Diversity of Salmonella isolated from Central Florida Surface Waters

Transcription

1 AEM Accepts, published online ahead of print on 29 August 2014 Appl. Environ. Microbiol. doi: /aem Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 Diversity of Salmonella isolated from Central Florida Surface Waters Rachel McEgan 1, Jeffrey C. Chandler 2, Lawrence D. Goodridge 2, and Michelle D. Danyluk 1* Department of Food Science and Human Nutrition, Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, Florida, USA 2 Center for Meat Safety and Quality, Food Safety Cluster, Department of Animal Sciences, Colorado State University, Fort Collins, Colorado, USA Abbreviated running headline: Salmonella from Florida Surface Waters Current Address: Department of Animal Sciences, University of Wyoming, Laramie, Wyoming, USA Current Address: Department of Food Science and Agricultural Chemistry, Faculty of Agricultural and Environmental Sciences, McGill University, Ste Anne de Bellevue, Quebec, Canada *Correspondent Footnote: Mailing address: University of Florida, Institute of Food and Agricultural Sciences, Department of Food Science and Human Nutrition, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA. Phone: Fax: Electronic mail address: mddanyluk@ufl.edu

2 Abstract Identification of Salmonella serotypes is important for understanding Salmonella environmental diversity. This study evaluates diversity of Salmonella isolated from 165 Salmonella-positive (165/202) Central Florida surface water samples and whether the serotype of the environmental Salmonella isolates can be predicted by a previously published Multiplex PCR assay (Kim et al., 2006). Multiplex PCR was performed on 562 Salmonella isolates; up to 36 isolates per water sample, to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings, prior to isolates being serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex pattern of 495 isolates (88.1%) did not match a previously reported pattern. The remaining 68 isolates matched reported patterns, but did not serotype to those patterns. Use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:-. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella in Central Florida surface waters are serotypically, genotypically, and phenotypically (antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella serotype.

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4 Introduction Salmonella, traditionally considered a zoonosis, was believed to originate from the gastrointestinal tract of animals, and outbreaks of salmonellosis were traced back to these reservoirs (1). More recently, the role of the non-host environment as a source for Salmonella and its role as a source of food contamination is being explored (2-4). One of the non-host reservoirs of Salmonella where considerable effort has focused is surface water, a potential public health risk through consumption, indirect consumption (for example irrigation of edible horticultural crops), or direct contact. A number of studies have characterized the distribution Salmonella isolates from different water sources (5-10). When evaluating surface water for Salmonella isolates, multiple serotypes are often identified from a single environmental sample indicating the diversity of this genus in the environment. Salmonella serotypes isolated from surface waters are often not clinically common (4-12) and often include those from the subspecies enterica, arizonae, and houtenae (5, 8). Serotypes from Salmonella enterica subsp. enterica that have been isolated in more than one surface water survey include: Anatum, Braenderup, Gaminara, Inverness, Muenchen, Newport, Rubislaw, Saintpaul, and Typhimurium (5-8). The number of different Salmonella serotypes isolated from one water sample can vary greatly, and may be influenced by sample volume and enrichment scheme (7, 9); up to 27 different Salmonella serotypes have been reported from a single surface water sampling site (9). The inclusion of multiple watersheds in studies may also lead to a large number of different Salmonella serotypes being isolated (7-9).

5 A current pitfall of Salmonella classification is length of time it takes to identify serotypes, commonly by serological discrimination classified according to the Kauffmann-White scheme (13) or through any number of emerging genetic analysis (14-18). Many of the rapid methods proposed for Salmonella classification used to reduce the time for classification are based and tested against the most clinically relevant serotypes (18), and their usefulness in predicting serotypes of environmental isolates is unknown. In order to begin to understand the diversity of Salmonella isolates in the non-host environment, it is crucial to serotype multiple isolates from each sample (9, 10). Using conventional serotyping for this purpose can be prohibitively expensive, laborious, and time consuming, making a rapid method desirable. The diversity of 562 Salmonella isolates previously recovered from Central Florida surface water sites sampled over 12 months (19) are presented here, along with an evaluation of a previously published (18) multiplex PCR method to rapidly serotype the isolates. Materials and Methods Water-Sample Collection and Isolation of Salmonella Salmonella isolation from Central Florida surface waters is described by McEgan et al. (19). Briefly, surface water samples were collected from 18 sites in Central Florida for 12 consecutive months (202 water samples total). All sites were in rural agricultural areas, away from animal-agricultural operations and are as described by McEgan et al. (19). Sampling sites included two lakes, one pond, six creeks, two streams, one river, and six canals. Salmonella was isolated using a modified United States Food and Drug

6 Administration Bacteriological Analytical Manual (FDA BAM) Salmonella enrichment (20). Salmonella was genetically confirmed by PCR of the inva and oric genes (21); a total of 562 Salmonella isolates were collected (19) and stored in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) supplemented with 15% glycerol at -80 C Multiplex PCR Analysis. Multiplex PCR analysis was performed on all Salmonella isolates. Frozen (- 80 C) isolates were streaked onto tryptic soy agar (TSA; Difco, Becton Dickinson) and incubated for 24±2 h at 35±2 C. A single colony was then transferred to TSB and incubated for 24±2 h at 35±2 C. From these cultures, DNA was extracted using the MoBio UltraClean DNA kit (MoBio, Carlsbad, CA, USA) according to the manufacturer s instructions. Isolated DNA was then subjected to the multiplex PCR reaction described by Kim et al. (18) with minor modifications. The assay uses the amplification of ten specific gene sections (in two separate PCR reactions) that result in the production of a serotype-specific pattern of bands to differentiate 30 of the most clinically relevant serotypes of S. enterica subspecies enterica (18). The presence absence pattern of the ten gene sections was compared to patterns reported by Kim et al. (18). The primers and reaction parameters were as previously described by Kim et al. (18), except that the PCR reagents were all obtained from the Fisher exactgene Complete PCR kit (Fisher Scientific, Pittsburgh, PA, USA). DNA amplicons were separated by agarose (2.5%) gel electrophoresis and visualized under UV light. Serogrouping and Serotyping

7 Frozen (-80 C) isolates were streaked onto TSA and incubated for 24±2 h at 35±2 C. A single colony from each isolate was applied to a glass slide with 5 μl sterile saline. Salmonella O Poly Antiserum (Becton Dickinson, Sparks, MD, USA) was applied and autoagglutination was reported after 1 min of rocking the glass slide. All isolates were serogrouped in order to determine if isolates that grouped together based on multiplex PCR patterns also belonged to the same serogroup. If isolates from the same water sample had identical presence absence patterns for the ten gene sections amplified during the multiplex PCR reaction and belonged to the same serogroup, only one was selected for further analysis. Selected isolates were sent to the National Veterinary Laboratory Service (USDA, Ames, Iowa, USA) for serotyping. Pulsed-field gel electrophoresis Isolates sent for serotyping were analyzed using DNA fingerprinting conducted using XbaI DNA macrorestriction followed by pulsed-field gel electrophoresis (PFGE). PFGE was performed according to the standard laboratory protocol recommended by the Centers for Disease Control and Prevention PulseNet program (22). The PulseNet universal standard strain Salmonella enterica Braenderup H9812 was used as the reference marker. Gel images were captured using a MultiDoc-It Imaging System (UVP, Upland, CA, USA), and images were analyzed using Bionumerics software version 6.6 (Applied Maths, NV, Austin, TX, USA). Similarity of each PFGE pattern was assessed using the unweighted pair group method with arithmetic averages with 2.0% band position tolerances. Dice coefficients had 1.5% optimization values. PFGE patterns that were

8 >80% similar were considered to be in the same genetic cluster; similarity coefficients were obtained by calculating Dice coefficients Antimicrobial Susceptibility Testing Isolates sent for serotyping and analyzed by PFGE, were assayed for susceptibility to 15 antibiotics by the calibrated dichotomous sensitivity method (23). The following antibiotics were tested: amikacin (An), amoxicillin-clavulanic acid (Amc), ampicillin (Am), cefoxitin (Fox), ceftriaxone (Cro), chloramphenicol (C), ciprofloxacin (Cip), gentamicin (G), imipenem (Imp), kanamycin (K), nalidixic acid (Na), streptomycin (S), sulfamethoxazole-trimethoprim (STx), and tetracycline (Te). Antimicrobial susceptibility was determined according to test standards presented in Bell et al. (23). Results Salmonella isolates. Salmonella isolates were collected from 165 (81.7%) of the 202 water samples analyzed (Figure 1). All collected Salmonella isolates displayed typical morphology and growth characteristics on XLT4 or Salmonella ChromAgar. A total of 562 isolates were obtained; up to 36 isolates were obtained and confirmed from a single sample. Multiplex PCR, serogrouping and serotyping. All 562 isolates were analyzed by multiplex PCR and serogrouping. Salmonella from the same sample (site and time) with identical presence absence multiplex PCR patterns, were assumed to be potentially the same isolate; Salmonella having different

9 multiplex PCR patterns were assumed to be different. In 68 (41.2%) of the 165 water samples from which Salmonella was isolated, all Salmonella isolates had identical multiplex PCR patterns as shown in Figure 2. In 52 (31.5%) water samples, two patterns were identified; in 21 (12.7%) water samples, three patterns were identified amongst the isolates. More than three patterns were identified in the remaining 24 (14.5%) water samples. If a multiplex pattern was previously reported by Kim et al. (18) as a specific serotype, that serotype is noted as the suggested serotype, and the corresponding serogroup was noted as the suggested serogroup (Table 1); 67 of the 562 isolates had multiplex patterns matching those reported by Kim et al (18); the multiplex pattern of 495 isolates (88.1%) did not match any pattern reported by Kim et al. (18). Of the 67 isolates matching reported multiplex patterns (18), suggested serotypes included: Bovismorbificans (22 isolates), Braenderup (9), Chester (1), Derby (1), Hadar (3), Meunchen (4), Newport (2), Ohio (9), Oranienburg (4), Poona (3), Saintpaul (3), Thompson (5), Typhimurium (1). Serogroups were determined for all 562 isolates and are reported in Table 1 as actual serogroups. In all water samples where Salmonella isolates were collected (n=165 samples), if isolates had matching multiplex patterns, as described above, the same isolates also had matching serogroups. However, within one water sample, not all isolates with matching serogroups had identical multiplex PCR patterns. Only 31/67 (46.3%) of the suggested serotypes from Kim et al. (18), matched the actual serogroup (Table 1).

10 To explore the possibility that the multiplex PCR could be an effective rapid method for determining serotypes of Salmonella collected from environmental sources, one representative Salmonella isolate from each water sample multiplex PCR pattern combination was sent for serotyping. Of the 562 Salmonella isolates analyzed by multiplex PCR and serogrouping, 223 were sent for Kauffmann-White serotyping. Thirty-one Salmonella isolates were not recovered following frozen storage and were unable to be analyzed further. None of the suggested serotypes based on the Kim et al. (18) multiplex PCR patterns matched the serotype determined by Kauffmann-White serotyping. Kauffmann-White serotyping identified a total of 33 different serotypes from the 233 isolates sent for serotyping. Water samples were collected over a continuous 12 month timeframe; the numbers of unique isolates from each water sample at each sampling time are detailed in Figure 1. The number of different serotypes isolated from any individual sampling site over the course of the 12 month study ranged from two at site fourteen to ten at sites eight and ten. The average number of serotypes isolated from a site was six. The frequency of isolation for each serotype across locations at all sample times is shown in Figure 3. No one serotype was isolated in all months of study from any of the sampling sites. The most frequently isolated serotypes, were Muenchen (11.5%), Rubislaw (9.5%), Anatum (8.8%), Gaminara (8.8%), and IV_50:z4,z23:- (6.8%). When the 33 serotypes isolated in this study were compared to the CDC s top 20 list of most clinically common serotypes, only seven were clinically common: serotypes Bareilly, Braenderup, Muenchen, Newport, Paratyphi B var.l-tartrate+, Saintpaul, and Typhimurium var 5 (Figure 3; 12). Salmonella serotypes isolated during the most

11 number of months include: Anatum (8/12), Muenchen (7/12), and Rubislaw (6/12). A temporal pattern of Salmonella serotype isolation was not observed PFGE Analysis Isolates sent for Kauffmann-White serotyping (n=223) were further analyzed using PFGE XbaI macro restriction banding patterns. Two isolates would not produce banding patterns from PFGE, even with the addition of thiourea: Salmonella Bareilly isolated from site five in August, 2010, that also did not produce a banding pattern from multiplex PCR, and Salmonella Paratyphi_B_var.L-tartrate+ from site twelve in March, 2011, that produced a banding pattern with multiplex PCR but did not match the other Paratyphi B isolate also recovered from the same site at the same time. Analysis of the PFGE patterns from 221 Salmonella isolates generated 11 major genotypic clusters (A-K; Figure S1) comprised of at least three isolates with a dice coefficient index cut-off point of 80% (Figure 4 and 5). Analyzing the Salmonella isolates separately into two dendrograms, (Figure 4 and 5) caused individual isolates to move position, compared to the complete dendrogram in S1. While the isolates within each cluster remained the same, cluster labels became disordered (Figure 5); for ease of interpretation, cluster labels were maintained as ordered in the complete dendrogram (Figure S1). The majority (141/223) of isolates, including at least one isolate from each sample site, clustered into one genotypic group (cluster A, Figures 3 and 4); 23 of the 33 total serotypes are represented in this cluster. Serotypes not found within cluster A included: Florida, Hartford, Lexington, Litchfield, Paratyphi_B, Saintpaul, 6,8:d:-, III_60:r:z, and Rough_O:d:1,7 (Figure 5). Sampling site one had the least genotypic diversity, with two

12 isolates in cluster A and three isolates outside the major genotypic clusters (Figure 3). Sampling sites eight, nine, eleven, and twelve had the greatest genotypic diversity, each with isolates in six of the 11 genotypic clusters (Figure 3). All typeable isolates of Salmonella Bareilly (n=2) cluster as identical by PFGE in cluster A; the only occurrence of this in the 221 isolates evaluated by PFGE. All Salmonella Gaminara (8.8% of isolates) isolates were found in cluster A. Isolates of serotypes Muenchen (11.5% of isolates) Rublislaw (9.5%), Anatum (8.8%), and IV_50:z4,z23:- (6.8%) clustered into: A, D and K; A, B and G; A; and A and H, respectively, with additional isolates outside of the major genotypic cluster groups. Over the entire sampling period, there were 19 instances where the same serotype from different sampling sites had identical PFGE patterns (100% similarity), containing between two and ten isolates. In six instances of isolates with identical PFGE patterns, some of the isolates were collected from the same sampling site and sampling month but had different multiplex PCR patterns. Ten Salmonella Anatum isolates with identical PFGE were isolated from seven different months and six different sites. Of the 23 total Salmonella Anatum isolates (Figure 3 and Figure 4), 20 in cluster A had 89.6% similar PFGE patterns. Nine Salmonella Braenderup isolates with identical PFGE came from September, 2010 and July, 2011 at sites two, four, five, and eighteen (including the four isolates from July, 2011, site four, with different multiplex PCR patterns). Of the total 13 Salmonella Braenderup isolates (Figure 3 and Figure 4), 11 in cluster A had 87.6% similar PFGE patterns. Twelve of the 19 instances (where identical PFGE patterns are reported) consist of only two isolates; in one case Salmonella Newport isolates from site ten in August, 2010 and December, 2010 had identical patterns. Isolates with identical

13 PFGE patterns were collected from different sites in the same month twice: Salmonella Bareilly in August, 2010 from sites four and five and Salmonella Rubislaw in December, 2010 from sites thirteen and sixteen Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing was performed on the 223 serotyped Salmonella isolates. Twenty-four isolates were susceptible to all tested antimicrobials, 41 were resistant to streptomycin, and 50 resistant to both streptomycin and kanamycin. Eighteen Salmonella isolates were resistant to more than five of the antibiotics tested; the antibiotic resistant profiles of these strains are shown in Table 2. None of the multidrug resistant (MDR) Salmonella isolates were resistant to ceftriaxone or ciprofloxacin. Three Salmonella Braenderup (3/13 total Salmonella Branderup strains isolated) and three Salmonella Florida (3/8 isolated) isolates were MDR. The Salmonella Braenderup were isolated from three sampling sites (two, four, and five) during July and were 87.6% similar by PFGE; the Salmonella Florida were isolated during August and October from sampling sites four, five, and nine and were 80.8% similar by PFGE. Two isolates of Salmonella Bareilly (4 total isolates), Salmonella Gaminara (13), Salmonella Hartford (5), and Salmonella Muenchen (17) were MDR. The Salmonella Bareilly were isolated during August, 2010 from sampling sites four and five and had identical PFGE patterns; the Salmonella Gaminara were isolated during November, 2010 from sampling sites fifteen and sixteen and also had identical PFGE patterns. The Salmonella Hartford were isolated during March from sampling sites three and four; and the Salmonella Muenchen were isolated during July, 2011 from sampling sites five and eleven; these isolates had

14 different PFGE patterns. Of the 18 MDR Salmonella isolates, four isolates were from sampling sites four and five; the remaining sampling sites had only one or no MDR Salmonella Discussion Studies evaluating the prevalence of Salmonella from surface waters vary in reported prevalence: 81.7% (165/202) in Central Florida (CFL; 19); 96% (106/110; 1,000 ml samples) in North Florida (NFL; 7); 79% (57/72; 555 ml) in Georgia (GA, 5); 62% (90/145; Moore swabs suspended in the water for 3-5 days) in Ontario, Canada (ON, 9); 57% (42/74; 25 ml) in North Carolina (NC, 6); 11% (16/145; 100 ml) in New York State (NY, 8); and 7% (18/252; 100 ml or three-day Moore swabs) in California (CA, 24). Even within one study of California Central coast waterways, Salmonella prevalence varied between 0 and 72% in 3330 ml water samples (25). Differences in water sampling volumes, and enrichment protocols complicate a direct comparison of the Salmonella prevalence reported here to those previously reported in other regions. To overcome this, Strawn et al., (10) isolated Salmonella out of the produce environment (including 250 ml water samples) in two locations using identical sample collection, detection and isolation schemes; Salmonella was isolated from 38% (15/40) of surface water samples in South Florida (SFL) and 10% (36/354; including those referenced in (8) above) in NY. While it is unfortunate that the number of water samples (40 in SFL vs. 354 in NY) are not the same, it is clear that differences in Salmonella prevalence exist on a regional level, independent of sample size or detection and isolation schemes. These differences may be due to other climatic factors, environmental pressures, wildlife or agricultural practices.

15 Future studies designed to specifically determine the exact factors that influence pathogen prevalence in surface waters between regions are warranted. As has previously been reported (5, 9, 10), more than one serotype of Salmonella is frequently isolated from a single Salmonella-positive surface water sample. Considering only differences in Kauffmann-White serotyping, multiple serotypes were identified in 32/165 Salmonella-positive water samples. When multiple Salmonella serotypes were identified from a single water sample, the typical number of serotypes identified was two; up to four different serotypes were isolated from a single water sample. Similarly, water samples from GA yeilded two serotypes from 15/72 water samples and three serotypes from 3/72 water samples (5). Strawn et al. (10) report 13/97 water samples with multiple serotypes, and one sample from SFL with three different serotypes. The varying number of isolates per sample or isolation scheme selected for further analysis complicates a direct comparison of the number of Salmonella serotypes from a single water sample. The potential for isolating multiple Salmonella serotypes from a single sample highlights the importance of using multiple enrichment and plating media to truly explore Salmonella diversity and to facilitate identification of isolates with atypical characteristics (7, 9, 10). As few as seven (New York State; 8) and as many as 38 different serotypes (Ontario, Canada; 9) have been isolated during surface water surveys, indicating a tremendous diversity of Salmonella serotypes in the environment. When comparing Salmonella isolates from water samples in SFL and NY using the identical isolation scheme, Strawn et al., (10) identified 9 and 12 Kaufmann-White serotypes, respectively, with 4 serovars from water samples in both locations. Surface water surveys in the

16 southeastern US report 5, 8, or 13 different serotypes from NC, NFL, and GA surface waters, respectively (5-7). In addition to sample size and isolation differences, the diversity of serotypes reported here (33 serotypes) may be a reflection of the study design (greater number of sampling locations and watersheds sampled) or other environmental pressures (wildlife, agricultural practices, etc.). Of the 33 Kauffmann-White serotypes isolated from Central Florida, 15 have been reported in other surface water studies (5-10). They include serotypes: Anatum from GA and ON; Baildon from SFL; Bareilly from GA; Braenderup from GA, NFL, and SFL; Gaminara from GA, NC, and SFL; Give from ON and NY; Hartford from ON; Inverness from NC and NFL; Litchfield from ON and SFL; Miami from NC; Muenchen from GA, NFL, and ON; Newport from NC, NFL, NY, ON and SFL; Rubislaw from GA, NFL, NY, and SFL; Saintpaul from GA and ON; and Typhimurium from NY and ON. None of the 16 serotypes, determined by multilocus sequencing, from the California Central-coastal waterway (25) match serotypes in this study. Independent of differences in sampling volume and enrichment protocols, 15/33 serotypes recovered here were also recovered during previous surveys of surface water, suggesting these serotypes may have an increased presence or fitness in surface waters compared with other Salmonella serotypes. Of the 11 Salmonella serotypes isolated in Central Florida and from at least two other locations, only five (Branderup, Meunchen, Rubislaw, Saintpaul and Typhimurium) are also reported on the CDC top 20 clinical isolates list (12). The frequency of Salmonella serotypes, over multiple surface water surveys, that are not often linked to human cases of salmonellosis suggests that the diversity of Salmonella in the environment includes many serotypes not commonly seen or clinically relevant. Future

17 studies to evaluate potential differences in environmental fitness, associations with hosts, and virulence of surface water Salmonella isolates from different regions are justified. The number of Salmonella serotypes differed significantly between the 18 sites tested. The fewest serotypes isolated from a single site over the 12 months of our study was two from site fourteen, a canal in a 55% agriculture land usage watershed (19). Two streams and one creek in watersheds with 32-33% agriculture, sites ten, four and six, had the highest number of serotypes (11, 10, and 9, respectively) isolated over the 12 months. Serotypes Anatum, Gaminara, Muenchen, and Newport were common to sites ten, six, and four, and were also reported in NFL (7) and SFL (10) surface waters. The repeated isolation of these serotypes from Florida surface water may speak to their increased fitness in this environment. The multiplex PCR reaction described by Kim et al. (18) could not serotype Salmonella isolates from Central Florida surface waters. Beaubrun et al. (26) report contradictory results, where 76.2% (48/63) of environmental Salmonella isolates from tomato farms in Virginia were correctly identified by the multiplex PCR (18) as serovars Newport, Braenderup, and Typhimurium. However, different patterns were reported for serotypes Berta, Derby, Hadar, Infantis, Montevideo, Muenchen, Newport, and Paratyphi, and more than one multiplex pattern for serotypes Seftenberg and Tennessee (26). While the multiplex PCR (18), did not predict the serotype of Salmonella isolates here, using multiplex patterns to group isolates collected from one water sample decreased the number of isolates for further characterization by more than half, decreasing the cost and time required to screen the numerous isolates. Use of the multiplex patterns (18) to group isolates introduces the risk of identifying isolates as identical when they are in fact

18 different serotypes, while allowing a greater number of isolates to be screened in a timely manner. PFGE analysis revealed 19 sets of Salmonella isolates with identical banding patterns from multiple months or sampling sites. Salmonella Anatum isolates with identical patterns were isolated from sampling site seven in November, December, and January; the three-month isolation of Salmonella Anatum from one sample site may indicate its survival in the surface water or a repetitive introduction from the same source. Subsequent surface water samples where Salmonella Anatum with identical PFGE patterns were identified has previously been seen in NC, in an area with a known influence from swine agriculture (6). Repeated isolation of Salmonella Heidelberg with identical PFGE patterns has also been reported in ON, where isolations from repeated samplings of stream water occurred from May through October (9). Genotypic clustering of Salmonella isolates by PFGE using only one enzyme (XbaI), as was performed here and by Patchnee et al. (9), may be further differentiated by repeating PFGE using a second enzyme or by alternate analysis. As such, the matches described here may be an artifact of the selected method of analysis, supported by the occurrence of more than one multiplex PCR pattern reported for isolates with identical PFGE patterns. The majority (115/223) of Central Florida isolates were susceptible to all antimicrobials tested or were resistant to only kanamycin and/or streptomycin, these are both secondary metabolites of Streptomyces spp., a common soil microbe and can be found in the environment, this resistance is not uncommon in environmental isolates (26, 27). Previously, Salmonella isolates from watersheds influenced by pig farms had increased antimicrobial resistance (6). Here, sites four and five had the greatest number

19 of MDR Salmonella isolates; while sampling sites selected in this study were not immediately adjacent to animal agriculture, the high prevalence of antimicrobial resistance isolates may be indicative of an animal agriculture influence to the watershed. Similar to previous reports (5-10, 25), a diverse group of Salmonella serotypes were isolated from Central Florida surface waters. Although the rapid serotyping of isolates could not be accomplished using the multiplex PCR (18), this technique allowed isolates from the same sample to be grouped as different or potentially the same, decreasing the effort and expense for subsequent typing. However, while the diversity of isolates was also reflected in the genetic profile by PFGE using XbaI, the occurrence of more than one multiplex PCR pattern reported for isolates with identical PFGE patterns, are indicative of a need for more discriminatory typing tools. Acknowledgements The authors thank the USDA CSREES Specialty Crops Research Initiative and the University of Florida Alumni Fellowship for the financial support of this project. The authors wish to thank Gwen Lundy, Luis Martinez, and Brian Buzzie for their technical assistance.

20 References 1. D Aoust JY Salmonella, p In M.P. Doyle (ed.), Foodborne Bacterial Pathogens. Marcel Dekker, New York, NY. 2. Jackson GJ, Langford CF, Archer DL Control of salmonellosis and similar foodborne infections. Food Control 2: Ijabadeniyi OA, Debusho LK, Vanderlinde M, Buys EM Irrigation water as a potential preharvest source of bacterial contamination of vegetables. J. Food Safety 31: Greene SK, Daly ER, Talbot EA, Demma LJ, Holzbauer S, Patel NJ, Hill TA, Walderhaug MO, Hoekstra RM, Lynch MF, Painter JA Recurrent multistate outbreak of Salmonella Newport associated with tomatoes from contaminated fields, Epidem. Infect. 136: Haley BJ, Cole DJ, Lipp EK Distribution, diversity, and seasonality of waterborne salmonellae in a rural watershed. Appl. Environ. Microbiol. 75: Patchanee P, Molla B, White N, Line DE. Gebreyes WA Tracking Salmonella contamination in various watersheds and phenotypic and genotypic diversity. Foodborne Pathog. Dis. 7: Rajabi M, Jones M, Hubbard M, Rodrick G, Wright AC Distribution and genetic diversity of Salmonella enterica in the Upper Suwannee River. Int. J. Microbiol. 2011; Strawn LK, Fortes ED, Bihn EA, Nightingale KK, Grohn YT, Worobo RW, Wiedmann M, Bergholz PW Landscape and meteorlogical factors affecting prevalence of three food-borne pathogens in fruit and vegetable farms. Appl. Environ. Microbiol. 79: Thomas JL, Slawson RM, Taylor WD Salmonella serotype diversity and seasonality in urban and rural streams. J. Appl. Microbiol. 114: Strawn LK, Danyluk MD, Worobo RW, Wiedmann M Distribution of Salmonella subtypes differ between two U.S. produce-growing regions. Appl. Environ. Microbiol. 80: Perch M, Braden CR, Bishop R, Fields P, Plikaytis R, Tauxe RV Salmonella surveillance summary, Centers for Disease Control and Prevention, Atlanta, GA. 12. Centers for Disease Control and Prevention National Salmonella surveillance annual summary, Centers for Disease Control and Prevention,

21 Atlanta, GA Bopp CA, Brenner FW, Fields PI, Wells JG, Stockbine NA Escherichia, Shigella, and Salmonella, p In Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds), Manual of Clinical Microbiology, 8 th ed. ASM Press, Washington, DC. 14. Achtman M, Wain J, Weill FX, Nair S, Zhou Z, Sangal V, Krauland MG, Hale JL, Harbottle H, Uesbeck A, Dougan G, Harrison LH, Brisse S Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLOS Pathogens 8: e Leader BT, Frye JG, Hu J, Fedorka-Cray PJ, Boyle DS High-throughput molecular determination of Salmonella enterica serovars by use of multiplex PCR and capillary electrophoresis analysis. J. Clin. Microbiol. 47: Yoshida C, Franklin K, Konczy P, McQuiston JR, Fields PI Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes. J. Microbiol. Methods 70: Fitzgerald C, Collins M, van DS, Mikoleit M, Brown T Multiplex, beadbased suspension array for molecular determination of common Salmonella serogroups. J. Clin. Microbiol. 45: Kim S, Frye JG, Hu J, Fedorka-Cray PJ, Gautom R, Boyle DS Multiplex PCR-based method for identification of common clinical serotypes of Salmonella enterica subsp enterica. J. Clin. Microbiol. 44: McEgan R, Mootian G, Goodridge LD, Schaffner DW, Danyluk MD Predicting Salmonella populations from biological, chemical, and physical indicators in Florida surface waters. Appl. Environ. Microbiol. 79: United States Food and Drug Administration Salmonella. In Bacteriological Analytical Manual. U.S. Food and Drug Administration, Silver Spring MD Malorny B, Hoorfar J, Bunge C, Helmuth R Multicenter validation of the analytical accuracy of Salmonella PCR: Towards an international standard. Appl. Environ. Microbiol. 69: Ribot E, Fair M, Gautom R, Cameron D, Hunter S, Swaminathan B, Barrett T Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7 Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 3:59-67.

22 Bell SM, Gatus BJ, Pham JN, Rafferty DL Antibiotic susceptibility testing by the CDS method, 7th ed. The Antibiotic Reference Laboratory, The Prince of Whales Hospital, Randwick, NSW, Australia. ualready%20sat%20finally%20+appendix.doc.pdf 24. Gorski L, Parker CT, Liang A, Cooley MB, Jay-Russell MT, Gordus AG, Atwill ER, Mandrell RE Prevalence, Distribution, and Diversity of Salmonella enterica in a major produce region of California. Appl. Environ. Microbiol. 77: Walters SP, Gonzalez-Escalona N, Son I, Melka DC, Sassoubre LM, Boehm AB Salmonella enterica diversity in Central Californian coastal waterways. Appl. Environ. Microbiol. 79: Beaubrun, JG, Cheng C, Chen K, Ewing L, Wang H, Agpaoa MC, Huang MJ, Dickey E, Du JM, Williams-Hill DM, Hamilton B, Micallef SA, Rosenberg Goldstein RE, George A, Joseph SW, Sapkota AR, Jacobson AP, Tall BD, Kothary MH, Dudley K, Hanes DE The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices. Food Microbiol. 31: Turpin P, Dhir V, Maycroft K, Rowlands C, Wellington E The effect of Streptomyces species on the survival of Salmonella in soil. FEMS Microbiol. Ecol. 101: Egan S, Wiener P, Kallifidas D, Wellington E Phylogeny of Streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters. Antonie Van Leeuwenhoek Intl. J. of General and Molecular Microbiol. 79:

23 Table 1. Suggested serotypes and corresponding serogroup of Salmonella isolated from Central Florida surface waters based on multiplex PCR pattern compared with isolate serogroup determined by autoagglutination. Suggested serotype is the serotype which corresponds to a multiplex PCR pattern reported by Kim et al. (18). Suggested Suggested No. Actual Serogroup % Serotype Serogroup Isolates A B C D E F G Matching Bovismorbificans B Braenderup B Chester A Derby A Hadar B Muenchen B Newport B Ohio B Oranienburg B Poona B Saintpaul A Thompson B Typhimurium A Other Downloaded from on April 29, 2018 by guest

24 Table 2. Antibiotic resistance profiles of Salmonella isolates from Central Florida surface waters, only isolates resistant to greater than five of the antibiotics screened are listed Serotype Site Month Antimicrobial Resistance Profile Anatum 13 February AmcAmFoxCfSTe Bareilly 4 August AmcAmFoxCfKSSTxTe Bareilly 5 August AmcAmFoxCfCKNaSSTxTe Braenderup 2 July AmcAmCfImpKS Braenderup 4 July AmcAmFoxKNaSSTx Braenderup 5 July AmcAmFoxCfCKNaStxTe Florida 4 August AmcAmFoxCfImpKS Florida 5 October AmcAmFoxCfKS Florida 9 August AmcAmFoxCfKS Gaminara 15 November AmcAmFoxCfCKNaSSTxTe Gaminara 16 November AmcAmFoxCfKS Hartford 3 March AmcFoxCfKNaSTe Hartford 4 March AmcAmFoxCfCKNaSTe Muenchen 5 July AmcCfCKNaSTxTe Muenchen 11 July AmcAmCfCKNaSTxTe Rough_O:d:1,7 14 September AmcAmFoxCfGmS Rubislaw 7 July AmFoxCfKSSTxTe Saintpaul 12 June AmcAmFoxCfKS Downloaded from on April 29, 2018 by guest

25 Figure 1. Unique Salmonella isolates from eighteen sampling sites over the 12 month survey (19). The bubble size represents the number of unique isolates collected at the specified sampling site and sample date. Figure 2. Unique multiplex PCR patterns (18), identified from a single surface water sample (site and time). Figure 3. Frequency of isolation of each serotype by locations across all 12 sample times. The bubble size represents the number of unique isolates collected at the specified sample site across all sample dates. Isolates that cluster into PFGE cluster A are represented by black bubbles, all other PFGE clusters are grey bubbles. Serotypes within the CDC top 20 clinically common serotypes are bolded on the y-axis. Figure 4. Dendrogram of Salmonella isolates recovered from Central Florida in group A, the largest >80% similar cluster, by PFGE-XbaI fingerprinting.. Figure 5. Dendrogram of PFGE-XbaI fingerprinting of Salmonella isolates recovered from Central Florida not grouped within cluster A. All isolates that are.>80% similar by PFGE are grouped into clusters; all clusters are labeled with B-K, as determined in the complete dendrogram produced from all Salmonella isolates recovered from Central Florida (S1). Lines connecting isolates are as used in complete dendrogram: two short dashes followed by a long dash, group B; all short dashes, group D; all medium dashes, group F; alternating short and medium dashes, group G; long dashes, group H, dotted line, group I (only groups with four or more isolates are labeled on the dendrogram). S1. Dendrogram of PFGE-XbaI fingerprinting of Salmonella isolates recovered from Central Florida. All isolates that are >80% similar by PFGE are connecting with a line next to the dendrogram: solid line, group A; two short dashes followed by a long dash, group B; all short dashes, group D; all medium dashes, group F; alternating short and medium dashes, group G; long dashes, group H, dotted line, group I (only groups with four or more isolates are labeled on the dendrogram).

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