DETECTION/ISOLATION OF

YERSINIA PESTIS BY COMPARISON

OF VIRULENCE PLASMID (pYV)-

ASSOCIATED PHENOTYPES IN

YERSINIA SPECIES

SAUMYA BHADURIMicrobial Food Safety Research Unit, Department of Agriculture,

Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane,

Wyndmoor, PA 19038

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.

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.

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.

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.

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 37oC. This

plasmid is unstable in nature.

Incubation at 37oC fosters the loss of plasmid and these

pathogens dissociate into avirulent clones.

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.

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 37oC 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).

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 37oC.

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.

Colony Morphology

The cells were grown on BHA at 37oC 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).

Crystal Violet Binding

(A) CV binding of YEP+ strain

showing small dark violet colonies.

(B) Avirulent YEP- strain showing

large white colonies.

The cells were grown on BHA at 37oC 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.

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 37oC 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).

Autoagglutination TestThe cells were grown in Eagle’s minimal tissue culture medium with 10% fetal bovine serum at 37oC

for 24 h without shaking.

Hydrophobicity Test

The cells were grown on BHA at 37oC 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.

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.

Requirement for Diagnostic Test

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. pestiscontaining 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.

Growth Conditions of

Yersinia Strains

The cells were grown in BHI broth at 25oC for 24-48 h and tested for the presence of pYV/pCD by PCR assay and pYV/pCD-associated phenotypic characteristics were determined.

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.

3

1

2

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.

Comparison of Selected Phenotypic Expression of

pYV/pCD-Bearing Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis

Organism Strain CM CV

bindingLcr

CR-

uptakeAA HP Plasmid

Y. enterocolitica GER + + + + + + +

Y. enterocolitica-C GER - - - - - - -

Y. pseudotuberculosis PB1/+ + + + + + + +

Y. pseudotuberculosis-C PB1/+ - - - - - - -

Y. pestis KIM 5 - + + + - - +

pYV/pCD-less

Y. pestisKuma - - - - - - -

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

Comparison of CR-Uptake by pYV/pCD

(+)ve and (-)ve strains

A number of derivatives of clinical strains of Y. pestis (CDC A1122, CO99.3015, 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.

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.