diversity of escherichia coli and salmonella spp isolates from playa … · 2017-02-09 ·...

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Diversity of Escherichia coli and Salmonella spp Isolates from Playa Waters and Sediments.

W. C. Rice, C. W. Purdy

The authors are William C Rice, Microbiologist, and Charles W Purdy, Microbiologist, United States Department of Agriculture, Agricultural Research Service, Conservation and Production Research Laboratory, Bushland, TX. Corresponding author: William C Rice, P. O. Drawer 10, 2300 Experiment Station Road, Bushland, TX 79012-0010; phone: 806-356-5706; fax: 806-356-5750; e-mail: [email protected].

ABSTRACT. Preliminary work indicates that the CAFO environment is populated by various strains of

Escherichia coli and Salmonella sp, numerous other members of the Enterobacteriaceae along with a wide variety of naturally occurring microorganisms. A central question to be addressed, are CAFO playa environments populated by resident genotypes and associated antibiotic resistant phenotypes or is there a shifting spectrum of genotypes and pathotypes? Answers to this question may influence management practices concerning the handling of animal waste. We report on the genetic structure of Escherichia coli and Salmonella sp that occupy the beef CAFO environment of the high plains of Texas. Research was conducted at seven feedyards and three control playas with periodic summer and winter samples collected over a two year period. Selective and nonselective media were used to isolate Escherichia coli and Salmonella sp and other microorganisms. Genetic analysis of Escherichia coli and Salmonella strains using for example repetitive element PCR (Rep-PCR) based assays indicate presence of five to seven dominate DNA fingerprint patterns for each genus. Serological and phenotypic analysis of 239 Salmonella sp indicates the presence of approximately 15 serotypes of Salmonella sp. Playa waters and sediments represent an environment suitable for the habitation of a small number of dominant Escherichia coli and Salmonella sp along with a wide variety of less frequently occurring strains as indicated by genotype.

Keywords. Pathogen diversity, DNA typing, serotype, beef cattle feedyard, playa

INTRODUCTION Escherichia coli O157:H7 and other pathogenic Escherichia coli strains are major food-borne

infectious pathogens and, due to their worldwide distribution, pose a serious threat to public health systems. Escherichia coli O157:H7 was first recognized following two outbreaks in Michigan and Oregon in 1982 (Riley et al 1983) and is capable of causing diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. In the United States close to 75,000 cases of O157:H7 infections are now estimated to occur annually (Mead et al. 1999). The genus Salmonella, comprises one of the most important pathogen groups involved in human foodborne illness, is divided into two species: S. enterica that is subdivided into over 2,000 serovars, and S. bongori. One serovar, S. enterica serovar Typhi CT18 is host restricted, infecting only humans (typhoid fever), while the other serovar, S. enterica serovar Typhimurium LT2, is a host generalist that infects humans (gastroenteritis) and other mammalian species. Domestic animals act as a reservoir of non-typhoid Salmonella infections and are responsible for millions of infections and multiple deaths in the human population costing billions of dollars (range 4 to 23) yearly (Todd 1990). Thus, the epidemiological relationships amongst various Salmonella serotypes; especially as related to outbreaks of human salmonellosis due to consumption of contaminated dairy, poultry and meat products; is a key concern in monitoring the presence and spread of disease.

One question of interest regarding beef cattle confined animal feeding operation (CAFO) environments is the population structure of various isolates of Escherichia coli and Salmonella spp. Are these CAFO environments dominated by a few resident genotypes or are there ever-shifting spectrums of genotypes? Playa water and sediment samples within the CAFO environment were

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obtained from seven feedyard and three control playas via the periodic collection of summer and winter samples over a four year period. Selective media were used for the isolation of members of the family Enterobacteriaceae, Escherichia coli and Salmonella sp. Research results indicate that beef CAFOs are populated by various strains of Escherichia coli and Salmonella sp along with numerous other members of the family Enterobacteriaceae (based on the use selective chromogenic media and biochemical evaluation). We report on the genetic structure of Escherichia coli and Salmonella strains that occupy the beef CAFO environment of the high plains of Texas. Genetic analysis of 221 Escherichia coli and 239 Salmonella strains was conducted using Rep-PCR assay employing the BOXA1R primer. Rep-PCR has proven to be a sensitive and rapid DNA typing method with broad applicability to analyzing the genetic variability of a broad range of microbial organisms (Versalovic et al. 1991, Olive and Bean 1999). Unknown playa Escherichia coli strains were referenced against the DEC A set of Escherichia coli strains obtained from the Dept. of Toxicology, Michigan State University. This reference collection is comprised of known enterohemorrhagic Escherichia coli (EHEC) isolates and enteropathogenic Escherichia coli (EPEC) isolates such as various serotypes; O157:, O128:, O111:, etc. Salmonella sp were serotype and compared on the basis of serology. Results of this study are reported below.

MATERIALS AND METHODS

ENVIRONMENTAL SAMPLING Environmental samples consisting of feedyard playa water and sediments were sampled at all

four cardinal locations (N, S, E, and W) within a feedyard playa primarily during the summer and winter of 2001 and 2002 (with the addition of a previously obtained 1998 sample). Duplicate one liter water samples were collected within 3 meters of one another at each location and pooled. Sediments were collected into 300 ml polypropylene containers using a round-nosed shovel at each location.

E. COLI ISOLATION PROCEDURE Water samples (100 ml aliquot) were diluted 1:1 in Novobiocin (25 µg/ml) – Tryptose broth

(NTB), incubated overnight at 37 ˚C followed by a 1:10 dilution in NTB and again incubated overnight at 37 ˚C. Aliquots (0.1 ml) of serial dilutions were plated out on sorbitol MacConkey agar plates and incubated overnight at 37 ˚C. Alternatively water samples were mixed 1:1 with 2X EC modified enrichment broth plus Novobiocin (25 µg/ml) / potassium tellurite (0.25 ug) and incubated overnight at 37 ˚C followed by direct plating on sorbitol MacConkey agar plates. Sediments were diluted 1:10 (10 g sediment / 90 ml NTB) and incubated overnight, and then aliquots (0.1 ml) of serial dilutions were plated out on sorbitol MacConkey agar plates and incubated overnight at 37 ˚C. White and pink/red colonies were selected, grown overnight and stored frozen in milks at -80 ˚C until further analysis. Suspect Escherichia coli strains stored at -80 ˚C were reisolated on HiCrome Agar™ (Sigma Chemical Co, St. Louis, MO) a chromogenic agar for differentiation of various Enterobacteriaceae species. All dark blue colonies (suspect E. coli species) were confirmed biochemically using the indole test.

SALMONELLA SP ISOLATION PROCEDURE Water samples (100 ml aliquot) were diluted 1:1 in 100 ml of 2X strength sulfur-brilliant-green

(SBG) enrichment broth plus novobiocin (25 µg/ml) and incubated for 24 h at 37 ˚C. A 100 ml aliquot of SBG enrichment culture was mixed with 100 ml of fresh 1X SBG plus novobiocin enrichment broth and cultured for 24 h at 37 ˚C. Sediments were diluted 1:10 (10 g sediment / 90 ml SBG plus

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novobiocin) and incubated in a shaker incubator at 37 ˚C for 24 h. The sediment-enrichment culture was transferred to 100 ml of 1X SBG plus novobiocin and incubated at 37 ˚C for 24 h. After the second incubation, samples were cultured for Salmonella spp on 3 types of differential media (MacConkey agar, brilliant green agar, and xylose-lysine-desoxycholate agar) at 37 ˚C for 24 h. Suspect Salmonella spp were evaluated on additional differential media (triple-sugar-iron, lysine-iron agar, motility indole ornithine, and urease). Suspect Salmonella spp revealed from these tests were evaluated on nutrient agar plates plus 5% bovine serum albumin for the absence of hemolytic activity. Suspect Salmonella spp were shipped to the National Veterinary Services Laboratory in Ames, IA for verification as Salmonella enterica sp and serotyping. Suspect Salmonella strains stored at -80 ˚C were reisolated on Rainbow Salmonella Agar™ (Biolog, Hayward, CA) a chromogenic agar for detection and evaluation of various Salmonella spp.

DNA PREPARATION AND BOXA1R PCR Bacterial genomic DNA preparations were made using PUREGENE® DNA isolation kits

according to manufactures instructions (Gentra Systems, Minneapolis, MN). PCR reactions (25 µl) contained the following: 40 ng of template DNA, 1X JumpStart reaction buffer (Sigma Chemical Co, St. Louis, MO), 1.5 mmol 1-1 MgCl2, 200 µmol 1-1 dNTP, 40 pmol 1-1 primer, and 1 U JumpStart Taq DNA polymerase (Sigma Chemical Co, St. Louis, MO). PCR temperature profile of 2 min at 94 ˚C followed by 30 cycles of 30 s at 94 ˚C, 30 s at 50 ˚C, and 1 min at 65 ˚C . The 30 cycles were followed by one cycle of 7.5 min at 72 ˚C. Aliquots (12 µl) of each reaction were run on 1.4% agarose gels and stained with ethidium bromide (Sambrook et al. 1989).

IMAGE ANALYSIS AND DENDROGRAM CONSTRUCTION Gel images were captured and analyzed by using a computerized video image analysis system

(Kodak image workstation 440CF, Eastman Kodak, Rochester, NY). Molecular weight standards were included in each gel to allow for normalization of gel images for valid between-gel comparisons of fingerprints. BOXA1R DNA fingerprint profiles were digitally compared using the Pearson and Dice coefficients and dendrograms were construct using the UPGMA method employing various programs within Bionumerics version 4.0 (Applied Maths, Austin, TX).

RESULTS AND DISCUSSION

Escherichia coli strains were recovered from all feedyard and one control playas while Salmonella spp were recovered only from feedyard playas. Over 800 suspect Escherichia coli strains were obtained from seven feedyards and one control playa during the sampling period. Approximately 37% were confirmed to be Escherichia coli isolates based on dark blue colony phenotype (Table 1) when plated on HiCrome Agar™ and confirmed by a positive indole assay result. Other Enterobacteriaceae bacterial strains noted are indicated by the following phenotypes that were observed on HiCrome Agar and they are indicated as follows: DBV, dark blue – E coli; SR, salmon red – Enterobacter cloacae or Citrobacter freundii; LP, light pink – Klebsiella pneumonia; CL, colorless – Salmonella enteritidis or Shigella flexneri and WH, white – undefined phenotype.

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Table 1. Phenotype of suspect E. coli strains*

Phenotype No of Isolates CL 125

DBV 224 LP 48

NG^ 235 SR 147 WH 46

*on HiCrome Agar ^no growth

A broad diversity of DNA fingerprint patterns and / or Salmonella serotypes were observed within the Escherichia coli and Salmonella strains isolated from the Texas high plains beef cattle CAFO playa environment. This is similar to other reports documenting a broad range of serotypes of Escherichia coli and Salmonella strains isolated from cattle-pastureland operations and beef cattle CAFO from different geographical locations and climatic environments (Beach et al. 2002; Dargatz et al. 2000; Purdy et al. 2004; Renter et al. 2003, 2004). Genetic analysis of 221 suspect Escherichia coli strains using Rep-PCR (BOXA1R) assay indicates the presence of at least five overall dominate DNA fingerprint types amongst those observed diverging from 30 % to 70 % similarity level (genetic variability) along with respective distinctive subtypes (Fig 1 and 2). A total of 42 subtypes comprising 100 isolates were observed. The number of strains per DNA fingerprint subtype ranged from 2 to 6. A total of 121 unique DNA fingerprint patterns were observed although some are similar to other dominant DNA fingerprints (range of 75 % to 95 % similarity). One DNA fingerprint pattern was confined primarily to playa F11 (Fig 1) while numerous DNA patterns were more broadly distributed amongst three to five playas (combinations of playas F2, F3, F5, F7 and F8) in both water and / or sediment samples (Fig 2). Identical DNA fingerprints were observed in different playas at different times, while playa F11 also contained several different DNA fingerprint broadly distributed throughout both water and sediment samples. Escherichia coli isolates from the feedyard playas were similar to the DEC A reference strain set in the range of 10 % to 95 % similarity (data not shown). No Escherichia coli isolate yielded an identical DNA fingerprint to known Escherichia coli O157:H7 strains. However, twelve Escherichia coli O157:H7 isolates were obtained by enrichment and the use of immunomagnetic separation technology from these playa water and sediment samples that were sent to other investigators (personal communication).

A total of 239 Salmonella strains were confirmed either by serological analysis or by growth and phenotypic analysis on Rainbow Salmonella agar. Phenotypic results on Rainbow Salmonella agar indicate the presence of either S gallinarum or S pullorum sp. A total of 15 confirmed Salmonella serotypes were determined by serological and or phenotypic analysis. Rep-PCR DNA fingerprint pattern analysis indicates the possibility of an additional three to five Salmonella sp (data not shown). The distribution of Salmonella serotypes across CAFO playas ranged from 4 serotypes that occur only in a single playa to two serotypes that are present in four playas (Table 2). With the exception of the serotype S typhimurium the genetic variability of the observed Salmonella spp ranged from the 23% to 81% similarity levels (Table 2).

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Table 2. Distribution of Salmonella serotypes across feedyard playas

No. of Similarity (%)

Salmonella sp strains DNA

types Playa (no of strains) amongst

Strains S agona 3 2 F1 (1), F5 (2) 49.3 S anatum 6 3 F1 (4), F2 (2) 78.0 S cerro 5 3 F11 (4), F3 (1) 47.7 S give 3 2 F11 79.8 S infantis 7 NA` F1 NA S kentucky 33 3* F3 69.4 S kiambu 10 3 F1 80.9

S mbandaka 51 5* F2 (8), F3 (16), F7 (21), F11

(6) 23.2 S montevideo 22 3* F1 (19), F7 (3) 42.2 S oranienburg 3 2 F2 66.5 S reading 12 3 F5 (3), F7 (5), F8 (1), F11 (3) 66.7 S thomasville 3 2 F5 53.3 S typhimurium 28 4 F8 (26), F11 (2) 0.1 `pending *no of major, several minor

S mbandaka was the most dominant serotype observed (51 isolates) while four serotypes occurred at a frequency of three isolates per serotype. The number of Salmonella serotypes occurring within a feedyard ranged from a minimum of three serotypes to a maximum of five serotypes per feedyard playa (Table 3). The number of recovered Salmonella isolates per playa ranged from 8 in playa F5 to 63 from playa F3.

Table 3. Distribution of Salmonella serotypes within feedyard playas

Feedyard playa

Salmonella sp Salmonella serotypes

F1 44 S agona, S anatum, S infantis, S kiambu, S montevideo F2 12 S anatum, S mbandaka, S oranienburg F3 63 S cerro, S kentucky, S mbandaka, F5 8 S agona, S reading, S thomasville F7 49 S mbandaka, S meleagridis, S montevideo, S reading F8 29 S montevideo, S reading, S typhimurium F11 25 S cerro, S give, S mbandaka, S reading, S typhimurium

S mbandaka was broadly distributed in playa waters and sediments of several playas and exhibited considerable genetic diversity (Fig 3). The genetic diversity of ten Salmonella serotypes recovered from three high population density playas indicates that identical genotypes are distributed across feedyard playas and that some serotypes are genetically diverse e.g. S mbandaka (Fig 4, 5, and 6). Feedyard playa waters and sediments represent an environment suitable for the habitation of a small number of dominant Escherichia coli and Salmonella spp along with a wide variety of less frequently occurring strains. It also appears that an ever-shifting spectrum of Escherichia coli and Salmonella spp can coexists along with a small number of dominant isolates. The long range temporal stability of dominant serotypes remains to be established.

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Conclusion

We have determined the genetic population structure of Escherichia coli strains and Salmonella spp present in feedyard playas by the use of repetitive DNA fingerprint method. A database of environmental isolates (reference strain collections) comprised of members of Enterobacteriaceae, Escherichia coli and Salmonella spp. has been established that will allow for the long term monitoring of microbiological populations associated with CAFO environments. We can evaluate this reference collection of Enterobacteriaceae, Escherichia coli and Salmonella strains from the Texas high plains to evaluate the movement of antibiotic resistance genes, the distribution of virulence genes, and to evaluate the fate and transport of pathogens in the environment. Thus, this ‘environmental library’ of microbial isolates will allow for the long term assessment of the impact of beef CAFO on various downstream agricultural production systems.

ACKNOWLEDGEMENTS The authors acknowledge the technical assistance of Mr. Colby Carter and Mr. Will Willis.

REFERENCES 1. Beach, J.C., Murano, E.A. and Acuff, G.R. (2002) Serotyping and antibiotic resistance profiling of

Salmonella in feedlot and nonfeedlot beef cattle. Journal of Food Protection 65, 1694-1699. 2. Dargatz, D.A., Fedorka-Cray, P.J., Ladely, S.R. and Ferris, K.E. (2000) Survey of Salmonella

serotypes shed in feces of beef cows and their antimicrobial susceptibility patterns. Journal of Food Protection 63, 1648-1653.

3. Mead, P.S. et al. Food-related illness and death in the United States. (1999) Emerging Infectious Diseases 5, 607-625.

4. Olive, D.M., and Bean, P. (1999) Principles and Applications of Methods for DNA-based Typing of Microbial Organisms. Journal of Clinical Microbiology 37, 1661-1669.

5. Purdy, C.W., Straus, D.C. and Clark, R.N. (2004) Diversity of Salmonella serovars in feedyard and nonfeedyard playas of the southern high plains in the summer and winter. American Journal of Veterinary Research 65, 40-44.

6. Renter, D.G., Sargeant, J.M., Oberst, R.D. and Samadpour, M. (2003) Diversity, frequency, and persistence of Escherichia coli O157 strains from range cattle environments. Applied and Environmental Microbiology 69, 542-547.

7. Renter, D.G., Sargeant, J.M. and Hungerford, L.L. (2004) Distribution of Escherichia coli O157:H7 within and among cattle operations in pasture-based agricultural areas. American Journal of Veterinary Research 65, 1367-1376.

8. Riley, L.W., Remis R.S., Helgerson, S.D. et al. (1983) Hemorrhagic colitis associated with a rare Escherichia coli serotype. New England Journal of Medicine 308, 681-685

9. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY, 6.3-6.35.

10. Todd, E. (1990) Epidemiology of foodborne illness: North America. Lancet. 336, 788-790. 11. Versalovic, J., Koeuth, T. and Lupski, J.R. (1991) Distribution of repetitive DNA sequences in

eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acid Research 19(24), 6823-6831.

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APPENDIX The following figures 1-6 consist of genetic analysis of bacterial DNA isolated from the

Escherichia coli and Salmonella spp. The column format is as follows: dendrogram, DNA fingerprint, strain designation, genus and species (Figs 3-6 only), feedyard playa, sample date, sample source and direction.

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Fig 1. Diversity of E coli isolates from feedyards F1, F11 and control playa C10.

Dice (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]ES_DBV-BoxA1R

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Fig 2. Diversity of E coli isolates from feedyards F2, F3, F5, F7, and F8

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F5

F5

F5

F5

F5

F5

F2

F2

F3

F5

F7

F2

F5

F2

F2

F3

F2

F2

F2

F2

F3

F2

F5

F5

F5

F7

F8

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F8

F8

F8

F7F8

F8

F8

F8

F8

F8

F8

F8

F8

F8

F8

F8

F7

F8

F8

F8

F8

F8

F7

F7

F3

F5

F8

F2

F8

F8

F2

F7

F8

F3

F3

F7

F5

F5

F5

F5

F5

F5

F5

F7

F2

F5

F3

F2

F2

F2

F2

F8

F3

F8

2002-03-25

2002-03-25

2002-03-25

2001-04-03

2001-04-03

2001-04-03

2001-04-03

2001-04-03

2002-02-11

2001-04-03

2001-04-03

2002-03-25

2001-05-30

2002-02-25

2002-02-25

2002-02-25

2001-04-03

2002-03-25

2002-03-25

2002-03-25

2001-04-03

2002-03-25

2002-03-25

2002-03-25

2001-06-18

2002-03-25

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-05-30

2001-05-30

2001-06-18

2001-06-18

2001-04-03

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-04-03

2002-02-11

2002-03-25

2002-03-25

2001-05-30

2001-04-03

2001-06-18

2001-04-03

2001-04-03

2002-03-25

2002-02-11

2001-04-03

2002-02-11

2001-04-03

2001-08-13

2002-02-11

2002-03-25

2002-03-25

2002-03-25

2001-05-30

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2001-05-30

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2001-05-30 2002-02-25

2002-02-25

2002-02-25

2002-02-25

2001-07-08

2001-07-08

2001-07-08

2002-02-25

2001-07-08

2001-07-08

2002-02-25

2001-07-08

2001-05-30

2002-02-25

2001-07-08

2001-07-08

2001-07-08

2001-07-08

2002-02-25

2001-05-30

2001-08-13

2002-03-25

2001-07-08

2001-04-03

2001-07-08

2001-07-08

2001-04-03

2001-05-30

2002-02-25

2002-03-25

2002-03-25

2001-05-30

2001-05-15

2001-05-15

2001-05-15

2001-06-18

2001-06-18

2001-06-18

2001-06-18

2001-05-30

2002-02-11

2001-06-18

2002-03-25

2002-02-11

2002-02-11

2001-04-03

2001-04-03

2002-02-25

2002-03-25

2001-07-08

Wa

Mu

Wa

Wa

Wa

Mu

Mu

Wa

Wa

Wa

Mu

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Wa

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Mu

Wa

Wa

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Mu

Mu

Wa

Wa

Wa

Mu

Mu

Wa

Mu

Wa

Wa

Mu

Wa

Mu

Mu

Mu

Mu

Mu

Wa

Wa

Mu

Mu

Wa

Wa

Wa

Mu

Wa

Wa

Mu

Wa

WaWa

Wa

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Mu

Mu

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Wa

Wa

Mu

Wa

Mu

Wa

Mu

Mu

Wa

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Wa

W

W

E

E

E

E

W

E

N

E

S

S

S

W

S

S

W

S

W

E

N

S

W

E

S

N

N

W

E

S

E

E

W

E

S

E

W

N

N

E

N

N

W

N

S

W

N

W

E

S

W

W

E

W

N

N

E

N

W

S

S

S

E

S

S

N

N

N

N

S

W

W

E

N

E

S

W

N

W

E

WE

E

S

W

S

W

W

W

W

E

N

S

E

N

N

N

S

S

W

E

W

S

N

N

W

N

S

W

N

N

N

W

N

W

W

N

N

N

W

N

E

N

W

N

W

W

N

E

E

N

10

Fig 3. Diversity of S mbandaka isolates from feedyard playas F2, F3, F7, and F11

100

9080706050403020

100

98.3

91.8

100

89.8

84.8

66.2

58.2

94.7

90.4

76.3

68.9

100

91.6

100

86

90

83.7

100

96.7

100

90.7

100

93.3

88.2

79.4

59.4

55.6

70.6

84.6

65.8

57.3

52

40.2

55.6

48.8

33.1

22.8

14.5

SS45

SS104SS62

SS159SS162

SS225SS108

SS171SS166

SS84SS90SS101

SS107SS98

SS42SS46

SS47SS48

SS49SS51

SS92SS94

SS83SS232

SS233SS105

SS159SS100SS153

SS142SS175

SS181SS182

SS184SS150

SS151SS154

SS158SS161

SS109SS163

SS234SS61SS99

SS227SS40

SS228SS177

SS186SS170

SS183

Salmonella

Salmonella Salmonella




Salmonella Salmonella Salmonella



















Salmonella

mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka


mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka


mbandaka mbandaka

mbandaka mbandaka

mbandaka mbandaka

mbandaka

F2

F3F3

F7F7

F11F3

F7F7

F3F3F3

F3F3

F2F2

F2F2

F2F2

F3F3

F3F11

F11F3

F7F3F7

F7F7

F7F7

F7F7

F7F7

F7F7

F3F7

F11F3F3

F11F2

F11F7

F7F7

F7

2002-02-11

2002-03-252001-08-13

2002-02-252002-02-25

2002-03-182002-03-25

2002-02-252002-02-25

2002-03-252002-03-252002-03-25

2002-03-252002-03-25

2002-02-112002-02-11

2002-02-112002-02-11

2002-02-112002-02-11

2002-03-252002-03-25

2002-03-252002-03-18

2002-03-182002-03-25

2002-02-252002-03-252002-02-25

2002-02-252002-02-25

2002-02-252002-02-25

2002-02-252002-02-25

2002-02-252002-02-25

2002-02-252002-02-25

2002-03-252002-02-25

2002-03-182001-08-132002-03-25

2002-03-182002-02-11

2002-03-182002-02-25

2002-02-252002-02-25

2002-02-25

Wa

MuWa

WaWa

WaMu

MuWa

WaWaMu

MuMu

WaWa

WaWa

WaWa

WaWa

WaWa

WaMu

WaMuWa

WaMu

MuMu

MuWa

WaWa

WaWa

MuWa

WaWaMu

WaWa

WaMu

MuMu

Mu

N

ES

SE

SW

NW

WSS

WN

WN

NW

WW

EE

WS

SE

SNN

NS

EE

WW

WN

SE

WE

SSN

SW

SS

WN

E

11

Fig 4. Diversity of Salmonella spp within F1 as determined by Rep-PCR

100

90807060504030

90

100

90.9

88.4

100

96.1

100

94.6

76.8

88

84.4

71

100

91.3

85.1

67.7

100

96

100

96.2

94.8

100

91.9

100

98.4

96.8

96.8

94

91

88.3

63.4

80

42.8

27.1

SS4

SS13

SS14

SS21

SS28

SS5

SS7

SS8

SS16

SS18

SS19

SS17

SS20

SS9

SS26

SS35

SS34

SS25

SS1

SS10

SS11

SS12

SS31

SS23

SS32

SS33

SS15

SS29

SS27

SS30

SS24

SS22

SS6

SS2

SS3

NG_SS13

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

montevideo

montevideo

montevideo

montevideo

montevideo

kiambu

kiambu

kiambu

kiambu

kiambu

kiambu

kiambu

kiambu

kiambu

montevideo

anatum

anatum

anatum

montevideo

montevideo

montevideo

montevideo

montevideo

montevideo

montevideo

anatum

kiambu

montevideo

montevideo

montevideo

montevideo

montevideo

montevideo

Suspect

Suspect

montevideo

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

F1

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-04

2002-02-13

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Mu

Mu

Mu

Wa

Wa

Wa

Wa

Mu

Wa

Mu

Mu

Wa

Mu

Mu

Mu

Mu

Wa

Wa

Wa

Wa

Wa

N

W

W

S

N

N

E

E

N

E

E

N

E

E

N

S

S

W

W

S

S

S

W

S

E

S

N

E

N

E

W

S

N

W

W

W

12


100

9590858075706560555045100

95.2

92.8

95.2

87.8

95.2

85.4

92.3

88.9

74.7

100

100

96.5

100

100

97.5

94.1

100

86.5

72.3

96

86.8

72.4

61.2

100

93.6

100

88.8

100

96.1

80.8

75.9

100

96.3

88.3

100

100

95.4

89.9

85

67.4

60.3

50.6

43.6

SS66

SS89

SS79

SS87

SS71

SS73

SS78

SS98

NG_SS151

NG_SS137

SS107

SS82

SS96

SS59

SS65

SS70

SS75

SS76

SS77

SS80

SS81

SS84

SS85

SS86

NG_SS138

SS91

NG_SS142

SS72

NG_SS148

SS90

SS97

SS101

SS111

SS112

NG_SS140

NG_SS131

NG_SS132

SS60

SS61

SS99

SS95

SS100

SS102

SS106

SS103

SS105

NG_SS134

SS64

SS92

SS94

SS83

SS109

SS62

SS104

SS110

SS108

SS58

SS74

SS56

SS57

SS55

SS88

SS155

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

Salmonella

NA

Salmonella

NA

Salmonella

Salmonella

Salmonella

NA

NA

NA

NA

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

mbandaka

NA

NA

mbandaka

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

mbandaka

kentucky

kentucky

NA

kentucky

NA

kentucky

NA

mbandaka

kentucky

mbandaka

NA

NA

NA

NA

NA

kentucky

mbandaka

mbandaka

kentucky

mbandaka

cerro

kentucky

kentucky

mbandaka

NA

kentucky

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

NA

mbandaka

kentucky

kentucky

kentucky

kentucky

kentucky

kentucky

NA

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

F3

2001-08-13

2002-03-25

2002-03-25

2002-03-25

2001-08-13

2002-03-25

2002-03-25

2002-03-25

2001-08-13

2001-08-30

2002-03-25

2002-03-25

2002-03-25

2001-08-13

2001-08-13

2001-08-13

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2001-08-30

2002-03-25

2001-08-13

2001-08-13

2001-08-30

2002-03-25

2002-03-25

2002-03-25

2001-08-30

2001-08-30

2001-08-13

2001-08-30

2001-08-30

2001-08-13

2001-08-13

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2001-08-30

2001-08-13

2002-03-25

2002-03-25

2002-03-25

2002-03-25

2001-08-13

2002-03-25

2001-08-30

2002-03-25

2001-08-13

2002-03-25

2001-08-13

2001-08-13

2001-08-13

2002-03-25

2001-08-30

Mu

Wa

Wa

Wa

Mu

Wa

Wa

Mu

M

M

Mu

Wa

Wa

Wa

Wa

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

M

Wa

W

Mu

W

Wa

Wa

Mu

W

W

M

W

W

Wa

Wa

Mu

Wa

Mu

Mu

Mu

Mu

Mu

W

Wa

Wa

Wa

Wa

Mu

Wa

Mu

W

Mu

Wa

Wa

Wa

Wa

Wa

Wa

W

N

S

E

N

W

N

N

N

S

W

W

W

W

S

W

E

N

S

S

E

E

W

N

N

W

E

E

W

S

S

W

S

E

W

N

N

N

S

S

N

W

N

S

E

S

E

S

W

E

E

W

W

S

E

S

W

N

N

N

N

N

S

W

13


100

908070605040302050

28.3

100

50

95.2

68.9

35.1

100

100

93.3

88.6

100

86.5

68.3

92.3

100

76.9

73.9

64.6

100

95.4

50.9

90.9

66.6

100

100

92.1

96.8

95.2

77.2

72.5

61.8

52.6

47.4

30.5

28.6

18.3

50

16.7

SS183

SS134

SS189

SS125

SS177

SS176

SS186

SS132

SS135

SS150

SS151

SS152

SS154

SS158

SS161

SS142

SS153

SS159

SS175

SS181

SS182

SS184

SS185

NG_SS170

SS163

SS190

SS140

SS145

SS146

SS148

SS164

SS165

SS166

SS139

SS147

SS171

SS128

SS129

SS133

SS162

SS126

SS127

SS159

SS123

SS137

SS138

SS136

SS170

SS131

Salmonella

NA

Salmonella

NA

Salmonella

Salmonella

Salmonella

NA

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

NA

NA

NA

Salmonella

NA

NA

Salmonella

NA

NA

NA

NA

Salmonella

NA

mbandaka

NA

suspect

NA

mbandaka

meleagridis

mbandaka

NA

NA

mbandaka

mbandaka

montevideo

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

mbandaka

meleagridis

NA

mbandaka

suspect

reading

reading

reading

montevideo

montevideo

montevideo

mbandaka

reading

reading

mbandaka

NA

NA

NA

mbandaka

NA

NA

mbandaka

NA

NA

NA

NA

mbandaka

NA

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

F7

2002-02-25

2001-05-29

1998-01-10

2001-05-29

2002-02-25

2002-02-25

2002-02-25

2001-05-29

2001-05-29

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2001-05-29

2002-02-25

1998-01-10

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2002-02-25

2001-05-29

2001-05-29

2001-05-29

2002-02-25

2001-05-29

2001-05-29

2002-02-25

2001-05-29

2001-05-29

2001-05-29

2001-05-29

2002-02-25

2001-05-29

Mu

Wa

na

Mu

Mu

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Mu

Mu

Mu

Mu

Wa

Wa

na

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Wa

Mu

Mu

Mu

Wa

Wa

Mu

Mu

Wa

Wa

Wa

Wa

Wa

Mu

Mu

E

E

E

N

S

S

W

N

W

W

W

N

N

S

E

N

N

S

S

E

E

W

W

W

E

E

N

E

E

W

W

W

W

N

E

N

S

E

S

E

W

N

S

S

S

E

N

N

W

diversity of escherichia coli and salmonella spp isolates from playa … · 2017-02-09 ·...

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