 50% [29,30,34], and 95% of all deaths resulting from seafood consumption in the US are caused by this bacterium . About 85% ofV. vulnificusinfections Microb Ecol DOI 10.1007/s00248-008-9413-8 Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-008-9413-8) contains supplementary material, which is available to authorized users. C. Baker-Austin :J. V. McArthur :A. H. Lindell :M. S. Wright Savannah River Ecology Laboratory, Drawer E, Aiken, SC, USA R. C. Tuckfield Savannah River National Laboratory, Bldg. 773-42A, Aiken, SC, USA J. Gooch National Oceanographic and Atmospheric Administration, Charleston, SC, USA L. Warner :J. Oliver Department of Biology, University of North Carolina at Charlotte, Charlotte, NC, USA R. Stepanauskas Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME, USA C. Baker-Austin (*) Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, Weymouth, Dorset DT4 8UB, UK e-mail: craig.baker-aus[email protected] occur between May and October, as this pathogen thrives in warm water (>20°C) . With advances in refrigeration, seafood originating from habitats whereV. vulnificusare most abundant may be consumed by at-risk individuals almost anywhere . It is believed that a large number ofV. vulnificusinfections may go unreported (CDC estimates ~50% reported), which could greatly contribute to under- estimates of the morbidity and mortality burden associated with this pathogen. V. vulnificusinfections are characterised by an extremely short time-span between the onset of symptoms and subsequent clinical outcome, and immediate antibiotic therapy for suspected cases is considered critical. If treatment is delayed greater than 72 h, the fatality rate of primary septicaemia-associatedV. vulnificusinfections is 100% . Interestingly,V. vulnificusis considered to have low levels of antibiotic resistance, and previous studies have shown sensitivity to tetracyclines, aminoglycosides, third- generation cephalosporins, chloramphenicol and newer fluoroquinolones [26,41,43]. Paradoxically, the twoV. vulnificusstrains CMCP6 and YJ016, for which genomes are publicly available, contain enzymatic-modification systems, active drug transporters and permeases suggestive of a genetic basis for antibiotic resistance. The significant morbidity and mortality associated withV. vulnificus infections, combined with the speed with which these infections progress and symptoms develop, underlie the need for a thorough analysis of the antibiotic resistance capabilities of this species. The occurrence, diversity and public health implications of environmental antibiotic resistance have only recently been appreciated [2,6,16,33,42]. Considering the proliferation of antibiotic resistance in clinical settings , an understanding of the selective pressures maintain- ing environmental reservoirs of resistance may have a direct impact on the treatment of infectious diseases, particularly for pathogens that persist in the environment. Given the considerable spatial and temporal heterogeneity in the distribution of resistance and the complex factors that affect its evolution, dissemination and persistence, antibiotic resistance is being increasingly viewed as an ecological problem . Addressing antibiotic resistance from an environmental standpoint can promote a better understand- ing of the ecology and evolution of antibiotic resistance, and may provide an early detection system for the development of antibiotic resistance mechanisms in clini- cally relevant bacteria . For example, several recent reports have implicated the role of industrial contamination, and in particular metal contamination, as an indirect selective agent for antibiotic resistance [2,24,39,40,46]. Of the few studies that have addressed antibiotic resistance in this important human pathogen, most have involved only a fewV. vulnificusisolates and relatively few antimicrobials. Morris and Tenney  analysed the antibiotic resistance capabilities of 19V. vulnificusstrains against seven antimicrobial agents. Zanetti et al.  tested sixV. vulnificusisolates taken from Italian coastal waters for sensitivity to 11 antimicrobials, while Ottaviani et al.  studied the resistance of eight environmental isolates to 27 antibiotics, including some antibiotics tested in this study. Thus, the aim of our study was to determine the antibiotic resistance capabilities across a much larger library ofV. vulnificusstrains (151 isolates) derived from three different estuarine environments and to determine the sensitivity of these isolates to a wide range of antimicrobial agents (26 drugs). Two of the sites analysed in this study are extensively contaminated with heavy metals (Shipyard Creek, Charleston, SC, USA and LCP Chemicals, Bruns- wick, GA, USA), whilst the reference site (the ACE Basin, Beaufort, SC, USA) is considered a pristine estuarine habitat. These habitats were chosen to assess the potential contribution of industrial contamination in driving environ- mental antibiotic resistance in this taxon. In addition, a small number of strains isolated fromV. vulnificus- associated primary septicaemia clinical cases were also screened. Our results suggest that the diversity and level of antibiotic resistance inV. vulnificusis far greater than previously thought, with potentially significant clinical ramifications. Materials and Methods Sample Collection Sediment and water samples were obtained from an industrially contaminated estuarine site (Shipyard Creek, Charleston, SC, USA), in March, June and October 2005 and an uncontaminated reference site (ACE Basin, SC, USA), in June and October 2005. Sediment and water samples were also obtained from the industrially contam- inated EPA Superfund site (LCP Chemical site, Brunswick, GA, USA) in October 2005. Triplicate 1-L surface water samples were obtained during maximum ebb and flood tides to isolate bacterial strains entering and leaving tidal creeks. During low tide, top 1-cm sediment samples were collected into sterile plastic bags from five equidistant locations along each creek, starting at creek mouths and ending at the upper reach of each creek. Bacterial isolation was performed within 24 h from sample collection, after refrigerated storage in the dark. Isolation and Genotyping ofV. vulnificusStrains A DNA non-radioactive probe (alkaline phosphatase labelled) targeting the species-specific hemolysin–cytolysin C. Baker-Austin et al. (vvh-AP) inVibrio vulnificuswas purchased from DNA Technology A/S (DK-8000 Aarhus C, Denmark) and used for identification and confirmation ofV. vulnificuscolonies on spread plates. Sediment and water samples were serially diluted in sterile phosphate buffered saline (PBS) [7.65 g NaCl, 0.724 g anhydrous Na 2HPO 4(Sigma), 0.21 g KH 2PO 4 (Sigma)/L, pH 7.4]. Aliquots of each sample were spread- plated onto two types of media for colony isolation. (a) Vibrio vulnificusagar (VVA) [30 g sodium chloride (Sigma), 10 g cellobiose (Sigma), 20 g peptone (Difco), 0.06 g bromthymol blue (Sigma) and 25 g agar (Difco)/L] and (b) CHROMagarVibrio(CAV; DRG International, Inc.) 74.7 g/L which contains 15 g/L agar, 8 g/L peptone and yeast extracts, 51.4 g/L salts and 0.3 g/L chromogenic mix . The chromogenic media yields presumptive identifications for specificVibriospecies. Turquoise colo- nies areV. vulnificus/Vibrio choleraein an approximate 50/ 50 ratio. Final species confirmation by the use of molecular tests utilisingV. vulnificusgene-specific markers  was subsequently performed. TypicalV. vulnificuscolonies on VVA plates and turquoise colonies from CAV plates were transferred with sterile toothpicks into individual wells of a 96-well plate containing 100μL of tryptic soy broth (TSB) plus 2% extra sodium chloride (total of 2.5% salt) and incubated 16 to 18h at 36°C. Cells were plated to VVA plates  and tested with thevvh-AP DNA oligo probe for species confirmation. Colony lift, hybridisation and colorimetric detection proce- dure were performed, essentially as previously described [7, 9,25,45]. Sediment samples (3 g each) were weighed into 50-mL sterile tubes and mixed with 27 mL 4° C PBS (to make a 10 −1 dilution). A 10 −3–10 −6 dilution series was set up with the sediment samples, inoculated on VVA plates and cultured overnight at 36°C. A number ofV. vulnificus isolation methodologies were simultaneously applied for unambiguous species identification. Firstly, an alkaline phosphatase-labelled DNA probe targeting the species- specific hemolysin–cytolysin (vvh-AP) was used for tenta- tiveV. vulnificusidentification mentioned above [8,41]. Isolates were then grown overnight in TSB (amended with 2.5% NaCl) at 36°C. DNA from individual isolates was extractedbyboiling(5min,95°C)1mLofculture followed by centrifugation, using the decanted supernatant as crude template . Colonies that positively hybridised with thevvh-AP DNA were later PCR amplified using the V. vulnificusspecificvvhA(hemolysin A) gene with forward primervvhA-F (CGCCGCTCACTGGGG CAGTGGCTG) and reverse primervvha-R (CCAGCCGT TAACCGAACCACCCGC) . For hemolysin gene PCR, cells were grown overnight at 22°C in heart infusion (HI) broth (Difco; Detroit, MI, USA) and cell lysatesprepared as follows: 200μL of the broth culture was centrifuged, resuspended in 200μL of filtered, autoclaved, deionised water and boiled for 5 min. PCR was conducted using a Genius thermal cycler (Techne; Princeton, NJ, USA). Briefly, cell lysates (5μL) were added to a master mix consisting of 17.75μL diethyl pyrocarbonate-treated water, 5 mM (8μL) dNTPs (Promega; Madison, WI, USA), 20 mM (0.09μL) of each primer (Bio-Synthesis; Lewis- ville, TX, USA), 5U (0.25μL) Taq polymerase (Promega), 25 mM (3.2μL) MgCl 2(Promega) and 10× (4μL) Mg-free buffer (Promega) for a final reaction volume of 40μL. The hemolysin gene was amplified using 24bp oligonucleotides that are specific for a 340-bp fragment located within this 1,416-bp gene unique toV. vulnificus. The primers utilised were Vv1 (CGC CGC TCA CTG GGG CAG TGG CTG) and Vv2 (CCA GCC GTT AAC CGA ACC ACC CGC). For all experiments, a negative control containing all PCR reagents and sterile HI broth was employed. The reaction mixture was overlaid with 20μL sterile mineral oil. For visualisation, gel electrophoresis was performed using a 2% agarose gel (NuSieve 3:1, BioWhittaker Molecular Appli- cations; Rockland, ME, USA) with PCR products that were stained with ethidium bromide (1.25μg/mL) . Finally, PCR amplification and sequencing of the 16S rRNA gene was performed on a subset of isolates for unambiguous species verification. Positively identifiedV. vulnificuscolonies were coded to conceal their source from the investigators, stored in TSB plus 2.5% salt with 30% glycerol and frozen at−80°C for later use. To putatively ascertain the potential virulence capabilities ofV. vulnificus isolates, a previously identified 200 bp randomly amplified polymorphic DNA (PCR) amplicon associated with clinical isolates was chosen for colony PCR screening for E- and C- genotypes . The presence of class 1 integrons was also ascertained by PCR [27,47]. Ten clinically derivedV. vulnificusstrains, from primary septicaemia clinical blood isolations and previously described , were also analysed to compare the public health implications of antimicrobial resistance in these bacteria. Antimicrobial Susceptibility Testing Minimal inhibitory concentrations (MICs) were determined by microdilution and 48-h incubation onto custom dehydrated 96-well MicroScan® panels (Dade Behring, Sacramento, USA) according to the manufacturer’s instructions and using Mueller–Hinton broth amended with 2.5% NaCl . The following antimicrobial agents (concentration ranges in mg/ L) were used in the MicroScan® panels, based on their mode of action, history of use and resistance, and clinical relevance: amikacin, 8–64; amoxicillin, 4–32; ampicillin, 4–32; apramycin, 8–32; azithromycin, 2–8; cefoxitin, 8–32; ceftriaxone, 8–64; cephalexin, 16–128; cephalothin, 16–128; Antibiotic Resistance in Vibrio vulnificus chloramphenicol, 8–32; ciprofloxacin, 1–4; erythromycin, 16–128; gentamicin, 2–16; imipenem, 2–16; meropenem, 2–16; moxifloxacin, 0.25–4; nalidixic acid, 4–32; nitrofur- antoin, 16–128; oxytetraycline, 4–32; ofloxacin, 1–8; peni- cillin, 16–128; streptomycin, 16–128; sulfathiazole, 250–500; tetracycline, 4–32; trimethoprim, 2–16; and trimethoprim– sulfamethoxazole, 2/38 and 4/76. Concentration levels within the specified range for each antibiotic were successive doublings from the range minimum to maximum. Resistance to doxycycline (which was not present on the MicroScan® panels) was later determined using both broth microdilution and agar dilution methods as outlined by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards ), using the most resistant 25% of the environmentally derivedV. vulnificus isolates. ATCCV. vulnificusstrain 27562 was used as a con- trol during the screening process. Isolates that could not be revived or grown over the course of the antimicrobial susceptibility testing were omitted from the final analyses. Randomised subsets of coded isolates were also re-screened at a separate laboratory to assess methodological reproducibility (data not shown). Resistance was defined as growth in the presence of the highest concentration of the antibiotic and partial resistance as growth in concentrations of the antibiotic up to, but not exceeding the highest concentrations used. Statistical Analysis The antibiotic response data were both left censored and right censored, that is, there are no response measurements below the minimum or above the maximum concentrations, respectively, found on the plates for each antibiotic tested. Left-censored observations were recorded as (MIN−MIN / 2) where MIN was the concentration range minimum per antibiotic. Right censored observations were recorded as (MAX + MAX / 2) where MAX is the concentration range maximum on the plates. There is no empirical justification for replacing a right censored value by a further doubling beyond the MAX concentration, as there is no empirical justification for replacing left censored values by zero. The only certainty for isolates that were inhibited by our lowest concentration or that grew at the highest concentration is that the true response is MAX, respectively. Our choice of left censored replacement values is widely acknowledged [15,28]. For consistency, we treated right censored values similarly. Two summary response measures were calculated, the total number of antibiotics for which an isolate was resistant to the MIN concentration or higher (N abRes ) and the trimmed mean antibiotic concentration producing a resistance effect and per isolate called the average resistance response metric (e C ab Re s ). The latter differs from the simple average resistance response con- centration among all 26 antibiotics in that the simpleaverage uses the same divisor (i.e. 26) for the sum (numerator) whether or not any term in the sum is Based on the concentration ranges previously established for each antibiotic,e C ab Re s is an average containing only those antibiotics with values≥MIN per isolate. Thus, the number of antibiotics represented in this sum was “trimmed”to a subset of those with evinced resistances. Because the concentration ranges for the different anti- biotics used varied by five orders of magnitude, all measurements among isolates within an antibiotic were first standardised; i.e. converted toZ-scores. Thereafter, all measurements regardless of antibiotic were on a trans- formed and common unitless scale and contributed equally to the average resistance response metrice C ab Re s . Simple linear regression was used to model the relationship betweenN abRes ande C ab Re s from which the standard Pearson’s(r) correlation coefficient was obtained. ANOVA methods were applied to these two summary response measures to examine the effects of sampling site (ACE, LCP and Shipyard Creek), month (March, June and October) and sample type (ebb, flood or sediment) and the respective interactions among these three main effects. Since the LCP site was only sampled in October and noV. vulnificusisolates were observed in March with the exception of SYC sediment samples, a summary ANOVA was performed using only two sites (ACE and SYC) and 2 months (June and October). Contingency tables were used to assess the frequency distribution differences between clinical and environmental genotypeV. vulnificusisolates among the three sites. All statistical methods were obtained from Steel and Torrie  and analyses were performed using the JMP™5.12 software. Results A total of 151V. vulnificusisolates were obtained from the three coastal sites (Table1). Of them, 29 (14.6%) were C- genotype (clinical) and 122 (85.4%) were E-genotype (environmental) according to the virulence typing method of Rosche et al.  described above. A greater proportion of C-genotypes were detected in October (26%) than June (9%). There were no significant differences in the frequen- Table 1Number ofV. vulnificusstrains isolated from the various sampling sites and sampling campaigns Site March June October ACE Basin, Beaufort, SC 0 a 21 27 Shipyard Creek, Charleston, SC 2 37 27 LCP, Brunswick, GA 0 a 0a 36 aACE Basin was not studied in March and the LCP Chemical site was not studied in March or June C. Baker-Austin et al. cy of E- and C-genotypes among the three sampling locations and three sample types (ebb, flood and sediment). On average, each isolate was resistant to 5.88 antibiotics, with a unimodal distribution of the number of resistances per isolate (Fig.1a). No discernible difference was found between the E- and C-genotypes with regard to either the average number of resistances per isolate (N abRes ) or the average concentration at which the isolate was resistant (e C ab Re s ). Resistances to aminoglycosides, cephams, folate pathway inhibitors and penicillins were the most common, varying between 0.6% (cefriaxone) and over 99% (apra- mycin) (Fig.2). Conversely, less than 1% of the isolates were resistant to chloramphenicol, quinolones, macrolides and carbapenams, except for nalidixic acid (7.3%) and azithrmycin (3.3%). Of the 26 antimicrobial agents tested, total sensitivity was evident for just three agents: imipenem, erythromycin and ciprofloxacin. In an ad hoc study, 38 isolates with the highestN abRes were screened for resistance to the synthetic tetracycline derivative doxycycline, of which one isolate was found resistant (2 mg/L). Of the entire environmental library (151 strains) 68 (45%) were resistant to three or more structural classes of antibiotic (Table S2).An initial three-way ANOVA showed no statistically significant sample type effect (i.e. no differences between sample types ebb, flood or sediment) for eitherN abRes or e C ab Re s . This main effect was dropped and a subsequent two-way ANOVA demonstrated a statistically significant difference (p< .05) in the antibiotic resistance profiles where bothN abRes ande C ab Re s were higher among ACE Basin isolates than Shipyard Creek isolates. This analysis also revealed a discernible (p< .05) interaction effect in that the difference between ACE and Shipyard isolates for either N abRes ore C ab Re s were larger in June than in October (Fig.1b, Fig. S1). Since sampling was conducted at all three sites in the month of October, a separate ANOVA was performed for this single month’s data and showed that bothN abRes ande C ab Re s were significantly higher (p< .05) among LCP isolates than either Shipyard Creek or ACE Basin isolates. There was a statistically significant (p<. 05) linear regression relationship betweene C ab Re s versusN abRes , and the two response measures were positively correlated (r¼ﬃﬃﬃﬃﬃ R 2 p ¼:70) (Fig.3). For a comparison with the environmental isolates, the same antimicrobial resistance testing procedures were applied on tenV. vulnificusstrains isolated from patients suffering fromV. vulnificus-associated septicaemia. These strains demonstrated varying levels of resistance, with N abRes =6.1(Table2). All strains were resistant to amikacin, apramycin, cephalexin and streptomycin; and with the exception of strain H3308, gentamicin. One strain (LSU 1606) demonstrated high-level resistance to 12 antimicrobials, including doxycycline (>100 mg/L). Elevated heavy metal concentrations were confirmed for the SYC and LCP sampling sites using ICP–MS  procedures. Compared to ACE, SYC sediments were enriched in Cu, Zn, Sr, Pb and Cd, while LCP sediments were contaminated with Hg (Table S1). Discussion This work represents one of the first large-scale surveys of antibiotic resistance and potential virulence in aVibrio species and provides further evidence for the presence of multi-antibiotic resistance in environmental bacteria. Only a handful of studies have addressed antibiotic resistance in this important human pathogen [13,26,41,43], and these have used a limited range of antimicrobial agents. We found an unexpectedly high frequency and level of resistance to diverse naturally derived as well as synthetic agents, including those often prescribed to treatV. vulnificus infections (tetracycline, gentamicin, ceftriaxone) [3,19,26], and even one of the frontline treatment agents, doxycycline (Figs.1,2, Table S2). Eleven structural classes of anti- biotics were used in our screening efforts (aminoglycosides, 0 10 20 30 40 50 60 70 80 90 0 5 10 15 20 25 30 350 1 2 3 4 5 6 7 8 9 10 11 12 13 0-1 2-3 4-5 6-7 8-9 10-11 12-13 Resistance per isolate (NabRes) a b C-Biotype E-Biotype October June Number of isolates Number of isolates Figure 1Distribution of the number of resistances per isolate (Nab Re s ) in the 151 environmentalV. vulnificusisolates divided by genotype (a) and by field sampling campaign (b). Since the LCP Chemical site was not sampled in October, only Shipyard Creek and the ACE Basin isolates were included in the seasonal comparison (b) Antibiotic Resistance in Vibrio vulnificus carbapenams, cephams, foliate pathway inhibitors, macro- lides, nitrofurans, penicillins, phenicols, quinolones, sul- phonamides and tetracyclines). Of these, 68 isolates (45%) were resistant to agents of three or more structural classes suggestive of widespread multi-antibiotic resistance. Simi- lar resistance patterns were found inV. vulnificusderived from primary septicaemia blood isolates (Table2). One of the ten analysed clinical isolates (LSU 1606) was resistant to 12 out of 26 antimicrobials. Strikingly, only oneV. vulnificusisolate from the 151 environmental and ten clinical strains was susceptible to all 26 antimicrobials (Table S2). These resistances may hinder the treatment ofV. vulnificusinfections, and could contribute directly to the high rates of mortality associated with these pathogens. Interestingly,V. vulnificusantibiotic resistance and genotype composition exhibited a marked temporal varia- tion, with higherN abRes ore C ab Re s and higher proportion of E-genotypes in June compared to October (Fig.1b). Figure 3Average resistance response ( e Cab Re s ) as a function of total number of antibiotic resistances per isolate (Nab Re s ). Shipyard Creek (SYC), LCP Chemicals (LCP) and the ACE Basin (ACE) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% Amikacin Gentamicin Streptomycin Apramycin Ampicillin Amoxicillin Penicillin Imipenem Meropenem Ceftriaxone Cefo xitin Cephalothin Cephal exin Trimethoprim/Sulfamethoxazole Trimethoprim Sulfathiazole Nitrofurantoin Tetracy cline Oxytetracycli ne Ciprofloxacin Moxifloxacin Ofloxacin Nalidixic Acid Erythromycin Azithromycin Chloramphenicol Percentage of isolates Resistant Partially resistant Sensitive AminoPen Ceph FPI Sul Nit Tet Quin MacCl Carb β 100% lactamases Figure 2Frequency of resistance to 26 antibiotics among the 151 environmentalV. vulnificusisolates. Resistance was defined as growth in the presence of the highest concentration of the antibiotic and partial resistance as growth in concentrations of the antibiotic up to, but not exceeding, the highest concentrations used. Data fordoxycycline resistance is not shown. Abbreviations used:Amino, aminoglycosides;Pen, penicillins;Carb, carbapenams;Ceph, ceph- ams;FPI, folate pathway inhibitors;Sul, sulfathiazole;Nit, nitro- furantion;Quin, quinolones;Mac, macrolides;Cl, chlorampenicol C. Baker-Austin et al. Notably, the highest clinical burden associated withV. vulnificusinfections usually takes place during the summer . More extensive studies, encompassing multiple years and a broader geographic scope, should be conducted to assess potential epidemiological implications of such potential seasonal trends. There were no consistent differences in antibiotic resistance (N abRes ande C ab Re s ) betweenV. vulnificusisolated from pristine (ACE Basin) and contaminated (Shipyard Creek and LCP Chemical) sites. The apparent lack of contamination effects on resistance patterns was further supported by the absence of resistance differences among flood, ebb and sediment samples in Shipyard Creek and LCP Chemical sites. This contradicts several prior studies, demonstrating elevated antibiotic resistance in heavy metal- contaminated environments, likely due to resistances to diverse metals and antibiotics being co-selected for [2,24, 39,40,46]. The apparent lack of indirect selection may be due to weak or non-existent genetic linkages between metal and antibiotic resistance in the studiedV. vulnificus. Alternatively, although the total concentration of several toxic metals was high in the sediments of Shipyard Creek (Cr and Cd) and the LCP Chemical site (Hg) (Table S1), the bioavailability of these toxic metals may be insufficient to impose significant indirect selection. Some heavy metals are capable of forming stable complexes in seawater [11, 12] reducing bioavailability and thus the potential foranthropogenic metal emissions to have a significant impact on antibiotic resistance. Most antibiotics, including many that are clinically relevant, are naturally produced by microorganisms in the environment [5,18,36], likely explaining the presence of antibiotic resistance genes and resistant microorganisms in pristine habitats (i.e. the ACE Basin) and in samples that predate the human use of antibiotics . Interestingly, we found a high frequency of resistances to aminoglycosides, cephams and penicillins (Fig.2), which are often encoded on plasmids in other gram-negative bacteria, including otherVibriospecies . However, no class 1 integrons, genetic elements frequently associated with the dissemina- tion of antibiotic resistance genes in clinical isolates, were detected in any of the 151 isolates. Conversely, we did find a positive correlation between plasmid DNA content and antibiotic resistance (data not shown), suggesting that horizontal gene transfer may be partly responsible for the observed resistances, involving mechanisms different from class 1 integron exchange. Furthermore,Vi b r i ospecies are known to harbour chromosomal integrons into which mobile gene cassettes potentially conferring antibiotic resistance can be inserted which constitutes an additional potential mechanism contributing to antibiotic resistance in V. vulnificus. It should be emphasised thatV. v u l n i f i c u s does not sustain prolonged presence in clinical settings, where it would undergo human-induced selection for antibiotic resistance, as in the well-known cases of obligate human and agricultural pathogens . Thus, high antimi- crobial resistance found in this study may either reflect innate resistance levels and/or horizontal transfer of resistance factors from anthropogenically derived taxa. Further work is clearly required to determine the precise molecular mechanisms of antibiotic resistance in this taxon. Of particular interest is the significant positive correlation between total number of antibiotic resistances (N abRes ) and average resistance response (e C ab Re s ) (Fig.3). It suggests that as the number of antibiotics to which an isolate is resistant increases, the concentration of the corresponding antibiotics that can be tolerated by the isolate increases as well. This seems to contradict the common assumption that the frequency and rates of antibiotic resistance in bacterial populations are directly related to the antibiotic exposure and inversely related to the cost that resistance imposes on the fitness of bacteria . The positive relationship betweenN abRes ande C ab Re s implies low fitness costs of the maintenance of antibiotic resistance genes in V. vulnificusand/or synergistic effects among the various antibiotic resistance factors. Likewise, a recent study indicates low or non-existent fitness impact imposed on an environmentally derivedE. colistrain via carriage of several mobile antibiotic resistance elements . Low Table 2Antimicrobial resistance information of the clinical and type strains Strain Source Genotype Resistance profile a C7184K2 Clinical C Ak, Apr, Cex, Gm, St LSU 763 Clinical C Ak, Apr, Cex, Gm, St LSU 1009 Clinical C Ak, Apr, Cex, Gm, St LSU 1365 Clinical C Ak, Apr, Cex, Gm, St LSU 1003 Clinical C Ak, Apr, Cex, Gm, St H3308 Clinical C Ak, Apr, Cex, NA, St CMCP6 Clinical C Ak, Apr, Cex, Cfx, Gm, St LSU 1015 Clinical C Ak, Apr, Cex, Gm, St, T LSU 1456 Clinical C Ak, Apr, Cex, Cfx, Gm, NA, St LSU 1606 Clinical C Ak, Apr, Azi, Cex, E, Fd, Gm, Otet, St, Sz, TE, Doxy V. vulnificusType C Ak, Apr, Cex, Cfx, Gm, St ATCC 27562 strain aAbbreviations used:Ak, amikacin;Am, ampicillin;Amx, amoxicillin; Apr, ampramycin;Azi, azithromycin;C, chloramphenicol;Cax, ceftriaxone;Cex, cephalexin;Cf, cephalothin;Cfx, cefoxitin;Deoxy, doxycycline;Fd, nitrofurantoin;Gm, gentamicin;Mer, meropenem; Mox, moxifloxacin;NA, nalidixic acid;Otet, oxytetracycline;P, penicillin;St, streptomycin;Sz, sulfathiazole,T, trimethoprim;TE, tetracycline Antibiotic Resistance in Vibrio vulnificus fitness cost of antibiotic resistance genes may explain slow recovery of antimicrobial sensitivity in obligate pathogens after the discontinuation of antimicrobials . To our knowledge this report constitutes the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the impor- tance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resis- tance factors. AcknowledgementsWe thank Blaine West, Charles Zemp, Kirk Kessler, Richard Gregory, and Marc Frischer for help accessing field sample collection sites. Brian Thompson and Brian Robinson are acknowledged for their assistance in isolate preparation. This work was supported by the National Oceanographic and Atmospheric Administration (NOAA) awards NA04OAR4600198 to Stepanauskas and NA05NOS4781244 to Oliver. Additional support was provided from the US Department of Energy Financial Assistance Award DE- FC09-96SR18546 to the University of Georgia Research Foundation. DisclaimerThis report was prepared as an account of work sponsored by an agency of the United States Government. 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A New Fungal Endophyte, Scolecobasidium humicola , Promotes Tomato Growth under Organic Nitrogen Conditions Rola S. Mahmoud 1* , Kazuhiko Narisawa 2 1 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo, Japan, 2 College of Agriculture, Ibaraki University, Ami machi, Ibaraki, Japan Abstract A new fungal endophyte, Scolecobasidium humicola , was identified as a common dark septate endophytic fungal (DSE) species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular, pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth with organic nitrogen sources. Citation: Mahmoud RS, Narisawa K (2013) A New Fungal Endophyte, Scolecobasidium humicola, Promotes Tomato Growth under Organic Nitrogen Conditions. PLoS ONE 8(11): e78746. doi:10.1371/journal.pone.0078746 Editor: Murad Ghanim, Volcani Center, Israel Received June 19, 2013; Accepted September 21, 2013; Published November 1, 2013 Copyright: © 2013 Mahmoud, Narisawa. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 23380190) from the Japan Society for the Promotion of Science; http://www.jsps.go.jp/english/e-grants/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction In the last few decades, interest in organic farming has increased all over the world; however, only 0.9% of the world’s agricultural lands are organic . Organic farming practices aim to establish stable production systems with concern for nature, in which the use of chemical nitrogen fertilizers, synthetic pesticides and growth-promoting chemicals is not allowed [ 2]. The input of nutrients in organic fields generally relies on organic fertilizers, manure, green manure, and/or crop residues; however, the balance of N mineralization/immobilization processes depends on the nature of these substrates and on the ecological conditions of each agro- system, which limit plant uptake and growth, especially at times of peak crop demand [ 3]. Recently, an interesting finding about plant nitrogen utilization in the forest ecosystem was reported [4 ]. It was shown that the largest pool of N is typically organic, e.g., amino acids and proteins are among the most abundant forms of organic N in the soil, comprising 80% of the soil N supply, while ammonium and nitrate contribute only 10%. This finding undoubtedly showed that symbiotic fungi, such as mycorhizal fungi, which can enhance host plant growth byimproving N nutrition, are extremely significant in the forest ecosystem. This can be achieved via an increase in theabsorptive surface area provided by fungal hyphae, greater uptake efficiency or by increasing access to various N sources that are unavailable to non-mycorrhizal plants [ 5,6]. However the ability of mycorrhizal fungi to maintain these benefits for their host plant under some field conditions common in industrialized agriculture is considered to be limited . Dark septate endophytic fungi (DSE), which are a miscellaneous group of ascomycetes colonize root tissues intracellularly and intercellularly without causing apparent negative effects on the host plant [ 8–10]. DSE associations have been recognized in approx. 600 plant species of 320 genera in 114 families, including non-mycorrhizal plant species [11 ]. DSE may benefit their host plants by facilitating the uptake of plant mineral nutrients, including P, N and water [ 12–15], and suppressing infection by plant pathogens [ 16–18]. Many aspects of their ecological roles remain unclear although several studies had focused on the abundance of DSE in different habitats. For that reason, the most suitable utilization method of DSE for organic farming is still unclear. In our study, we hypothesized that natural and agriculture systems might share similar DSE species, and DSE could play a role in supporting plant growth under these agriculture systems, especially the organic farming system. In order to prove this hypothesis, key DSE species were isolated andPLOS ONE | www.plosone.org1 November 2013 | Volume 8 | Issue 11 | e78746 identified in both natural and agriculture ecosystems. Here, we describe the features or identity of the selected key DSE species that were effective in supporting tomato growth with different organic nitrogen sources. Materials and Methods Sample collection and fungal isolation Soil samples were collected in October 2010 from a forest, organic field and conventional field at the Field Science Center of College of Agriculture, Ibaraki University Japan. Three soilsamples (approximately 300 ml) were collected at 0-20 cmdepth from each site and were kept in polyethylene bags and stored at 4°C for a maximum of one month prior to utilization. Composite soil was prepared to bait endophytic fungi as described by Narisawa et al. [ 17]. Each soil sample was combined and mixed with autoclaved potting soil (Peat pot; Kureha Chemical Industry Co., Tokyo, Japan) at the ratio of 1:2 (v/v). The seeds of tomato Solanum lycopersicum cv. Gohobi (Sakata Seed, Yokohama, Japan) and Chinese cabbage Brassica campestris cv. Musou (Takii Seed, Kyoto, Japan) were surface sterilized by immersion in a 70% solution of ethanol for 30 seconds, followed by a solution of sodium hypochlorite (2% available chlorine) for 1.5 minutes. Then seeds were rinsed three times with sterilized distillated water, dried overnight and placed on 1.5% water agar medium (15 g Bacto agar [Difco Laboratories, Detroit, MI] for 1 liter distilled water) in Petri dishes. After 4 days, the axenically grown seedlings were transplanted (three seedlings per pot) in to 90- mm diameter pots containing 100 ml composite soil. Each collection site was considered as a bloc containing three replicates for each soil sample and plant species. Seedlings were grown under greenhouse conditions with the temperature reaching 25°C. After three months, the roots collected from young tomato and Chinese cabbage plants in each replicate were washed with running tap water to remove debris and cutinto approximately 1-cm segments. Fifteen root segments werechosen in random from each bait plant, washed three times in a 0.005% solution of Tween 20 (J.T. Baker Chemical Co.,Philipsburg, NJ), and rinsed three times with sterile distilled water (SDW). The segments were air-dried overnight and plated on nutrient agar containing 25 g. L -1 corn meal (infusion form; Difco Laboratories) and 15 g. L -1 Bacto agar (Difco Laboratories). These plated roots were incubated for 3 weeks at room temperature (approximately 23°C). Morphological observation and identification Fungal isolates were identified on the basis of microscopic morphology. The pure fungal culture was grown at room temperature on 55-mm diameter Petri dishes containing half-strength cornmeal malt yeast extract agar (1/2 CMMY, 25 g corn meal (infusion from Difco), 15 g Bacto agar (Difco), 10g malt extract (Difco), 2g yeast extract (Difco), for 1 L distilled water). To provide good observation conditions, slide cultures were made. Small pieces, approximately 3 x 3 mm, of Publum agar [Mead Johnson mixed Publum; Canadian Post Corporation, Ontario, Canada, 25 g; Bacto agar, 5 g; MilliQ water, 250 ml] were sandwiched between two 18 x 18-mmcover glasses (Matsunami Class Ind., Osaka, Japan) and placed in a 9-cm water agar plate to provide humidity. After 2-4 weeks, when the culture had grown sufficiently, the Publum agar was carefully removed and cover glasses were mounted on 76 x 26 mm micro slide glasses using PVLG (Polyvinyl alcohol, 16.6 g; Lactic acid (Wako Chemical Ind., Osaka, Japan), 100 ml; glycerin (Wako Chemical Ind., Osaka, Japan), 10 ml; MilliQ water, 100 ml) mounting medium. Conidiogenous cells and conidia were measured under a light microscope (BX51; Olympus, Tokyo, Japan) with UPlanFLN FLN100x/1.30 Oil. DNA extraction, amplification, sequencing and analysis Fungal DNA were extracted using a PrepMan TM Ultra Extraction Kit (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s protocol, The fungal isolate was identified by sequencing the internal transcribed region (ITS) of the 18S rDNA, using universal primers ITS-1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′). Fifty microliters of PCR mixture contained 0.2 μM concentration of each primer, 0.2 mM of eachdeoxynucleoside triphosphate, 10× Ex Taq buffer (TaKaRa Bio, Otsu, Japan) and 0.25 U of Ex Taq DNA polymerase (TaKaRa Bio), and 50ng DNA template. The reaction cycle consisted of initial denaturation at 94 °C for 4 min followed by 35 cycles of denaturation at 94 °C for 35 sec, annealing at 52 °C for 55 sec and extension at 72 °C for 2 min, and final extension at 72 °Cfor 10 min. The PCR products were sequenced using a model 3130x DNA sequencer (Applied Biosystems, Foster City, CA,USA) and the ABI PRISM TM Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The primers used for sequence determination were ITS1F and ITS4R. The determined sequences were analyzed usingMEGA version 5.05 and compared with similar DNA sequencesretrieved from the DDBJ/EMBL/GenBank databases using the NCBIBLAST program. Pathogenicity screening In order to distinguish non-pathogenic fungi from pathogenic and other saprotrophic fungi, 15 fungal isolates showing diverse morphology were grown on oat meal agar medium (OMA; 10 g oatmeal and 18 g Bacto agar) enriched withnutrients (1 g MgSO 4·7H 2O, 1.5 g KH 2PO 4, and 1 g NaNO 3) per liter in Petri dishes (55-mm diameter). They were incubated at room temperature (approximately 23°C). After two weeks, 2- day-old Chinese cabbage seedlings (three seedlings per plate)were transplanted onto each fungal colony. The seedlings transplanted onto non-inoculated medium were used as a control. The whole set was placed into sterile culture pots (CB-1; As One, Osaka, Japan) and incubated in a growth chamber at 23°C under a 16-h photoperiod (180 mol m -2 s -1 ) for 2 weeks. Symptoms were evaluated according to an index of 0 to 3 (0: no visible symptoms; 1: light yellowing; 2: yellowing and late growth; 3: wilting or death). Plants were harvested and oven-dried at 60°C for 48 h and their dry weight was measuredfor comparison with control plants. Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org2 November 2013 | Volume 8 | Issue 11 | e78746 Endophyte screeningAfter successful pathogenicity testing, the efficacy of selected isolates to promote tomato growth was observed. The fungal isolates were grown on Petri dishes filled with oat meal agar medium supplemented with nutrients (MgSO 4.7H 2O, 1 g, KH 2PO 4 1.5 g; and NaNO 3 1 g L -1 ). Surface sterilization of tomato seeds was performed as described for the pathogenicity testing. Inoculation was preformed as described above. Endophyte screening for organic nitrogen sources In order to identify the effect of nitrogen on fungal infection, a nitrogen source test was conducted. The selected fungal isolates were grown for 2 weeks in 6-cm Petri dishes filled with oat meal agar medium as described above but NaNO 3, 1g L -1 was replaced by one of the selected nitrogen sources of amino acids, such as L-Valin, L-Phenylalanine, and L-Leucine (Wako Industries, Ltd., Japan), at the concentration of 20 mg L -1 . Tomato seedlings (three seedlings per plate) were transplanted and inoculation was preformed as described previously. To determine the endophytic nature of the fungal isolate, infected hyphae of the inoculated fungi in 3-week-old tomato seedling roots were observed after washing, cross sectioned, and stained in 50% acetic acid solution containing 0.005% cotton blue under an Olympus BX50 microscope with UplanFI20 and 40/0.30 objectives (Olympus). Data analysis The mean dry biomass of each treatment was calculated and analyzed with one-way ANOVA. Differences among treatment means were detected with Tukey’s honestly significant difference test (Tukey HSD). Results and Discussion Fungal isolation Two hundred and five fungal isolates were obtained from 270 root segments of tomato and Chinese cabbage grown as bait plants in a mixed soil. Fifty-six percent of fungal isolates were isolated from Chinese cabbage, and 44 % were isolated from tomato. The dominant isolated fungi are species of Fusarium (22%). They were mostly isolated within 4 days of placing root segments on the medium. Other taxa, including Scolecobasidium humicola (4%), Leptodontidium orchidicola (1%), and Phialocephala fortinii (1%) were isolated in much smaller numbers from both plants. They were mostly isolated after one to three weeks. Scolecobasidium humicola were found at all three sites but L. orchidicola and P. fortinii only in the forest. Phialocephala fortinii and L.orchidicola are DSE fungi distributed in a wide geographical area in many alpine and subalpine habitats. In addition, P. fortinii is considered to be the dominant root endophyte in forests [ 19]. The genus of Scolecobasidium was first described by Abbott in 1927 as two species, S. terreum and S. constrictum . The genus includes soil-borne and saprotrophytic species from plant litter [ 20–23]. Our study indicates that S. humicola is a dark septate endophyte in forests and agriculture fields, and is able toimprove plant growth in comparison with the control. A new endophytic species, S. humicola, was described for the first time in this paper. Endophyte screening To eliminate saprotrophic and/or pathogenic fungal isolates, Chinese cabbage seedlings were inoculated with 15 randomly selected isolates showing diverse morphology from each colonial morphology group. Seven isolates were originally obtained from the forest, 5 from a conventional field and 3 from an organic field. In the results of the inoculation test, only two of the 15 isolates tested (approximately 13 %) were notpathogenic to the Chinese cabbage seedlings. These two isolates, H2-2 and F1-3, caused no visible sign of disease or decay of the seedlings. The weight of dried plants was 56 ± 11 and 59 ± 14 mg for H2-2 and F1-3, respectively, and showed no significant difference compared to the control plants (68 ± 5 mg) (Figure 1). The most ineffective isolates (over 86%), once re-inoculated in to axenically-grown Chinese cabbage seedlings, caused extremely yellowing of leaves and suppression of plant growth (Figure 1). Colonies of 3 isolates, H2-2, F1-3 and O-MH, on OMA medium were similarly dark brown. The colonial growth of the 3 isolates was similarly slow (up to 15 mm in diameter after 4- week incubation at 23 °C) (Figure 2a). The conidiophores were micronematous, and the conidia rough-walled with one to three septata (Figure 2b). Mecelial hyphae were hyaline to brown, septate-forming densely coiled hyphae, resembling microsclerotia. These morphological characteristics were identical among the 3 isolates and were in agreement with the genus of Scolecobasidium . The ITS sequence of the three isolates showed 99%-100% similarity to Scolecobasidium humicola (NCBI/GenBank Accession No. DQ307332.1). Nitrogen source utilization and anatomic observations Uninoculated (control) plants could use NaNO 3, but could not effectively use three amino acids (valin, leucine, and phenylalanine). Alternatively, plants treated with three isolates, H2-2, F1-3, and O-MH, of S. humicola were able to use all 3 amino acids (Figure 3). The treated plants with H2-2 and F1-3 isolates were able to grow well on the medium amended with NaNO 3 as N source, but the treated plants with O-MH isolate showed yellowing leaves and decreased biomass by 50% on the same medium. The dry weight of treated plants with 3 isolates in Valin treatment was significantly high compared to the control. When phenylalanine was used with isolate F1-3, tomato plant biomass was significantly improved but not with 2 isolates H2-2 and O-MH. Optimum plant growth was observed when the plants were treated with H2-2 and F1-3 amended with leucine as a nitrogen source at 2% concentration. Many studies have suggested the involvement of DSE fungi in nutrient uptake to host plants and improved plant growth under natural conditions, i.e. a forest [ 12,13,19]. In addition, DSE fungi have been isolated from agriculture fields under nutrient-stressed conditions [ 24,25]. We successfully showed that tomato plants treated with S. humicola H2-2 and F1-3 were able to use amino acids and the plant biomass increased in Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org3 November 2013 | Volume 8 | Issue 11 | e78746 comparison with the control. This finding suggested that the ability of S. humicola as a plant growth-promoting fungus increased under organic nitrogen sources. This is not a unique feature of S. humicola as Heteroconium chaetospira could significantly improve plant biomass by transporting organic nitrogen to host plant [ 26]. Newsham [ 27] confirmed that the inoculated host plant with the selected DSE fungi responds more positively if there is no supply of available inorganic nitrogen. To determine the endophytic nature of S. humicola isolate H2-2, anatomical observation of tomato roots under NaNO 3 or leucine treatment was conducted. The hyphae of S. humicola colonized in epidermal cells heavily, but fungal hyphea were lightly colonized in outer cortical cells. No hyphae could be observed in the inner cortical cells or in the vascular cylinder under both treatments ( Figure 4). The fungal hyphae only produced conidia and microsclerotia-like-structures on the root surface under NaNO 3 treatment. In conclusion, although future detailed research is necessary to fully support our hypothesis, our study found for the first time that S. humicola is a common DSE species and may act as a key DSE species under both natural and agricultural conditions. Figure 1. Dry weight of Chinese cabbage inoculated with different fungal isolates. Dry weight of Chinese cabbage seedlings grown on basal media (10 g oatmeal and 18 g Bacto agar, enriched with nutrients 1 g MgSO 4·7H 2O, 1.5 g KH 2PO 4, and 1 g NaNO 3) inoculated with different fungal isolates. The filled columns represent the groups of selected fungi. Data are the means ± SE, n = 5. Asterisks represent significant differences between each treatment and the control (** P <0.01, * P <0.05) following Tukey’s honestlysignificant difference test. doi: 10.1371/journal.pone.0078746.g001 Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org4 November 2013 | Volume 8 | Issue 11 | e78746 Figure 2. Scolecobasidium humicola colony and conidia. Colony (A) and a light micrograph (B) of Scolecobasidium humicola after 14 days at 23°C grown on OMA medium. Arrowhead indicates a micronematous conidiophore with rough-walled septate conidia (arrows). Bars: A = 14 mm, B = 20 µm. (C) Tomato seedlings grown on basal media (OMA) amended with L-Leucine amino acid, the control on the right , and inoculated tomato seedlings with S.humicola on the left. doi: 10.1371/journal.pone.0078746.g002 Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org5 November 2013 | Volume 8 | Issue 11 | e78746 Figure 3. Dry weights of tomato inoculated with Scolecobasidium humicola . Dry weights of tomato seedlings grown on basal media (OMA) amended with four different nitrogen sources. white bar: control, black bar: Scolecobasidium humicola H2-2, black lines bar: S. humicola F1-3, and gray lines bar: S. humicola O-MH treatments. Data are the means ± SE, n = 5. Asterisks represent significant differences between each treatment and the control (P <0.05) following Tukey’s honestly significant difference test. doi: 10.1371/journal.pone.0078746.g003 Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org6 November 2013 | Volume 8 | Issue 11 | e78746 Figure 4. Interaction between tomato roots and Scolecobasidium humicola. Cross section of a tomato root stained with 0.005% cotton blue in 50% acetic acid 3 weeks after inoculation. The cortex (Co) mostly consists of three cell layers. Fungal hyphae can be seen on the root surface (arrows), within epidermal (Ep) cells. Vc = vascular cylinder. Bar = 20 µm. doi: 10.1371/journal.pone.0078746.g004 Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org7 November 2013 | Volume 8 | Issue 11 | e78746 Author ContributionsConceived and designed the experiments: RSM KN. Performedthe experiments: RSM. Analyzed the data: RSM. 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Finlay RD, Frostegård Å, Sonnerfeldt AM (1992) Utilization of organic and inorganic nitrogen sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl. ex Loud. New Phytol 120: 105–115. doi:10.1111/j.1469-8137.1992.tb01063.x. 15. Caldwell BA, Jumpponen A (2003) Arylsufatase production by mycorrhizal fungi. In: Fourth International Conference on Mycorrhizae, Montreal, Canada 312. 16. Narisawa K, Tokumasu S, Hashiba T (1998) Suppression of clubroot formation in Chinese cabbage by root endophytic fungus, Heteroconium Chaetospira . Plant Pathol 47: 206-210. doi: 10.1046/j. 1365-3059.1998.00225.x. 17. Narisawa K, Kawamata H, Currah RS, Hashiba T (2002) Suppression of Verticillium wilt in eggplant by some fungal root endophytes.Eur J Plant Pathol 108: 103-109. doi:10.1023/A:1015080311041. 18. Narisawa K, Usuki F, Hashiba T (2004) Control of Verticillium yellows in Chinese cabbage by the dark septate endophytic fungus LtVB3. Phytopathology 94: 412-418. doi: 10.1094/PHYTO.2004.94.5.412. PubMed: 18943758. 19. Addy HD, Hambleton S, Currah RS (2000) Distribution and molecular characterization of the root endophyte Phialocephala fortinii along an environmental gradient in the boreal forest of Alberta. Mycol Res104. 20. Abott EV (1927) Scolecobasidium, a new genus of soil fungi. Mycologia: 29-31. 21. Barron GL, Busch LV (1962) Studies on the soil hyphomycete Scolecobasidium . Can J Bot. 40: 77–84. doi:10.1139/b62-009. 22. Roy RY, Dwivedi RS, Mishra RR (1962) Two new species of Scolecobasidium from soil. Lloydia. 25: 164–166. 23. Grandi RAP, Gusmão LFP (2002) Decomposing hyphomycetes on leaf litter of Tibouchina pulchra . Cogn. Rev Bras Bot. 25: 79–87. 24. Priyadharsini P, Pandey RR, Muthukumar T (2012) Arbuscular mycorrhizal and dark septate fungal associations in shallot ( Allium cepa L. var. aggregatum ) under conventional agriculture. Acta Bot Croat 71 (1): 159–175. 25. Zubek S, Stefanowicz AM, Błaszkowski J (2012) Arbuscular mycorrhizal fungi and soil microbial communities under contrasting fertilization of three medicinal plants. Appl Soil Ecol 59: 106–115. doi: 10.1016/j.apsoil.2012.04.008. 26. Usuki F, Narisawa K (2007) A mutualistic symbiosis between a dark septate endophytic fungus, Heteroconium Chaetospira, and a nonmycorrhizal plant, Chinese cabbage. Mycologia, 99(2): 175–184. doi:10.3852/mycologia.99.2.175. PubMed: 17682770. 27. Newsham KK (2011) A meta-analysis of plant responses to dark septate root endophytes. New Phytol. doi: 10.1111/j. 1469-8137.2010.03611.x. Scolecobasidium humicola Promotes Tomato Growth PLOS ONE | www.plosone.org8 November 2013 | Volume 8 | Issue 11 | e78746
R E S E A R C H A R T I C L E Open Access Antibiotic use for Vibrio infections: important insights from surveillance data Kam Cheong Wong 1,2, Anthony M. Brown 2, Georgina M. Luscombe 2, Shin Jie Wong 1and Kumara Mendis 1* Abstract Background: There is a paucity of data on the in vivo efficacy of antibiotics for lethal Vibrio species. Analyses of long-term surveillance datasets may provide insights into use of antibiotics to decrease mortality. Methods: The United States Centers for Disease Control and Prevention (CDC) Cholera and Other Vibrio Illness Surveillance (COVIS) dataset from 1990 to 2010, with 8056 records, was analysed to ascertain trends in antibiotics use and mortality. Results: Two-thirds of patients (5243) were pr escribed antibiotics – quinolones (56.1 %), cephalosporins (24.1 %), tetracyclines (23.5 %), and penicillins (15.4 %). Considering all Vibrio species, the only class of antibiotic associated with reduced odds of mortality was quinolone (od ds ratio 0.56, 95 % CI 0.46-0.67). Patients with V. vulnificus treated according to CDC recommendations had lower mortality (quinolone alone: 16.7 %, 95 % CI 10.2-26.1; tetracycline plus cephalosporin: 21.7 %, 16.8-27.5; no antibiotic: 51.1 %, 45.6-56.7; each p < 0.001). Cephalosporin alone was associated with higher mortality (36.8 %, 28.2-46.3). For V. cholerae non-O1, non-O139, mortality rates were lower for quinolone (0 %, 0 –2.0) or tetracycline (4.3 %, 1.2-14.5) compared to no antibiotic (9.3 %, 6.4-13.3). For all Vibrio species, mortality rates increased with number of antibiotics in the treatment regimen (p < 0.001). Treatment regimens that included quinolone were associ ated with lower mortality rates regardless of the number of antibiotics used. The main cli nical syndromes of patients with V. vulnificus infection were septicaemia (53.1 %) and wound infections (30.6 %). Mortality among V. vulnificus patients with septicaemia was significantly higher than for other clinical syndromes (p < 0.001). In a multivar iate regression model, mortality in cases with V. vulnificus was associated with presence of pre-existing conditions (ORs ranged from 4. 52 to 10.30), septicaemia (OR 2.64, 95 % CI 1.92-3.63) and no antibiotic treatment (OR 7.89, 95 % CI 3.94-15.80). Conclusion: In view of the lack of randomized control trials, surveillance data may inform treatment decisions for potentially lethal Vibriosis. Considering all Vibrio species, use of quinolones is associated with lower mortality and penicillin alone is not particularly effective. For the most lethal species, V. vulnificus , treatment that includes either quinolone or tetracycline is associated wi th lower mortality than cephalosporin alone . We recommend treating patients who present with a clinical syndrome suggestive of V. vulnificus infection with a treatment reg imen that includes a quinolone. Keywords: Vibrio infection, Vibriosis, Antibiotics, Antimicrobials, Surveillance * Correspondence: [email protected] 1Bathurst Rural Clinical School, School of Medicine, University of Western Sydney, PO Box 9008, Bathurst, NSW 2795, AustraliaFull list of author information is available at the end of the article © 2015 Wong et al. BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated. Wong et al. BMC Infectious Diseases (2015) 15:226 DOI 10.1186/s12879-015-0959-z Background The incidence of Vibrio infections, which can cause acute diarrhoea and potentially serious complications such as hypovolemic shock and septicaemia, continues to rise in the United States . Vibrio vulnificus is the most lethal species, and there are limited data on the ef- fectiveness of antibiotic use in V. vulnificus infections . In 1988, the Centers for Disease Control and Pre- vention (CDC) in the United States established a surveil- lance system for human infections caused by all species of Vibrio known as “Cholera and Other Vibrio Illness Surveillance ”(COVIS) . This surveillance information has been used to inform healthcare providers and edu- cate the public . Clinical trials indicate that using an effective antibiotic as an adjuvant therapy to treat V. cholerae reduces the duration of diarrhoea [5, 6] and illness by almost 50 % in patients with moderate and severe dehydration . Stud- ies of treatment efficacy for lethal Vibrio species such as V. vulnificus , have inherent ethical difficulties and conse- quently there are no randomised control trials (RCT) of V. vulnificus in humans . Animal model in vivo stud- ies of antibiotic sensitivities to Vibrio infection may not all be applicable to humans due to differences in phar- macokinetic parameters . Recommendations for V. vulnificus infections from a recent review are largely based on case reports or animal models . The CDC provides recommendations on treatment regimens for V. vulnificus ,V. parahaemolyticus and V. cholerae non-O1, non-O139 . However only recommendations on treat- ing V. cholerae are based on a human RCT . Observational studies on lethal Vibrio infections, using established surveillance data over a long defined period of time, may provide insights about the associations be- tween the use of antibiotics and patient outcomes. For example, Purcell et al. conducted a systematic review of the use of prophylactic antibiotics in the prevention of meningococcal disease and had to rely almost entirely on surveillance data to support their use . Dechet and colleagues reviewed non-foodborne Vibrio infections using COVIS data (1997 to 2006) and concluded that the optimal antibiotic treatment for Vibrio infections re- mains unknown . We have analysed COVIS data from 1990 to 2010: (a) to ascertain whether the COVIS data on antibiotic use were consistent with the CDC treatment recommenda- tions, and (b) to determine the relationship between antibiotic treatment and mortality. Methods Study design and participants Data were obtained from the CDC ’s Enteric Diseases Epidemiology Branch from case report forms submitted to the COVIS during 1990 to 2010 . COVIS data are collected using a standardised form . The COVIS dataset included information on demographics, the Vibrio species isolated and the source of the specimen, clinical features (symptoms and signs), mortality, and pre-existing conditions and treatments during the 30 days prior to the Vibrio illness. The dataset also in- cluded information on whether or not the patient had an antibiotic as treatment for the Vibrio illness (i.e. yes, no, unknown), and if so, the name of the antibiotic/s. Free text records of antibiotic names and some pre- existing conditions and treatments (specifically malig- nancy, immune disorders, proton pump inhibitors and antacids) were reviewed by authors KCW and SJW inde- pendently and classified using the Systematized Nomen- clature of Medicine – Clinical Terms (SNOMED CT®) system (SNOMED Premium Version 1.0) and then reviewed jointly to reach consensus . Pre-existing conditions included heart disease, diabetes, liver disease, alcoholism, malignancy, renal disease, haematological disease and immune disorders. SNOMED CT® codes were used to classify antibiotics into antimicrobial classes (e.g. quinolone, cephalosporin), antimicrobial subclasses (e.g. first, second and third generation cephalosporins) and to standardise and review pre-existing conditions and treatments (e.g. free text records of gastric surgery were reviewed and those considered irrelevant, such as hernia repair, were removed). There were 8950 patients in the COVIS dataset, 6137 (68.6 %) of whom were recorded as having had antibi- otics as treatment for their Vibrio illness. However, the specific name of the antibiotic was not recorded for 894 (14.6 %). A sensitivity analysis, whereby results were compared for analyses which either included or excluded the 894 patients who had an unnamed antibiotic for the Vibrio illness, was performed. There were no substantial differences in terms of their epidemiological information and mortality rates, and consequently the patients with- out a named antibiotic were excluded from subsequent analyses, resulting in a final sample size of N = 8056. Statistical analysis For the purposes of analysis, where antibiotic use was recorded in COVIS as ‘unknown ’this was recoded as ‘ no ’antibiotic used. Similarly, where data were missing or recorded as ‘unknown ’for clinical signs or symptoms, pre-existing conditions or treatments, or mortality, they were recoded as ‘absent ’or ‘no ’for analysis. Based on re- ported symptoms, patients with V. vulnificus were classi- fied into the following clinical syndrome groups: (i) septicaemia, characterised by the isolation of the organ- ism from blood AND the presence of either fever or shock; (ii) gastroenteritis, defined as the presence of blood in stool, OR both diarrhoea and vomiting, OR any gastrointestinal symptom AND the isolation of organism Wong et al. BMC Infectious Diseases (2015) 15:226 Page 2 of 9 from stool; (iii) wound infection, characterised by cellu- litis OR bullae OR fever in the absence of septicaemia, where the organism was isolated from a wound only; or (iv) other (not meeting any of the other criteria). Where a patient met the criteria for more than one clinical syn- drome, septicaemia took precedence over gastroenteritis, and gastroenteritis over a wound infection. Skewed continuous data were reported as medians with an interquartile range (IQR). Associations between demographics, antibiotic use and mortality were ex- plored using unadjusted odds ratios with 95 % confi- dence intervals. Chi-square analyses explored univariate associations between factors such as antibiotic use and mortality. The association between the total number of antibiotics used and mortality was explored using the linear-by-linear association chi-square test. A series of univariate logistic regression models were conducted on the sub-sample of patients with V. vulnificus to deter- mine predictors of mortality. Variables in these regres- sion analyses included age, gender, year of notification, number of pre-existing conditions, clinical syndrome presentation and type of antibiotic regimen (quinolone only; quinolone and another antibiotic; at least one anti- biotic, but not quinolone; or no antibiotic at all). Un- adjusted odds ratios (OR) and 95 % confidence intervals (CIs) were produced. A multivariate logistic regression analysis was also conducted, including all of these vari- ables, to produce adjusted ORs and 95 % CIs. All analyses were conducted using SPSS (version 21; IBM, 2012) and αwas set at p < 0.05. Ethics committee approval All data were de-identified and the CDC confirmed that ethics approval and informed consent were not applic- able for this research. Results Of the 8056 patients, the median age was 47 years (IQR 33 to 62 years; N = 7773). Over two-thirds of the patients were male (68.6 %; 5424/7905) and 98.3 % (7921/8056) of the patients had only one Vibrio species identified. 127 (1.6 %) patients had two species and eight (0.1 %) patients had three Vibrio species identified. The most commonly identified species were V. parahaemolyticus, V. vulnificus, V. alginolyticus and the non-O group strains of V. cholerae (Table 1). The proportion of fatal cases differed by Vibrio species, the greatest being in those with V. vulnificus (Table 1). Cases of V. parahaemolyticus in- creased dramatically over time, with peaks in 1998, 2004, 2006 and 2009 –10, and there was a steady increase in cases of V. alginolyticus (Fig. 1). Peak incidence typically occurred during the summer months (June to August, 52.6 % of cases). There were 5243 (65.1 %) patients who had an anti- biotic as treatment for their Vibrio illness. The most commonly used antibiotics were quinolones (56.1 %), followed by cephalosporins (24.1 %), tetracyclines (23.5 %) and penicillins (15.4 %). The use of quinolones in- creased considerably after 1996 with peaks around 1997, 2005, and 2010, while the use of cephalosporin, tetracycline and penicillin rose only slowly from 1990 to 2010 (Fig. 2). A total of 602 (7.5 %) patients with a Vibrio illness were recorded as deceased. Mortality was significantly associated with being male (OR 2.10, 95 % CI 1.70-2.59). Bullae (present in 7.3 % of cases), and shock (6.5 %) were associated with significantly increased odds of mortality (OR 8.67, 95 % CI 7.11-10.58; OR 28.49, 95 % CI 23.18- 35.01 respectively). Presence of at least one of the eight pre-existing conditions (33.2 %) significantly increased risk of death (OR 16.83, 95 % CI 13.18-21.49); in par- ticular, a history of liver disease (10.6 %; OR 18.81, 95 % CI 15.63-22.63) and alcoholism (8.9 %; OR 13.50, 95 % CI 11.19-16.27). The association between antibiotic classes (use of at least one antibiotic of a particular class in a treatment regimen) and mortality is presented by Vibrio species in Table 2. Considering all patients, regardless of Vibrio species, the only class of antibiotic associated with re- duced odds of mortality was quinolone (OR 0.56, 95 % Table 1 Vibrio species, mortality and gender distribution; United States cases during 1990 to 2010 (N = 8056) Vibrio species Total n (%) Mortality n (%) Male n (%) V. parahaemolyticus 3474 (43.1) 29 (0.8) 2243 (66.1) V. vulnificus 1599 (19.8) 491 (30.7) 1367 (86.4) V. alginolyticus 874 (10.8) 11 (1.3) 589 (69.1) V. cholerae non-O1, non-O139 763 (9.5) 41 (5.4) 474 (62.9) V. fluvialis 445 (5.5) 13 (2.9) 243 (56.0) V. cholerae O1 269 (3.3) 4 (1.5) 137 (51.1) V. mimicus 211 (2.6) 5 (2.4) 116 (55.0) V. hollisae a 131 (1.6) 1 (0.8) 79 (61.2) P. damsela b 63 (0.8) 3 (4.8) 45 (71.4) V. cholerae O139 28 (0.3) 0 (0.0) 15 (57.7) V. furnissii 19 (0.2) 1 (5.3) 13 (76.5) V. metschnikovii 14 (0.2) 1 (7.1) 7 (53.8) V. cincinnatiensis 2 (<0.1) 0 (0.0) 1 (50.0) Species not identified 279 (3.5) 9 (3.2) 169 (61.7) Other, no further information 28 (0.3) 0 (0.0) 16 (64.0) Total n/a 602 (7.5) 5424 (67.3) N.B. some patients had more than one species isolated, so numbers total to N=8199Proportions here are of the total patients, not total species identifiedInformation on gender was missing for n=151aFormerly Vibrio hollisae , now Grimontia hollisae  bFormerly Vibrio damsela , now Photobacterium damsela Wong et al. BMC Infectious Diseases (2015) 15:226 Page 3 of 9 CI 0.46-0.67). This association held for those patients with V. vulnificus (OR 0.58, 95 % CI 0.46-0.73), and V. cholerae (non-O1, non-O139) (OR 0.12, 95 % CI 0.04- 0.40). Cephalosporin was associated with an increased odds of mortality (OR 2.60, 95 % CI 2.16-3.13) overall, and notably for V. parahaemolyticus (OR 6.41, 95 % CI 2.89-14.24). For those patients with V. vulnificus infec- tion, use of the antibiotics quinolone, cephalosporin and tetracycline was associated with significantly lower mor- tality rate while use of penicillin was equivocal. There was a statistically si gnificant positive associ- ation between the mortality rates and the number of antibiotics used in a treatment regimen (linear by linear association = 172.90, p < 0.001). Irrespective of Vibrio species, a treatment regimen that included a quinolone was always associated with lower mortality rate regard- less of the total number of antibiotics in the treatment regimen (Fig. 3a). Whether or not the treatment regimen included a cephalosporin, tetracycline, or penicillin was not associated consistently with the mortality rate (Fig. 3b, c, d). Tables 3 and 4 show the mortality associated with vari- ous antibiotic regimens (including CDC recommenda- tions) for treating Vibrio vulnificus and Vibrio cholerae non-O1, non-O139 infections. Mortality rates for V. vul- nificus were significantly lower in those patients taking quinolone only or tetracycline combined with a third generation cephalosporin as per CDC recommendations (Table 3). Using quinolone as a reference, comparison between the CDC recommended combination (i.e. tetra- cycline and third generation cephalosporin) and other combinations that included quinolone, showed no statis- tically significant differences, except the comparison with taking a cephalosporin alone or a penicillin alone or no antibiotic (p = 0.002, p = 0.024, p < 0.001 respect- ively; Table 3). For V. cholerae (non O1 and non-O139) , only use of quinolone alone was associated with a sig- nificantly lower mortality rate (Table 4). 0 50 100 150 200 250 300 350 400 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Number of cases V. parahaemolyticus V. vulnificus V. alginolyticus V. cholerae non-O1, non-O139 V. cholerae O1 V. cholerae O139 Fig. 1 Patients with Vibrio infections over time, 1990 –2010, in United States 0 50 100 150 200 250 300 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Number of patients Year Quinolones Cephalosporins Tetracyclines Penicillins Fig. 2 Patterns of antibiotic use in the United States for patients with Vibrio illness: 1990 to 2010 (N = 5243) Wong et al. BMC Infectious Diseases (2015) 15:226 Page 4 of 9 Considering only V. vulnificus, 53.1 % (839/1581) had septicaemia, 4.5 % (71) had gastroenteritis, 30.6 % (484) had a wound infection, and 11.8 % (187) had other clin- ical presentation. Eighteen cases were excluded because they could not be classified as information on the sam- ple source was missing. Cases with septicaemia were more likely to fatal (40.6 %, 341/839), followed by 28.9 % (54) of those with other presentation, 28.2 % (20) of those with gastroenteritis and 14.3 % (69) of those with wound infections (p < 0.001). A series of logistic regres- sion analyses with single predictor variables was con- ducted, with year of notification dichotomised into 1990 to 1996 versus 1997 to 2010 to reflect the significant in- crease in quinolone use from 1996 observed in Fig. 2. The crude odds of mortality amongst those with V. vul- nificus increased significantly with the number of pre- existing conditions (see Table 5). In comparison to wound infections, all other clinical syndromes conferred increased odds of dying. Compared to quinolone alone, an antibiotic regimen without quinolone, and no anti- biotic use at all were associated with increased odds of mortality. The multivariate analysis on mortality for pa- tients with V. vulnificus infection showed the same patterns for number of pre-existing conditions. Gastroenteritis no longer conferred significantly greater odds of dying over wound infections, but septicaemia and other clin- ical syndrome did. In the adjusted model, quinolone alone remained superior in comparison to no antibiotic treatment. Discussion Using this large surveillance dataset covering more than two decades, we determined that the two most import- ant factors associated with mortality are the particular Vibrio species and the class of antibiotic used. Quin- olone is the only class of antibiotic associated with lower mortality in all Vibrio species, regardless of the number of antibiotics used in a treatment regimen. In potentially lethal V. vulnificus infections, the use of quinolone alone or the combination of tetracycline and third generation cephalosporin had the lowest mortality which is in-line with the current CDC recommendations. Penicillin was the least effective antibiotic for vibriosis according to our analysis . In 1984, the USA National Institutes of Health recommended use of penicillin or tetracycline to treat V. vulnificus based on in vitro sen- sitivity studies . Morris cautioned that in vitro data can be misleading, and recommended against using penicillin as a single antibiotic to treat V. vulnificus in- fections in humans . In 2002, Tang conducted a study on mice and reported that quinolones as single antibiotics were as effective as cefotaxime-minocycline (third generation cephalosporin and tetracycline) in combination . However Tang cautioned that results of animal models may not be applicable to humans due to differences in pharmacokinetic parameters . Our findings support Morris ’s caution against penicillin as a single antibiotic (i.e. penicillin was not associated with reduced mortality) and substantiate Tang ’s previous find- ings in animals (i.e. third generation cephalosporin and Table 2 Mortality and antibiotic use, by type of Vibrio illness (N = 8056) All Vibrio species Alive n = 7454 Deceased n = 602 Crude Odds Ratio (95% CI) Received at least one antibiotic 65.1% 64.6% 0.98 (0.82-1.16) At least one quinolone 37.4% 24.9% 0.56 (0.46-0.67) At least one cephalosporin 14.5% 30.6% 2.60 (2.16-3.13) At least one tetracycline 14.5% 25.1% 1.98 (1.63-2.40) At least one penicillin 9.5% 15.8% 1.78 (1.41-2.25) V. parahaemolyticus n = 3445 n = 29 Received at least one antibiotic 56.5% 65.5% 1.46 (0.68-3.15) At least one quinolone 39.8% 37.9% 0.93 (0.44-1.97) At least one cephalosporin 6.6% 31.0% 6·41 (2.89-14.24) At least one tetracycline 7.3% 20.7% 3.29 (1.33-8.16) At least one penicillin 4.6% 3.4% 0.75 (0.10-5.53) V. vulnificus n = 1108 n = 491 Received at least one antibiotic 86.6% 68.2% 0.33 (0.26-0.43) At least one quinolone 37.1% 25.5% 0.58 (0.46-0.73) At least one cephalosporin 42.3% 33.2% 0.68 (0.54-0.85) At least one tetracycline 47.5% 27.3% 0.42 (0.33-0.52) At least one penicillin 18.5% 17.7% 0.95 (0.72-1.25) V. alginolyticus n = 863 n = 11 Received at least one antibiotic 76.0% 36.4% 0.18 (0.05-0.62) At least one quinolone 29.7% 18.2% 0.53 (0.11-2.46) At least one cephalosporin 23.6% 18.2% 0.72 (0.15-3.35) At least one tetracycline 10.0% 0.0% n/a At least one penicillin 19.1% 9.1% 0.42 (0.05-3.33) V. cholerae non-O1, non- O139 n = 722 n = 41 Received at least one antibiotic 66.1% 39.0% 0.33 (0.17-0.63) At least one quinolone 39.3% 7.3% 0.12 (0.04-0.40) At least one cephalosporin 9.4% 19.5% 2.33 (1.04-5.25) At least one tetracycline 12.3% 19.5% 1.72 (0.77-3.85) At least one penicillin 9.6% 7.3% 0.75 (0.23-2.48) n/a not applicable Wong et al. BMC Infectious Diseases (2015) 15:226 Page 5 of 9 tetracycline in combination are more effective). More re- cently Shaw and colleagues evaluated the antimicrobial susceptibility of V. vulnificus recovered from two commer- cial environmental areas and found that V. vulnificus dem- onstrated resistance to penicillin , which may further confirm the inefficacy of penicillin. For V. cholerae (non-O1, non-O139), quinolone was the only class of antibiotic associated with lower mortality rate. For V. parahaemolyticus, quinolone and penicillin appeared to have equivocal odds ratios for mortality; while cephalosporin and tetracycline were associated with higher mortality. However, the total number of deaths from V. cholerae was 41 and for V. parahaemolyticus was 29, so conclusions are limited. Generally, the mortality rate increased along with the number of antibiotics used in the treatment regimen. We postulated that seriously ill patients were given more than one antibiotic and were associated with increased Fig. 3 aMortality by use of quinolone in treatment regimen. bMortality by use of cephalosporin in treatment regimen. cMortality by use of tetracycline in treatment regimen. dMortality by use of penicillin in treatment regimen. ** indicates a statistically significant difference at p < 0.001, * indicates a statistically significant difference at p < 0.05. n = 3132 used one antibiotic; 1305 used two antibiotics; 652 used three antibiotics; 154 used four antibiotics Table 3 Mortality associated with various antibiotic regimens in the treatment of V. vulnificus (n=1599) Na Crude mortality % (95% CI) Comparison with Quinolone only p value CDC treatment recommendation b Quinolone only 14/84 16.7 (10.2-26.1) – Tetracycline + cephalosporin (all generations) 49/226 21.7 (16.8-27.5) 0.329 Tetracycline + 3 rdgeneration cephalosporin 39/182 c 21.4 (16.1-28.0) 0.366 Other combinations Quinolone + cephalosporin (all generations) 24/98 24.5 (17.1-33.9) 0.195 Quinolone + tetracycline 15/95 15.8 (9.8-24.4) 0.874 Quinolone + cephalosporin + tetracycline 15/87 17.2 (10.7-26.5) 0.920 Other single antibiotics Tetracycline alone 30/145 20.7 (14.9-28.0) 0.456 Cephalosporin alone (all generations) 39/106 36.8 (28.2-46.3) 0.002 Penicillin alone 18/54 33.3 (22.2-46.6) 0.024 No antibiotic 156/305 51.1 (45.6-56.7) <0.001 an=399 patients with less common antibiotic regimens not included here, thus numbers do not total to n=1599bhttp://www.cdc.gov/vibrio/vibriov.html (accessed 27 June 2014): “Doxycycline (100 mg PO/IV twice a day for 7-14 days) and a third-generation cephalosporin (e.g. ceftazidime 1-2 g IV/IM every eight hours) are generally recommended. A single agent regimen with a fluoroquinolone such as levofloxacin, ciproflox acin or gatifloxacin, has been reported to be at least as effective in an animal model as combination drug regimens with doxycycline and a cephalosporin ” cn=182 patients are a subset of the n=226 patients with a tetracycline combined with a cephalosporin Wong et al. BMC Infectious Diseases (2015) 15:226 Page 6 of 9 number of antibiotics in their treatment; hence, their mortality rate was higher possibly because they were sicker instead of due to the larger number of antibiotics used. We found that quinolone was the only antibiotic associated with reduced mortality rate regardless of the number of antibiotics in the patient ’s regimen. We have shown an increase in use of quinolone after 1996 but this may be a reflection of increased use in the wider community. Linder and colleagues have reported a three-fold increase in prescribing quinolone between 1995 and 2005 in the United States adult population . This increase in quinolone prescription may not necessarily reflect increased recognition of the efficacy of this antibiotic amongst prescribers or the promulga- tion of treatment guidelines. It is likely that marketing, advertising and provision of sample antibiotics might have partly contributed to the increase in prescribing of newer antibiotics [15 –17]. We found associations between liver disease, alcohol- ism and previous ill health and mortality with all species. For V. vulnificus the number of pre-existing conditions was associated with increased odds of mortality in both unadjusted and adjusted models. Others have found that patients with liver disease or alcoholism are at higher risk of V. vulnificus infection [10, 18, 19]. This may be Table 4 Mortality associated with various antibiotic regimens in the treatment of V. cholerae non-O1, non-O139 (n=763 a) N Crude mortality % (95% CI) Comparison with Tetracycline aloneFET p value CDC treatment recommendation b Tetracycline alone 2/46 4.3 (1.2-14.5) – Other single antibiotics Quinolone alone 0/ 192 0.0 (0.0-2.0) 0.037 Penicillin alone 1/38 2.6 (0.5-13.5) 1.000 Cephalosporin alone 2/29 6.9 (1.9-22.0) 0.638 No antibiotic 25/ 270 9.3 (6.4-13.3) 0.395 FET fisher ’s exact test an=188 patients with less common antibiotic regimens not included here, thus numbers do not total to n=763bhttp://www.cdc.gov/cholera/treatment/antibiotic-treatment.html (accessed 27 June 2014): “Antibiotic choices should be informed by local antibiotic susceptibility patterns. In most countries, doxycycline is recommended asfirst-line treatment for adults, while azithromycin is recommended as first-linetreatment for children and pregnant women ” Table 5 Predictors of death in cases of V. vulnificus in the United States during 1990 to 2010 Fatal N (%) Non-fatal N (%) Crude OR (95% CI) Adjusted OR (95% CI) Age, mean (SD) 56.6 (13.5) 58.0 (17.8) 0.995 (0.989-1.001) 0.99 (0.98-0.998) Gender Female 72 (15.2) 135 (12.6) reference reference Male 402 (84.8) 939 (87.4) 0.80 (0.59-1.09) 0.70 (0.49-1.000) Year of notification 1990 –1996 103 (21.7) 194 (18.1) reference reference 1997 –2010 371 (78.3) 880 (81.9) 0.79 (0.61-1.04) 0.99 (0.73-1.34) Pre-existing conditions 0 41 (8.6) 397 (37.0) reference reference 1 107 (22.6) 277 (25.8) 3.74 (2.53-5.53) 4.52 (2.92-6.99) 2 194 (40.9) 227 (21.1) 8.28 (5.69-12.03) 10.30 (6.72-15.78) 3 or more 132 (27.8) 173 (16.1) 7.39 (4.99-10.95) 9.31 (5.93-14.62) Clinical presentation Wound 68 (14.3) 406 (37.8) reference reference Gastroenteritis 20 (4.2) 49 (4.6) 2.44 (1.36-4.35) 1.52 (0.77-3.01) Septicaemia 333 (70.3) 493 (45.9) 4.03 (3.01-5.40) 2.64 (1.92-3.63) Other 53 (11.2) 126 (11.7) 2.51 (1.67-3.79) 1.69 (1.04-2.74) Antibiotic treatment Quinolone only 13 (2.7) 68 (6.3) reference reference Quinolone and other/s 106 (22.4) 335 (31.2) 1.66 (0.88-3.12) 1.44 (0.73-2.84) Antibiotic/s, not quinolone 208 (43.9) 534 (49.7) 2.04 (1.10-3.77) 1.84 (0.95-3.56) None 147 (31.0) 137 (12.8) 5.61 (2.97-10.62) 7.89 (3.94-15.80) Wong et al. BMC Infectious Diseases (2015) 15:226 Page 7 of 9 because V. vulnificus uses transferrin-bound iron, which is usually abundant in these patients, for growth [18, 20]. Another hypothesis about the increase mortality in the presence of liver disease is that the shunting of por- tal blood containing V. vulnificus infection around a dis- eased liver may lead to septicaemia . The main clinical syndromes in patients with V. vulnificus infec- tion were septicaemia and wound infections. Mortality among V. vulnificus patients with septicaemia was sig- nificantly higher than for other clinical syndromes. A treatment regimen that included quinolone was associ- ated with lower mortality compared with cephalosporin alone or penicillin alone or no antibiotic at all. We rec- ommend that patients who present with a clinical syn- drome suggestive of V. vulnificus infection be treated with a regimen that includes a quinolone. There are a number of limitations to this study that re- late to the underlying surveillance data collection. For example, non-cholera vibriosis only became nationally notifiable in USA from 2007 onwards . Several per- tinent details regarding use of antibiotics were not re- corded systematically such as timing or order of antibiotic use. There were 894 patients who had an un- named antibiotic for the Vibrio infection. Cephalospo- rins could not be categorised into the four generations due to the small subgroup size. This may have contrib- uted to the paradoxical observation in Table 2 where a treatment regimen that had included a cephalosporin was associated with increased mortality rate in V. chol- era (non-O1 and non-O139) and V. parahaemolyticus in- fections, but reduced mortality elsewhere. The dataset did not detail specific cause of death, but an infection such as Vibrio is likely to have been significant. Further characterisation of liver disease (type and severity) was not possible due to the inconsistency of these data in the COVIS dataset. Finally, it is possible that the use and ef- fectiveness of different antibiotics reflect changes in the antibiotic sensitivities of the organism, however we are unable to explore this with the data available in the COVIS dataset. Determining the optimal antibiotic regimen for a po- tentially lethal infection is difficult because randomised controlled trials may not be possible and in vitro or ani- mal models may not be easily applied in patients. In this context, systematic experience from detailed surveillance data may inform treatment decisions. However informa- tion from surveillance data is only as good as the data collected. The COVIS dataset may not have included every Vibrio cases and reporting may have been biased towards more severe cases . It is important that cli- nicians provide detailed and timely data to surveillance programs such as COVIS. Publication of findings from surveillance data may help encourage clinicians to pro- vide specific data regarding antibiotic use. Surveillance authorities must be encouraged to simplify and refine the data collection tools and seek more specific informa- tion on classes of antibiotics and the time frame of their use so that the influence of these important factors can be reported and analysed. Conclusions Surveillance of large numbers of affected individuals over longer periods of time appears to be a reasonable method of determining antibiotic use and outcome pat- terns. Vibrio infection remains a serious condition with significant mortality. Adjuvant antibiotic therapy in addition to basic care with fluids has an important role. The use of quinolones may reduce risk of death in pa- tients with V. vulnificus and V. cholerae (non-O1 and non-O139). For V. vulnificus , which has the highest mortality rate, a treatment regimen which is in line with the CDC recommendations i.e. that includes either quin- olone alone, or tetracycline and a third generation ceph- alosporin is associated with lower mortality. Penicillin alone is not particularly effective. We recommend treat- ing patients who present with a clinical syndrome sug- gestive of V. vulnificus infection with a treatment regimen that includes a quinolone. Competing interests No authors have any commercial or other association that might pose aconflict of interest (e.g., pharmaceutical stock ownership, consultancy,advisory board membership, relevant patents, or research funding). The University of Western Sydney and the University of Sydney have no conflict of interest in the content and publication of this paper. Authors ’contributions All authors participated in the conceptualisation of the research objectives and methods. KCW and SJW performed the coding of variables with freetext fields using SNOMED-CT. GL managed and analysed the data. KCW, AB,and GL wrote the manuscript; and all authors reviewed and approved the manuscript. Acknowledgements We would like to thank Dr Hazel Dalton at the School of Rural Health, University of Sydney for her assistance in the research grant application; Ms Anna Newton at the CDC for providing access to the COVIS dataset; and theUniversity of Western Sydney (UWS) for providing an Early Career ResearchGrant to KCW; and Professor Annemarie Hennessy (Dean of the School of Medicine, University of Western Sydney) for her valuable comments to our manuscript. Declaration The content of this manuscript has not been presented in any meeting at the time of submission of this manuscript to the journal. Funding This work was supported by the University of Western Sydney (Conjoint Early Career Research Grant awarded to author KCW). Author details1Bathurst Rural Clinical School, School of Medicine, University of Western Sydney, PO Box 9008, Bathurst, NSW 2795, Australia. 2School of Rural Health, Sydney Medical School, University of Sydney, PO Box 1191, Orange 2800 NSW,Australia. Received: 12 November 2014 Accepted: 21 May 2015 Wong et al. BMC Infectious Diseases (2015) 15:226 Page 8 of 9 References1. Newton A et al. Increasing rates of vibriosis in the United States, 1996 –2010: review of surveillance data from 2 systems. Clin Infect Dis.2012;54 Suppl 5:S391 –5. 2. Daniels NA. Vibrio vulnificus oysters: pearls and perils. Clin Infect Dis. 2011;52(6):788 –92. 3. Centers for Disease Control and Prevention. National Cholera and Vibriosis Surveillance . 2014 27 June 2014]; Available from: http://www.cdc.gov/ nationalsurveillance/cholera-vibrio-surveillance.html. 4. Centers for Disease Control and Prevention. Vibrio Illness ( Vibriosis) . 2013 27 June 2014]; Available from: http://www.cdc.gov/vibrio/index.html. 5. Greenough 3rd W et al. Tetracycline in the treatment of cholera. Lancet. 1964;1(7329):355. 6. Nelson EJ et al. Antibiotics for both moderate and severe cholera. N Engl J Med. 2011;364(1):5 –7. 7. Tang HJ et al. In vitro and in vivo activities of newer fluoroquinolones against Vibrio vulnificus. Antimicrob Agents Chemother. 2002;46(11):3580 –4. 8. Horseman, M.A. and S. Surani. A comprehensive review of Vibrio vulnificus: an important cause of severe sepsis and skin and soft-tissue infection .Int J Infect Dis. 15(3): p. e157-66. 9. Purcell B et al. Effectiveness of antibiotics in preventing meningococcal disease after a case: systematic review. BMJ. 2004;328(7452):1339. 10. Dechet AM et al. Nonfoodborne Vibrio infections: an important cause of morbidity and mortality in the United States, 1997 –2006. Clin Infect Dis. 2008;46(7):970 –6. 11. International Health Terminology Standards Development Organisation. SNOMED CT . 2014 27 June 2014]; Available from: http://www.ihtsdo.org/ snomed-ct/. 12. Highly invasive new bacterium isolated from US east coast waters. JAMA, National Institutes of Health. 1984. 251(3): p. 323 –5. 13. Morris Jr JG, Tenney J. Antibiotic therapy for Vibrio vulnificus infection. JAMA. 1985;253(8):1121 –2. 14. Shaw, K.S., et al. Antimicrobial susceptibility of vibrio vulnificus and vibrio parahaemolyticus recovered from recreational and commercial areas of chesapeake bay and maryland coastal bays PLOS One, 2014. 9(2):e89616. 15. Linder JA et al. Fluoroquinolone prescribing in the United States: 1995 to 2002. Am J Med. 2005;118(3):259 –68. 16. Wazana A. Physicians and the pharmaceutical industry: is a gift ever just a gift? JAMA. 2000;283(3):373 –80. 17. Chew LD et al. A physician survey of the effect of drug sample availability on physicians ’behavior. J Gen Intern Med. 2000;15(7):478 –83. 18. Bullen JJ et al. Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch Intern Med. 1991;151(8):1606 –9. 19. Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981 –1993. J Infect Dis. 1996;173(5):1176 –83. 20. Brennt CE et al. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J Infect Dis.1991;164(5):1030 –2. 21. Blake PA et al. Disease caused by a marine Vibrio Clinical characteristics and epidemiology. N Engl J Med. 1979;300(1):1 –5. 22. Weis, K.E., et al. Vibrio illness in Florida, 1998 –2007 .Epidemiol Infect. 2011;139(4): p. 591 –8. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Wong et al. BMC Infectious Diseases (2015) 15:226 Page 9 of 9
Vibrio illness in Florida, 1998–2007 K. E. W E I S 1,2*, R. M. H A M M O N D 2,R.HUTCHINSON 2 AND C. G. M. B L A C K M O R E 2 1Council of State and Territorial Epidemiologists Applied Epidemiology Fellowship, Atlanta, GA, USA2Bureau of Environmental Public Health Medicine, Division of Environmental Health, Florida Department of Health, Tallahassee, FL, USA (Accepted 18 May 2010; ﬁrst published online 14 June 2010) SUMMARY This study characterized the current epidemiology of vibrio infections in Florida and examined cases reported from 1998 to 2007. Logistic regression was used to determine risk of death. There were 834 vibrio infections in 825 individuals (average annual incidence rate 4. 8/1 000 000). CommonVibriospecies reported wereVibrio vulniﬁcus(33 %),V. parahaemolyticus(29 %), and V. alginolyticus(16 %). Most exposures were attributed to wounds (42 %), and the most common clinical syndromes were wound infections (45 %) and gastroenteritis (42 %). Almost half of individuals reported an underlying health condition. Risk of death was associated with any underlying condition and increased with the number of conditions (P<0. 0001). In Florida, incidence of vibriosis associated with raw oyster consumption has decreased while incidence associated with wound infections has increased. Most prevention eﬀorts to date have focused on oyster consumption. New educational messages focusing on the risk of vibriosis from wound infections should target high-risk populations. Key words: Epidemiology, infectious disease,Vibrio vulniﬁcus. INTRODUCTION Vibrio are Gram-negative, rod-shaped bacteria com- monly found in warm coastal waters worldwide [1, 2]. The Centers for Disease Control and Preven- tion (CDC) estimates thatVibriospp. cause over 8000 infections annually , and the incidence of vibriosis has increased for all species since 1996 [4, 5], due in part to better surveillance and reporting. In the USA, most cases of vibriosis are reported from Gulf Coast states, where vibrios are commonly found in theirnatural habitat [6–8]. Infections are acquired by consuming contaminated food or water or exposing wounds and abrasions to marine environments [1, 2]. Cases typically occur in the warm summer months. The more common species in the USA include Vibrio vulniﬁcusandV. parahaemolyticus. V. vulniﬁ- cusis often associated with raw oyster consumption, wound infections, and primary septicaemia . V. parahaemolyticusis typically associated with gas- troenteritis. Other common species includeV. chol- eraenon-O1 (gastroenteritis) andV. alginolyticus (wound infections) . Vibriosis has the highest case-fatality rate (CFR) of any enteric disease, mainly attributable toV. vul- niﬁcus, making this a very important public health * Author for correspondence : K. E. Weis, Ph.D., MPH, Division of Environmental Health, Florida Department of Health, 4052 Bald Cypress Way, Bin A08, Tallahassee, FL 32399-1712, USA. (Email : [email protected]) Epidemiol. Infect.(2011),139, 591–598.fCambridge University Press 2010 doi:10.1017/S0950268810001354 concern despite the low incidence reported annually. Based on FoodNet data from 2007, the incidence of vibriosis was 0. 2 cases/100 000 individuals, and the CFR was 3. 6 % . Incidence rates for other common foodborne bacterial enteric diseases in the USA are higher, although CFRs are much lower. Based on these same data, rates for the most common foodborne diseases are (incidence per 100 000, case fatality) :Salmonella(14. 9, 0. 4 %),Campylobacter (12. 8, 0. 1 %),Shigella(6. 2, 0. 1 %), andEscherichia coliO157 :H7 (1. 2, 0. 2 %) . The highest overall CFR for common foodborne enteric diseases (Sal- monella,0. 4 %) is almost 90 % less than the overall CFR for vibriosis (3. 6 %). Historically, Florida’s incidence rate for vibriosis (0. 4/100 000) is double and the CFR (10. 0 %) is almost triple the national rate . Because of the high CFRs associated withV. vul- niﬁcusfrom consumption of raw oysters, there has been a focus in the Gulf Coast states to increase awareness in high-risk individuals, i.e. those with underlying health conditions. As required by Florida law since 1993, all food establishments selling raw oysters must have a visible warning posted to make the consumer aware of health risks associated with their consumption. The Florida Department of Health (FDOH) also regularly distributes educational materials and presents data and risk reduction mess- ages at venues statewide to a variety of audiences, from health professionals to consumers. The goal of these eﬀorts is to increase awareness in high-risk population groups and healthcare providers in order to reduce incidence and case fatality. The vast majority of the current messaging is related to the risk of infection withV. vulniﬁcusassociated with raw oyster consumption. Vibriosis has been a notiﬁable disease in Florida since 1981, in the Gulf Coast states since 1988, and nationally since 2007. However, most states have been voluntarily reporting since 1997. The epidemiology of vibrio infections in Florida has been described previously [10, 11]. From 1981 to 1993, the annual incidence of vibriosis was 4. 3/1 000 000 individuals. The most common species reported during this period wereV. parahaemolyticus,V. vulniﬁcus,andV. chol- eraenon-O1, respectively, with gastroenteritis being the most commonly reported clinical syndrome, with raw oyster consumption in the week prior to illness reported in 45 % of cases . To determine whether trends in vibriosis have changed with time, we sought to describe the current epidemiology of vibrioinfections in Florida using data collected from 1998 to 2007. METHODS We examined cases of vibriosis reported to FDOH with onset dates from 1998 to 2007. Data were col- lected using the ‘ Cholera and Other Vibrio Illness Surveillance Report ’ (CDC Form 52.79). The average annual incidence rate for cases of vibriosis was cal- culated using yearly population data from Florida Charts  based on data from the Florida Legislature, Oﬃce of Economic and Demographic Research. Descriptive analyses were performed for all re- ported cases and examined by species. Medians (ranges) were reported for non-normally distributed continuous variables and frequencies (percentages) for categorical variables. Kruskal–Wallis tests were used to compare median values. Crude odds ratios (ORs) and 95 % conﬁdence intervals (CIs) were re- ported for bivariate analyses. Logistic regression was used to assess the associ- ation between various demographic and disease characteristics and death from vibriosis. Predictor variables that were considered in our analyses in- cluded age, race/ethnicity, gender, clinical syndrome, exposure, and presence and numbers of underlying health conditions. The clinical syndrome ﬁeld in- cluded the following categories : septicaemia, the presence of bacteria in the blood which can be characterized by fever and chills to hypotension and shock ; wound infection, either from injuries sustained in aquatic environments or from pre-existing wounds and characterized by fever, cellulitis, and pain around the site of infection ; and gastroenteritis, an inﬂam- mation of the stomach and intestines characterized by diarrhoea and vomiting. Adjusted ORs and 95 % CIs were reported. Data were analysed using SAS version 9.1 (SAS Institute, USA). RESULTS All species There were 834 cases of vibriosis in 825 individuals reported to FDOH from 1998 to 2007 (median 82. 5/ year). The average annual incidence rate was 4. 8 cases/1 000 000 individuals. Infections were most common in males (71. 8 %), whites (84. 5 %), and non- Hispanics (84. 9 %). The median age of reported cases 592 K. E. Weis and others was 50. 0 years (range 0–96 years), with the majority (74. 9 %) between the ages of 30 and 79 years. Seven individuals were simultaneously infected with mul- tiple species. The most common species wereV. vulniﬁcus, V. parahaemolyticus, andV. alginolyticus(Table 1). The frequency of isolated species varied by year of diagnosis (Fig. 1), with most cases occurring during months when the water temperature and weather are warmer (Fig. 2). Seasonality did not vary by species ; however, age at diagnosis was diﬀerent (P<0. 0001). The median age at diagnosis for cases ofV. vulniﬁcus was 58 years, compared to 36 years in cases ofV. al- ginolyticusand 42 years forV. parahaemolyticus. Wound-related symptoms were reported for 373 (44. 7 %) cases, followed by 350 (42. 0 %) with gastro- enteritis, 45 (5. 4 %) with septicaemia, and 66 (7. 9%) with other reported symptoms (Fig. 3). There were 220 (26. 4 %) cases attributed to raw oyster consump- tion, 313 (37. 5 %) to wound infections, 96 (11. 5%) to other seafood exposures, and 205 (24. 6 %) had no reported exposure (Fig. 4).Presence of any underlying health condition was reported for 400 (48. 5 %) cases. Of these, 228 (57. 0%) had multiple conditions reported. Heart disease, al- coholism, and liver disease were the most common (Table 2). There were 82 deaths reported, yielding a CFR of 9. 9 %. Age at onset, gender, presence of any underly- ing health condition and total number of conditions, mode of exposure, and clinical syndrome were sig- niﬁcantly associated with death. The odds of death from vibriosis increased by 2. 3 % for every 1 year in- crease in age (P=0. 0001). Median age at diagnosis was higher (P=0. 0001) for cases resulting in death Table 1.Number of reported cases of vibriosis by species, Florida, 1998–2007 Vibriospecies No.* % V. vulniﬁcus276 33. 1 V. parahaemolyticus245 29. 4 V. alginolyticus131 15. 7 V. choleraenon-O1 55 6. 6 V. ﬂuvialis46 5. 5 V. hollisae30 3. 6 V. damsela14 1. 7 V. mimicus13 1. 6 Other vibrios 5 0. 6 Species not identiﬁed 19 2. 3 Total 834 100 * Seven individuals were infected with multipleVibrio species. 140 120 100 80 60 40 20 0 Number of cases 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 V. vulnificus V. parahaemolyticus V. alginolyticus Other vibrios Fig. 1.Cases of vibriosis by species and year of diagnosis, Florida, 1998–2007. 160140 120 100 80 60 40 20 0 Number of cases Jan. Feb. Mar. Apr. May June July Aug. Sep. Oct. Nov. Dec. V. vulnificus V. parahaemolyticus V. alginolyticus Other vibrios Month of onset Fig. 2.Seasonality of vibriosis by species, Florida, 1998–2007. 400 350 300 250 200 150 100 50 0 Number of cases Wound Gastroenteritis Septicaemia Other V. vulnificus V. parahaemolyticus V. alginolyticus Other vibrios Fig. 3.Clinical syndromes of vibriosis by species, Florida, 1998–2007. 350 300 250 200 150 100 50 0 Wound Oyster Other seafood* Unknown Number of cases V. vulnificus V. parahaemolyticus V. alginolyticus Other vibrios Fig. 4.Exposures associated with cases of vibriosis by species, Florida, 1988–2007. * Other seafood includes clams, mussels, shrimp, crab, ﬁsh, etc. Vibriosis in Florida, 1998–2007 593 than those that did not. Being male was associated with death (crude OR 2. 92, 95 % CI 1. 48–5. 78). Most deaths (95. 1 %) had one or more underlying health conditions reported. The odds of death in cases with one or more underlying health conditions were 25. 5 (95 % CI 9. 2–70. 4) times that of cases with no under- lying condition. The odds increased with the total number of underlying health conditions (P<0. 0001). Odds of death also diﬀered by exposure and clinical symptoms, with oyster consumption, gastroenteritis, and septicaemia being signiﬁcantly associated with death (Table 3). In multivariable analyses, the best predictive model for death from vibriosis included total number of underlying health conditions and exposure, based on model ﬁt statistics. The odds of death increased with increasing number of underlying conditions : one (OR 9. 8, 95 % CI 2. 7–35. 6), two (OR 18. 7, 95 % CI 5. 3–66. 3), and three or more conditions (OR 50. 7, 95 % CI 14. 5–177. 1). The odds of death were greater for exposure through oyster consumption (OR 6. 7, 95 % CI 3. 2–14. 0) compared to wound infection. V. vulniﬁcus From 1998 to 2007, there were 276 cases ofV. vul- niﬁcusreported (average annual incidence 1. 6/ 1 000 000). There were 141 (50. 7 %) cases presentingwith wound-related symptoms, 79 (28. 6 %) cases pres- enting with gastroenteritis, 40 (14. 5 %) with septi- caemia, and 16 (5. 8 %) with unknown syndrome reported (Fig. 3). The most common cause was wound infection (125 cases, 45. 3 %). Ninety-one (33. 0 %) cases were associated with oyster consump- tion, 12 (4. 3 %) with other seafood consumption, and 48 (17. 4 %) had unknown cause (Fig. 4). At least one underlying health condition was reported for 75. 0% of cases, with most (70. 0 %) reporting multiple con- ditions. There were 76 deaths amongV. vulniﬁcusinfections (CFR 27. 5 %), with 33 deaths in those presenting with septicaemia (CFR 82. 5 %), and 73 in those with underlying health conditions (CFR 96. 1 %). Most (56 %) deaths were associated with raw oyster con- sumption, and 14 % were associated with wound in- fections. The odds of death in cases ofV. vulniﬁcus with one or more underlying conditions were 11. 8 (95 % CI 3. 6–38. 8) times the odds of death in those with none. V. parahaemolyticus There were 245 cases ofV. parahaemolyticusreported (average annual incidence 1. 4/1 000 000). The most common presenting clinical syndromes were gastro- enteritis (137 cases, 55. 9 %) and wound infection (96 cases, 39. 2 %) (Fig. 3). Most (50. 2 %) cases were associated with consumption of seafood : 73 (29. 8%) with oysters and 50 (20. 4 %) with other seafood (e.g. crabs, shrimp). There were 86 (35. 1 %) cases associ- ated with wound infections (Fig. 4). Eighty-four (32. 6 %) cases had an underlying health condition reported. There was one death in cases ofV. para- haemolyticusinfection. From 1998 to 2007, there were 28 documented outbreaks ofV. parahaemolyticusthat included 301 individuals [average 11 cases (range 2–115)]. Of the outbreaks, 35. 7 % were laboratory conﬁrmed. Most (82. 1 %) were associated with seafood consumption at a restaurant. Crustacean shellﬁsh were the most commonly implicated source (50. 0 %), followed by molluscan shellﬁsh (28. 6 %), with speciﬁc vehicles in- cluding shrimp (28. 6 %), crabs (25. 0 %), and oysters (21. 4 %). The most frequent contamination factors were cross-contamination from raw ingredients of animal origin, bare-handed contact, and inadequate cleaning. Almost all of the reported proliferation factors were related to time-temperature abuse, in- cluding inadequate cold-holding and slow cooling. Table 2.Underlying conditions in cases of vibriosis, Florida, 1998–2007 Characteristics of underlying conditions No. % Total cases 825 100. 0 Number of underlying conditions per case 0 425 51. 5 1 172 20. 9 2 122 14. 8 3759. 1 o4313. 8 Type of underlying condition Heart disease 141 17. 1 Alcoholism 132 16. 0 Liver disease 131 15. 9 Diabetes 105 12. 7 Malignancy 62 7. 5 Renal disease 49 5. 9 Immunodeﬁciency 45 5. 5 Haematological disease 42 5. 1 Gastric surgery 35 4. 2 Peptic ulcer 34 4. 1 594 K. E. Weis and others V. alginolyticus There were 131 cases ofV. alginolyticusreported (average annual incidence 0. 7/1 000 000). Most cases presented with wound infections (97 cases, 74. 1%) or other symptoms (27 cases, 20. 6 %) (Fig. 3), and most were associated with exposure from wounds (74 cases, 56. 5 %) (Fig. 4). All cases in individuals aged 0–19 years presented with wound or other symptoms. Forty-one (31. 3 %) cases had at least one underlying health condition. There were no outbreaks and no deaths associated withV. alginolyticusduring this 10-year period. DISCUSSION This analysis updates a previous summary of reported vibrio infections in Florida . The clinical and epi- demiological features of vibriosis have changed since the 1996 report. Most notably, we have seen a change in common species and risk factors and an increase in incidence and CFRs. The most commonly reported species changed fromV. parahaemolyticus(1981– 1993) toV. vulniﬁcus(1998–2007). Cases of vibriosis attributed to raw oyster consumption have decreased from 45 % to 26 % over the same time-frame. Woundinfections were the most common clinical syndrome in the current analysis compared to gastroenteritis as reported by Hladyet al. . Incidence of vibriosis has increased in Florida from 4. 3/1 000 000 in- dividuals (1981–1993) to 4. 8/1 000 000 (1998–2007). CFRs also increased for vibriosis presenting with gastroenteritis (2–9 %) and septicaemia (47–76 %). Due to changes in the way risk factor and background population data were recorded over the years, we were unable to assess the relative risks in certain high-risk groups, such as people with AIDS and raw oyster-consuming adults, as done previously . V. vulniﬁcuswas the most common species in Florida, unlike other areas of the country where V. parahaemolyticuswas the most common . V. vulniﬁcusis associated with more serious illness [1, 4, 7], and is the leading cause of death related to seafood consumption [6, 7].V. parahaemolyticusis re- cognized as the leading cause of gastroenteritis associ- ated with seafood consumption in the USA [14–16], and is often associated with foodborne outbreaks [1, 14, 17]. The median number of cases reported an- nually in Florida has increased from 16 (1981–1993)  to 20 cases (1998–2007). An increase in incidence ofV. parahaemolyticushas also been noted through- out the USA and in other countries [1, 14]. Rising Table 3.Predictors of death in cases of vibriosis, Florida, 1998–2007 Non-fatal Fatal Crude associations No. % No. % OR 95 % CI Presence of any underlying condition* 322 43. 378 95. 125. 5(9. 2–70. 4) Number of underlying conditions 0 421 56. 74 4. 91. 0 (reference) 1 155 20. 917 20. 711. 5(3. 8–34. 8) 2 101 13. 621 25. 621. 9(7. 3–65. 2) o3668. 940 48. 863. 8 (22. 1–184. 1) Exposure# Wound infection 300 53. 411 14. 11. 0 (reference) Oyster consumption 174 31. 044 56. 46. 9(3. 5–13. 7) Other seafood consumption 88 15. 75 6. 11. 6(0. 5–4. 6) Clinical syndromes Wound infection 355 47. 814 17. 11. 0 (reference) Gastroenteritis 316 42. 530 36. 62. 4(1. 3–4. 6) Septicaemia 11 1. 534 41. 578. 4 (33. 0–186. 1) Other 61 8. 24 4. 91. 7(0. 5–5. 2) Gender Female 218 29. 310 12. 21. 0 (reference) Male 522 70. 370 85. 42. 9(1. 5–5. 8) OR, Odds ratio ; CI, conﬁdence interval. * Compared to no underlying condition. #Excludes cases with unknown exposure (n=203). Vibriosis in Florida, 1998–2007 595 water temperatures [1, 17] and the emergence of new strains ofV. parahaemolyticusthroughout the world, speciﬁcally the O3 :K6 strain and its serovari- ants  have been suggested as possible causes. Finally,V. alginolyticusis commonly associated with ear infections and illness in younger individuals , with similar associations found in Florida. Wound infections and seafood consumption were an equal source of exposure toVibriospp. in Florida, whereas seafood consumption is most often associated with vibriosis throughout the USA . A possible cause for increased wound-related exposures is that Florida has over 2000 miles of shoreline . This access may lead to increased participation in water activities, possibly yielding a higher number of wound exposures. Further, Florida has higher average ambi- ent air and water temperatures year-round than many parts of the country, which may increase the likeli- hood of such exposures because of the longer season for water activities and higher vibrio bacterial counts. In accord with other studies, incidence and mor- tality rates associated with vibriosis in Florida were higher in summer months [4, 17] and in males [1, 8], consistent with historical Florida trends . Male gender was signiﬁcantly associated with death in crude analysis ; however, gender was not a signiﬁcant predictor or confounder in adjusted analyses. Another important feature of vibriosis is that the greatest risk of illness and death is in those with underlying health conditions. We also found a strong association between underlying health conditions and death fromV. vulniﬁcus. Individuals with alcoholism and liver or heart disease have increased risk of in- fection, as well as increased risk of death. Increases in incidence and CFRs seen in Florida may be partially attributable to increased awareness of vibriosis in healthcare providers and increased surveillance. In addition, we have experienced several large outbreaks ofV. parahaemolyticusin Florida during this period. However, increases in incidence have also been found in FoodNet states . Even after excluding cases ofV. parahaemolyticusthat occurred during known outbreaks, incidence in this species in the FoodNet states has also increased . It is alarming that there are higher incidence and fatality rates in Florida despite increased educational campaigns aimed at high-risk groups. Other Gulf Coast states have seen similar increases in incidence over time, as well. Louisiana had an average annual incidence rate in 2000–2001 of 5. 9 cases/1 000 000 compared to 6. 7 in 2006–2007, while Mississippiincreased from 2. 3to2. 9/1 000 000 and Texas in- creased from 1. 4to2. 3/1 000 000 during those same years . Florida law requires a warning notice in all food service establishments and retail markets serving or selling raw oysters. The FDOH interacts with state- wide medical organizations and health support groups to present prevention messages to the health- care community, and distributes educational pam- phlets and materials at health fairs. Further, there are several ongoing national educational campaigns sponsored by the Interstate Shellﬁsh Sanitation Conference (ISSC)  and the Gulf & South Atlantic Fisheries Foundation  that aim to raise awareness in people with underlying health conditions regarding their risk ofV. vulniﬁcusinfection associated with raw oyster consumption . Given the increasing inci- dence seen nationwide, the ISSC is currently also working on regulatory policies related to molluscan shellﬁsh in order to prevent vibriosis. Florida education eﬀorts seem to have been suc- cessful, with an almost 50 % reduction in total number of cases associated with raw oyster consumption compared to that reported previously . Despite this success, these campaigns are mainly funded to focus on one high-risk group, raw oyster consumers, and exclude those with wound infections. Florida has seen an increase in the number of cases associated with wound infections. While there is a continued need to warn consumers about the risk from consuming raw oysters, future prevention eﬀorts should focus on those at highest risk for wound infections such as those with diabetes. Outreach eﬀorts should target specialist groups for wounds such as wound care treatment centres, endocrinologists, and other providers that treat diabetic patients, as well as local diabetes support groups. These new educational campaigns should include risk messaging related to wounds sustained during water-related recreational activities or ex- posure to aquatic environments. Outreach pro- grammes should focus onV. vulniﬁcus-related wound infections. Although it is possible to get wound infec- tions from other vibrios,V. vulniﬁcusis particularly virulent, and messaging should focus on the recog- nition, treatment, and prevention of these infections. Little research has focused speciﬁcally on wound- related cases of vibriosis which are more numerous than oyster-relatedV. vulniﬁcusinfections ; therefore, another direction would be to examine the extent of wound infections and treatment of these cases, including wound debridement and amputation. 596 K. E. Weis and others Research is also needed to address the potential im- pact that the environment has on increasing incidence of vibriosis, particularly the role that environmental conditions in oyster harvesting areas have on the bacterial count of harvested oysters. There are a few limitations that should be noted. We chose to examine the 10 most recent years with complete vibrio data available. The previous study included data up to 1993 . However, we did not include the years 1994–1997 in our analysis due to concerns regarding data quality for those years. As with all foodborne illnesses, cases of vibriosis are un- derreported, and reporting is probably biased towards more severe cases. Cases of non-cholera vibriosis were not nationally notiﬁable until 2007 but have been re- ported in Florida since 1981. This may account for some of the diﬀerences between the epidemiology of vibriosis in Florida and elsewhere in the USA. Data were also not always complete on each individual. In Florida, case investigation and reporting is typically performed at the county-level, and there may be inconsistencies in the way data were collected. Moreover, patients or their families were not always able to recall details of food histories or other ex- posures. Almost 25 % of reported cases had no known exposure, a huge gap in epidemiological data. Finally, we were unable to assess some factors related to vib- riosis (e.g. hospitalization, treatment) since these variables were not included in our database. The changing patterns of vibriosis noted in this and other studies may be related to increased awareness and surveillance, as well as higher water temperatures and other environmental factors. However, the in- creased incidence in vibrio infections highlights the need for continued and improved education and risk reduction for at-risk populations, with a new focus on wound-related infections. Because certainVibriospp. are associated with signiﬁcant morbidity and mor- tality, especially in those with underlying health con- ditions, a continuing focus on increasing awareness and understanding of vibriosis in healthcare pro- viders, high-risk populations with speciﬁc underlying health conditions, seafood consumers, and those with occupational or recreational exposures to seawater is needed. ACKNOWLEDGEMENTS The Council of State and Territorial Epidemiologists provided ﬁnancial support for K. Weis during her Applied Epidemiology Fellowship.DECLARATION OF INTEREST None. REFERENCES 1.Morris Jr. JG.Cholera and other types of vibriosis : a story of human pandemics and oysters on the half shell. 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