nontyphoidal salmonellosis is the leading cause of bacterial foodborne illness in the us and contributed to approximately 33% of all foodborne - related deaths in 2009 . the disease is characterized by a gastroenteritis that is associated with a wide range ( > 2500 ) of salmonella enterica serotypes . historically , disease reservoirs for salmonella were primarily attributed to contaminated poultry meat and eggs , but other sources include soil , factory surfaces , animal feces , and raw meats [ 3 , 4 ] . more recently , orange juice [ 58 ] and other produce products [ 912 ] have been increasingly implicated as the source of salmonellosis outbreaks . moreover , the number of cases per outbreak is greater for vegetables than for any other product . irrigation water may play an important role in contaminating soil and produce with salmonella [ 14 , 15 ] . storm water runoff and septic tanks are known contributors of pathogens to surface water [ 16 , 17 ] , but rain events may also aid in the transport of pathogens from environmental sources in forested and grassed buffer zones into farm ponds . laboratory assays have demonstrated the potentiality of pathogen uptake through the roots   and flowers of edible plants , and salmonella from irrigation sources has been shown to adhere to plants and survive for long periods of time . the suwannee river is a central feature of the suwannee river watershed and is the largest source of free - flowing fresh water to the gulf of mexico . the region is characterized by relatively minimal human impact and spans the coastal plains of southern georgia and north central florida . a recent survey of surface water collected within the southwest georgia portion of the watershed reported the presence of s. enterica . this region is considered a  hot spot  for environmental sources of salmonellosis , as case rates within this region were 1.5 times higher than the national average [ 14 , 23 ] , and river water collected locally over a 12-month period was positive for s. enterica in 79.2% of samples . s. enterica densities directly correlated with water temperature ( r = 0.49 ; p < 0.05 ) and precipitation levels ( r = 0.68 ; p < 0.05 ) and also increased 62% in summer compared to spring months . this survey also identified serovars with environmental sources that were associated with disease in this region . however , the genetic relationship of strains from clinical and environmental sources was not examined . s. enterica isolates from aquatic environments show a wider diversity of serotypes than those recovered from animal sources [ 2426 ] , but the genetic diversity and distribution of s. enterica from environmental sources is still relatively unknown . therefore , the present study examined the genetic profiles of s. enterica recovered from the suwannee river to those of strains from clinical and other environmental sources . the diversilab repetitive extragenic palindromic pcr ( rep - pcr ) analysis was used to evaluate genetic similarity of salmonella strains because it shows enhanced discrimination compared to other methods such as pulsed field gel electrophoresis [ 2729 ] . results showed that s. enterica from suwannee river surface water samples comprised a diverse population that was genetically distinct from strains from other environmental sources and from most strains of clinical origin . this research establishes an s. enterica database that should serve as reference for routine monitoring and source tracking in future outbreaks . s. enterica isolates were recovered from surface water samples of the suwannee river ( n = 110 ) , as described below . reference strains included a total of 186 s. enterica isolates from both clinical ( n = 31 ) and environmental ( n = 45 ) sources and strains that were kindly provided by abc research , inc . , dr . mickie parish and margaret richards , as described in supplemental table 1 ( available online at doi:10.1155/2011/461321 ) . all strains were stored at 80c in luria bertani nacl ( 1% ) broth ( lbn ) and 50% glycerol and subcultured on lbn agar ( la ) for genetic characterization . sampling locations included public access sites at the big shoals state park , fl ( bp ) , stephen foster state folk culture center in white springs , fl ( ws ) , spirit of suwannee campground boat ramp ( sp ) , and the florida sheriffs boys ranch boat ramp ( br ) . samples were collected in sterile , glass containers by skimming the surface water and transported in coolers on ice to the laboratory , stored for no more than 24 hours at 4c , and processed as described below . salmonella water samples ( 500 , 100 , 50 , 10 , and 1  ml ) were used to determine most probable number ( mpn ) by addition to equal volumes of sterile 1% buffered peptone water ( bpw ) at 2x or 1x ( for 1  ml samples ) concentration in triplicate . broth cultures were incubated at 37c overnight with shaking ( new brunswick scientific incubator ) . these cultures ( 1  ml of each ) were subsequently transferred into 9  ml of tetrathionate broth ( tt ) for selective enrichment at 37c overnight with shaking . tt broth cultures were then streaked for isolation onto xld ( oxoid ) agar plates , spotted onto la plates , and incubated at 37c overnight shaking ( fisher scientific incubator ) . salmonella - positive samples were confirmed by species - specific pcr or dna probe identification of presumptive positive colonies in xld and la , as described below . the mpn/100  ml of each sample from the suwannee river was determined by the number of replicate enrichment cultures for each dilution that was confirmed by species - specific identification of presumptive positive colonies , as described below . all samples with typical colony morphology for salmonella on xld agar plates were confirmed by dna probe colony hybridization and by pcr . for dna probe assay , colonies were grown overnight on la at 37c and transferred by overlay to filter paper ( whatman no . 541 ) , as previously described . briefly , colonies on filters were lysed in 0.5  m naoh and 1.5  m nacl solutions , neutralized in 2  m ammonium acetate ( fisher , pittsburgh , pa ) , and washed in 1x ssc buffer ( 0.003  m sodium citrate and 0.03  m nacl ) . filters were treated in proteinase k ( 20  g / ml ) in ssc and washed in ssc . filters were hybridized at 56c in buffer with an alkaline - phosphatase - labeled ( dna technologies , denmark ) dna probe ( 5>ctggttgatttcctgatcgc>3 ) derived from the s. enterica inva gene . filters were washed with 1x ssc at 56c , followed by washes at room temperature to remove unbound probe , and developed in the presence of nbt / bcip substrate ( fisher , pittsburgh , pa ) . for pcr confirmation , broth cultures ( 1  ml ) were extracted by boiling cells from a suspension of one colony in 400  l of pbs and incubated for 7  min at 100c . samples were centrifuged at 13,000  rpm for 3  min , and the supernatant was transferred to a new microcentrifuge tube . extracted dna in the supernatant ( 2  l ) was combined with 23  l of master mix ( eppendorf ) , forward primer inve ( 5-tgcctacaagcatgaaatgg-3 ) , and reverse primer inva ( 5-aaactggaccacggtgacaa-3 ) for pcr amplification on a mastercycler gradient thermal cycler ( eppendorf ) . the following conditions were used : initial denaturation for 3  min . at 94c , followed by 30 cycles of 1  min . at 94c , 1  min . at 56c , and 1  min . at 72c , with the final extension of 15  min . at 72c . samples were combined with 2 l of 6x loading dye ( promega ) and run on 1% agarose gels for visualization of pcr products by ethidium bromide staining . diversilab rep - pcr analysis of s. enterica isolates was performed according to manufacturer 's specifications for the salmonella ( biomerieux ) . briefly , genomic dna from isolates grown on la plates was extracted using ultraclean microbial dna isolation kit ( mo bio laboratories , inc . ) . dna concentration was determined by spectrometry ( spectra max plus 384 , molecular devices , sunnyvale , calif ) and stored at 20c . pcr amplification used s. enterica rep - pcr dna fingerprinting kit ( biomerieux ) with amplitaq dna polymerase and geneamp 10x pcr buffer i with mg  cl2 ( applied biosystems , inc . ) . each pcr reaction ( 25 l ) included 2 l of the s. enterica primer mix , 0.5  l of the amplitaq dna polymerase , 2.5  l of the buffer i , 18  l of the master mix , and approximately 2  l ( 50  ng/l ) of template dna . amplification was performed in a mastercycler gradient thermal cycler ( eppendorf ) according to the conditions indicated in the s. enterica rep - pcr fingerprinting kit . all rep - pcr amplicons were screened by electrophoresis in a 1.5% agarose gel ( fisher scientific ) in tris - acetate - edta buffer containing ethidium bromide . sample analysis was performed using amplicons ( 1  l ) loaded onto the dna microfluidics labchip and for capillary electrophoresis by the agilent 2100 . electrophoretograms were analyzed by diversilab system software ( biomeriuex ) for strain comparison of the dna similarity to the diversilab rep - pcr online library for s. enterica ( n = 352 ) . the chi - square test was used to compare segregation of rep - pcr genogroups with strain origin ( clinical versus environmental ) and serovar . salmonella was isolated from the surface water at all sites that were sampled on the suwannee river ( figure 1 ) . these sites include the big shoals state park ( bs ) , stephen foster state folk culture center in white springs , fl ( ws ) , spirit of suwannee campground ( sp ) , and florida sheriffs boy 's ranch ( br ) as shown in figure 1 . big shoals has the closest proximity to the source of the river in the okeefenokee swamp , while the other downstream sites were in closer proximity to higher - density human activity . we found that 96% of samples were positive for s. enterica as indicated by recovery of isolates with the inva gene . although statistics were not possible because mpn was not performed from replicate samples from each site , the results did show that salmonella levels ranged from not detectable to 5400  mpn/100  ml ( table 1 ) . interestingly , the two upstream sites , big shoals and white springs , consistently maintained about 10- to 100-fold lower mpn levels january through april , compared to downstream sites , which were from more densely populated regions or located adjacent to agricultural activity . with the exception of the sample collected at white springs in january , at least 30 isolates were recovered from each time point from all sites and were stored as frozen stocks for additional testing . total heterotrophic aerobic bacterial counts on la from suwannee river water samples during this time period ranged between 1.0  10 and 6.9  10  cfu ml ( data not shown ) . the genetic relatedness among isolates of s. enterica recovered from the suwannee river was evaluated by the diversilab rep - pcr system . these isolates were compared to other s. enterica strains from both clinical and environmental sources and to the diversilab s. enterica library . this method divides s. enterica strains into two main clusters that segregate subspecies iii from subspecies i strains ( figure 2 ) . replicate analysis ( n = 10 ) of the same strains ( n = 6 ) generally showed at least 95% similarity ( data not shown ) . therefore , isolates with > 95% similarity were considered to be clonal by this assay . a total of 499 strains were examined by rep - pcr , and all strains were > 60% similar by this assay . strains segregated into 16 genogroups using the criteria of > 85% dna similarity for more than two strains , while 14 strains were ungrouped ( supplemental figure 1s ) . overall , s. enterica strains recovered from the suwannee river were quite diverse and were distributed among 10 genogroups , while 6 isolates did not cluster with other strains ( supplemental figure 1s ) . none of the suwannee isolates clustered with subspecies 3 ( arizonae ) . significant ( p < 0.001 ) genetic relatedness was observed among the suwannee river isolates by rep - pcr , and these strains were generally distinct from strains derived from other sources . most ( 74% ) of the suwannee river strains segregated into only two genogroups , namely , 10 and 15 . each of these groups contained only one isolate from non - suwannee sources , including one from a clinical case and another from a tomato . conversely , 69% of strains from the diversilab library , consisting of isolates from clinical sources , were distributed into genogroups 5 , 6 , 11 , and 13 , while only 12% of suwannee river isolates were included in any of these groups . interestingly , suwannee river isolates were also distinct from strains derived from other environmental sources including those from central florida , such as frogs , oranges , or lakes ( table 2 ) . for example , the isolates from central florida lakes clustered into genogroups 5 and 11 that were predominantly populated with strains of clinical origin . examination of the distribution of suwannee river isolates by sampling site showed that the different genogroups were found among various sampling locations , and the genogroups that were most commonly populated with suwannee isolates ( 10 and 15 ) were found at all sites ( table 3 ) . although strains were isolated from enriched samples , clonal isolates ( > 95% similarity ) did not necessarily correspond to the same sample site or time . however , because of the low number and unequal distribution of isolates collected from each site or time point , statistical analysis was not performed with respect to sample site or seasonality , and no clear relationship was established . s. enterica isolates ( n = 30 ) recovered from the suwannee river in a prior study in 1998 - 1999   were serotyped , and serotypes included inverness ( n = 9 ) , muenchen ( n = 3 ) , rubislaw ( n = 6 ) , braenderup ( n = 2 ) , and one strain each of montevideo , newport , johannesburg , and cubana . some strains were untypeable strains ( n = 3 ) or rough ( n = 3 ) . rep - pcr analysis showed agreement with serology of s. enterica derived from the suwannee river and elsewhere , and strains ( 70% ) with the same serovar were frequently clonal ( > 95% similar ) with one or more strains from independent sources . however , rep - pcr also revealed genetic distinctions among strains of the same serovar , as serotypes frequently segregated into multiple genogroups ( table 6 ) . furthermore , the genetic relatedness of isolates that were un - typeable by serology was revealed by rep - pcr . for example , an un - typeable suwannee river isolate ( uf-14 ) was not identified as s. enterica by serotyping but demonstrated 96% dna similarity to other suwannee river isolates that were serotyped as braenderup ( uf-8 and uf-9 ) in genogroup 9 ( supplemental figure 1(s ) ) . 95% dna similarity with two suwannee river isolates serotyped as inverness ( uf-28 and uf-30 ) within genogroup 10 . a high level of dna similarity ( > 90% ) was also noted among several suwannee river isolates with serotype rubislaw and another un - typeable strain ( uf-3 ) within genogroup15 . however , two un - typeable ( uf-12 , uf-15 ) strains did not cluster with any other isolates at the 85% level of dna similarity . a single clinical s. enterica inverness serotype clustered with 39 suwannee river isolates in genogroup 10 , including 8 suwannee river isolates that were also inverness serotype . the remaining suwannee river isolate that serotyped as inverness was found in genogroup 5 that also included suwannee river isolates which serotyped as muenchen and johannesburg . several of the suwannee river strains indicated very high levels ( 90% ) of dna relatedness to important clinical serovars such as typhimurium , montevideo , and muenchen . s. enterica was widely distributed in the upper suwannee river and was recovered from multiple locations at different points of time . serotyping and genetic analyses illustrated the diversity of these strains but also showed that some genotypes may be more prevalent in the suwannee river than others , as > 70% of strains clustered into only two genogroups . furthermore , these genogroups were distinct as most strains derived from clinical and other environmental sources were excluded . interestingly , strains recovered from central florida lakes in a preliminary study using similar methodology were much more likely to cluster with strains from clinical sources . although the number of strains in this study is too small to draw conclusions about these populations , results suggest that genotypes of s. enterica may be differentially distributed in florida aquifers . the diversilab rep - pcr assay showed some agreement with serotyping , as serotypes from different sources frequently clustered together . however , genetic typing also indicated that some stains with the same serotype were genetically diverse . for example , strains from the suwannee river with serotype inverness were mostly ( 8 of 9 ) in genogroup 10 , which also included a clinical inverness strain , but one of the suwannee river inverness isolates diverged to genogroup 5 and was more genetically related to strains with serotype muenchen . weigel et al .   noted greater discrimination power for rep - pcr compared to pfge , but kerouanton et al . chmielewski et al .   found rep - pcr and eric - pcr to be highly discriminatory among isolates from poland , but reproducibility was an issue with band number and positioning varying with the model of the thermocycler . the diversilab system ( biomerieux ) for rep - pcr used in the present study is distinctive from other rep - pcr assays due to the standardized reagents , online database , internal controls for peak size and height , and the use of capillary electrophoresis for characterization of pcr products . we found that independent samples of the same salmonella strain were highly reproducible and always clustered with > 95% similarity . recently found this system was an excellent predictor of serotype , and kilic et al . the top 10 disease - associated serotypes in 2009 included newport , typhimurium , javiana , muenchen , heidelberg , montevideo , oranienburg , and st . while the true diversity of salmonella serovars in the florida aquifers is still unknown , the present study identified 8 serovars ( inverness , muenchen , rubislaw , braenderup , montevideo , newport , johannesburg , and cubana ) and 10 genogroups from this region . a prior investigation in 1989 reported 14 salmonella serovars derived from the suwannee river that included allandale , aqua , braenderup , daytona , gaminara , hartford , inverness , montevideo , muenchen , paratyphi b , saintpaul , subspecies iv , tallahassee , and typhimurium . another study in 1997 reported 7 serovars ( rubislaw , tallahassee , gaminara , javiana , muenchen , inverness , and hartford ) , and in 1998 , 10 serovars were reported ( allandale , montevideo , gaminara , hartford , inverness , javiana , muenchen , rubislaw , tallahassee , and arizonae ) by the florida department of agriculture consumer services . thus , muenchen and inverness were common to all studies ( including the present study ) , while braenderup , montevideo , and rubislaw were found in more than one study . the present study conducted in north florida showed some similarity to a recent survey of salmonella from the little river in southern georgia portion of the upper suwannee river watershed . both reports identified muenchen and rubislaw among the predominant serotypes , but 40.6% of strains in the prior study were identified as s. enterica subsp . arizonae , which was not recovered from the suwannee river either by serotype or rep - pcr genotype . they concluded that recovery of muenchen in the watershed was epidemiologically significant , as its incidence is increasing in human cases in georgia ( 34% increase over 10 years ) . furthermore , the muenchen strains from the suwannee river were associated with a predominantly clinical ( genogroup 5 ) genotype . no javiana was included in this study , and the braenderup identified from the suwannee river formed a unique rep - pcr cluster ( genogroup 9 ) . neither study identified the most common serotype - associated salmonellosis , namely , serotype enteritidis , from water samples . furthermore the rep - pcr genogroup 6 that was identified with clinical enteritidis strains did not contain any isolates from the suwannee river . however , it should be noted the genogroup 7 that included clinical typhimurium strains did cluster with 4 isolates from the suwannee river . thus , perhaps the most intriguing finding of the present study was the identification of salmonella strains in the suwannee river that were genetically associated with strains from clinical sources . although these investigations are not a systematic or exhaustive survey of suwannee river watershed , they demonstrate the diversity of salmonella in possible irrigation sources and suggest the presence of potential pathogens . franz and van bruggen   reported that the prevalence of pathogens in the environment is inversely proportional to the genetic diversity of the biome and that eutrophic environments promote decreased diversity . while the present investigation did not examine environmental parameters with respect to distribution of salmonella in florida , there are striking differences in the human population densities associated with upstream versus downstream sites . the upstream sites are in closer proximity to the source of this river and greater distance from agriculture and other human impact . for example , the most upstream site at big shoals is in a state park and is essentially unpopulated with no agriculture in close proximity to the river , while the other sites are in direct contact with human populations . these differences may explain the 10- to 100-fold increase in the mpn ml at downstream sites relative to upstream . however , there were no striking differences observed in terms of diversity of salmonella from the different sites on suwannee river . future studies are needed to examine the complex environmental parameters , especially in relationship to nutrient availability , agricultural input , wildlife distribution , flow rates , rainfall and other factors , that may impact the microbial diversity and survival of salmonella .
the suwannee river spans the florida / georgia border to the gulf of mexico , and contributes to regional irrigation and recreational activities . 
 association of salmonella enterica with these resources may result in the contamination of produce and disease outbreaks . 
 therefore , surface water was examined for the distribution of s. enterica at multiple time points from 4 sites on the upper suwannee river . 
 isolates were confirmed by detection of the inva gene , and 96% of all samples were positive for the bacterium . 
 most probable number enumeration ranged from < 18 to 5400  mpn/100  ml . 
 genetic diversity of these isolates ( n=110 ) was compared to other environmental ( n=47 ) or clinical ( n=28 ) strains and to an online library ( n=314 ) using diversilab rep - pcr . 
 all strains showed > 60% similarity and distributed into 16 rep - pcr genogroups . 
 most ( 74% ) of the suwannee river isolates were clustered into two genogroups that were comprised almost exclusively ( 97% ) of just these isolates . 
 conversely , 85% of the clinical reference strains clustered into other genogroups . however , some suwannee river isolates ( 12% ) were clustered with these primarily clinically - associated genogroups , supporting the hypothesis that river water can serve as a disease reservoir and that pathogenic strains may persist or possibly originate from environmental sources .