DETECTION/ISOLATION OF YERSINIA PESTIS BY COMPARISON OF VIRULENCE PLASMID (pyv)- ASSOCIATED PHENOTYPES IN YERSINIA SPECIES

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1 DETECTION/ISOLATION OF YERSINIA PESTIS BY COMPARISON OF VIRULENCE PLASMID (pyv)- ASSOCIATED PHENOTYPES IN YERSINIA SPECIES SAUMYA BHADURI Microbial Food Safety Research Unit, Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038

2 The Medically Significant yersiniae Yersinia pestis is an etiological agent of plague transmitted primarily through fleas from infected rodents. Closely related enteropathogenic species Yersinia enterocolitica and Yersinia pseudotuberculosis cause gastrointestinal disease characterized by diarrhea and are associated with consumption of contaminated food.

3 Yersinia pestis & Food Epidemiological reports demonstrated that the consumption of inadequately cooked goat and camel meat cause oro-pharyngeal plague. The identification of multidrug-resistant strains could pose serious public health threat if they cause plague in large population in the United States by deliberate contamination of food.

4 Historical Perspective of Isolation At present, brain heart infusion (BHA) sheep blood agar and MaConkey agar are recommended for the isolation of Y. pestis by the World Health Organization. But the isolation is complicated by the presence of background flora. The selective cefsulodin-irgasan-novobiocin (CIN) agar and irgasan-nystatin agar restricts the growth of Y. pestis. These media require additional testing for the identification of this pathogen. These tests are time consuming, costly and labor intensive since a large numbers of presumptive colonies have to be screened.

5 Present Detection Method The chromosomally-encoded pigmentation phenotype (Pgm + ) was used for detection of Y. pestis. But due to high frequency of spontaneous deletion of Pgm locus this detection method could result false-negative. Hence, there is no working classical microbiological method for the detection/isolation of Y. pestis.

6 Virulence Plasmid (pyv/pcd) All three pathogenic species target lymph tissues and have genetic determinants essential for infection in these tissues, as well as to overcome host defense mechanisms which are located on a virulence plasmid (pyv/pcd) of about 70-kb. The pyv/pcd genes are expressed only at 37 o C. This plasmid is unstable in nature. Incubation at 37 o C fosters the loss of plasmid and these pathogens dissociate into avirulent clones.

7 Calcium-Dependent Growth Phenotype of pyv/pcd In all three pathogens, carriage of pyv/pcd imparts the calcium-dependent growth phenotype (low calcium response) when cultured at 37 C. Low calcium response (Lcr) is expressed phenotypically on calcium-deficient/low calcium solid media by the formation of pinpoint colonies (0.36 mm in diameter) due to inhibition of cell division.

8 Low Calcium Response of pyv/pcd in Yersinia species The cells were grown on calcium-deficient by chelating calcium with sodium oxalate or low calcium (238 µm calcium) solid media at 37 o C for 24 h. (A) pyv-pcd + strain appeared as pin point colonies (0.36 mm in diameter). (B) Avirulent pyv-pcd - strain was used as negative control showing large colonies (1.37 mm in diameter).

9 pyv/pcd-encoded V & W Antigens and Released Proteins The Lcr also results in the production of pyv/pcd-encoded virulence-associated antigens (V and W) and a series of released proteins (Yops) at 37 o C.

10 pyv/pcd Encoded Phenotypes in Y. enterocolitica The pyv/pcd in Y. enterocolitica (YEP + ) has been correlated with several other in vitro characteristics which are phenotypically expressed at 37 C. These well characterized pyv/pcd-associated virulence determinants had been used for isolation and detection of various serotypes of pyv/pcd-bearing Y. enterocolitica in food.

11 Colony Morphology The cells were grown on BHA at 37 o C for 24 h. (A) Virulent YEP + strain appeared as small colonies (1.13 mm in diameter). (B) Avirulent pyv/pcd less (YEP - ) strain used as negative control showing large colonies (2.4 mm in diameter).

12 Crystal Violet Binding The cells were grown on BHA at 37 o C for 24 h. The plates were gently flooded with 10 ml of a 100 µg/ml of crystal violet (CV) solution for 2 min and decanted. (A) CV binding of YEP + strain showing small dark violet colonies. (B) Avirulent YEP - strain showing large white colonies.

13 Congo Red (CR)-Uptake The cells were grown on calcium-deficient by chelating calcium with sodium oxalate or low calcium (238 µm calcium) solid media with 75 µg/ml of CR at 37 o C for 24 h. (A) Virulent YEP + strain appeared as red pin point colonies (0.36 mm in diameter). (B) Avirulent YEP - strain was used as negative control showing large white colonies (1.37 mm in diameter).

14 Autoagglutination Test The cells were grown in Eagle s minimal tissue culture medium with 10% fetal bovine serum at 37 o C for 24 h without shaking.

15 Hydrophobicity Test The cells were grown on BHA at 37 o C for 24 h. A loop of cells from a colony was mixed with latex particle on a slide. (A) Virulent YEP + cells formed clumps. (B) Avirulent YEP - cells remained dispersed.

16 Objective The objective of this study was to determine whether the phenotypic characteristics of pyv/pcd, including CV binding, colony morphology/size, Lcr, CR-uptake, AA, and HP are expressed in Y. pestis and the differential expression of these phenotypes can be utilized for the isolation/detection of Y. pestis in foods.

17 Requirement for Diagnostic Test

18 Yersinia Strains A derivative (KIM 5) of pyv/pcd- bearing clinical strain of KIM (Kurdistan Iran man) of Y. pestis (YP) lacking the chromosomally-encoded pigmentation virulence determinants (Pgm - ) was used in this study to show that the CR binding was encoded specifically by pyv/pcd. Strain Kuma, a derivative of a clinical strain of Y. pestis containing the Pgm locus but lacking pyv/pcd was also used to differentiate CR-uptake encoded by the Pgm locus and pyv/pcd, respectively. The pyv/pcd- bearing clinical isolates of Y. pseudotuberculosis (YPST) (serotype O:1b; strain PB1/+) and Y. enterocolitica (YE) (serotype O:3; strain GER) were also used in the current study.

19 Growth Conditions of Yersinia Strains The cells were grown in BHI broth at 25 o C for h and tested for the presence of pyv/pcd by PCR assay and pyv/pcd-associated phenotypic characteristics were determined.

20 PCR Assay of pyv/pcd Lanes 1 (YE), 5 (YPST) and 9 (YP): Absence of virf in cells from white border surrounding red pinpoint colony. Lanes 2 (YE), 6 (YPST), and 10 (YP): Presence of virf in cells from red pinpoint colony (center) surrounded by white border. Lanes 3 (YE), 7 (YPST) and 11 (YP): Presence of virf in cells from red pinpoint colony with no surrounding white border. Lanes 4 (YE), 8 (YPST), and 12 (YP): Presence of virf in original strains.

21 Review of pyv/pcd-associated Phenotypes of Yersinia Species pyv/pcd-bearing cells are designated as virulent YEP + strain. pyv/pcd-negative cells are designated as avirulent YEP - strain.

22 Comparison of Selected Phenotypic Expression of pyv/pcd-bearing Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis Organism Strain CM CV binding Lcr CRuptake AA HP Plasmid Y. enterocolitica GER Y. enterocolitica-c GER Y. pseudotuberculosis PB1/ Y. pseudotuberculosis-c PB1/ Y. pestis KIM pyv/pcd-less Y. pestis Kuma

23 Effect of Media on Congo Red-Uptake in pyv/pcd- Bearing Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis Organism Strain CR-BHO CR-MOX Y. enterocolitica GER + + Y. enterocolitica-c GER - - Y. pseudotuberculosis PB1/+ + + Y. pseudotuberculosis C PB1/+ - - Y. pestis KIM5 - + pyv/pcd-less Y. pestis Kuma - - Low calcium Calcium deficient

24 Comparison of CR-Uptake by pyv/pcd (+)ve and (-)ve strains A number of derivatives of clinical strains of Y. pestis (CDC A1122, CO , Yokohama, P12, D1, D3, D5, D7, D9, D13, and D17) containing Pgm locus but lacking the pyv/pcd were also used to show absence of pyv/pcd-encoded CR-uptake by plating them on CR-MOX. These strains did not bind CR and thus confirmed that the CR-uptake is expressed by pyv/pcd. These observations indicate that the CR-uptake in Y. pestis grown on CR-MOX is independent of Pgm locus and is not expressed under this condition. This phenotype is encoded by pyv/pcd only on calcium depleted medium.

25 Conclusion Out of six pyv/pcd-associated phenotypes examined, only three phenotypes (Lcr, CR-uptake, and CV binding) were expressed in Y. pestis, while all six properties were expressed in Y. enterocolitica and Y. pseudotuberculosis. The specific CR-uptake of Y. pestis in the calcium-deficient CR-MOX medium provides a screening medium to isolate, detect and to differentiate this pathogen from Y. enterocolitica and Y. pseudotuberculosis. This method of isolation/detection for Y. pestis in food was verified by recovering the organism from artificially contaminated sterilized ground beef. The delayed expression of Lcr and CV binding and the non-expression of colony morphology, HP and AA provide a diagnostic tool for differentiating Y. pestis from Y. enterocolitica and Y. pseudotuberculosis. The combination of these six pyv/pcd-associated phenotypes expression provides the means to identify Y. pestis colonies in clinical samples, animals, and food.