ISOLATION/DETECTION OF YERSINIA PESTIS BY … · agar and MaConkey agar are recommended for the ......
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
ISOLATION/DETECTION OF YERSINIA PESTIS BY … · agar and MaConkey agar are recommended for the ... (CIN) agar and irgasan-nystatin agar restricts the growth of Y. pestis. These media
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
Closely related enteropathogenic species Yersinia
enterocolitica and Yersinia pseudotuberculosis cause
gastrointestinal disease characterized by diarrhea
and are associated with consumption of
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.
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
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.
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.
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.
The objective of this study was to determine whether
the phenotypic characteristics of pYV/pCD, including
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
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.
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
uptakeAA HP Plasmid
Y. enterocolitica GER + + + + + + +
Y. enterocolitica-C GER - - - - - - -
Y. pseudotuberculosis PB1/+ + + + + + + +
Y. pseudotuberculosis-C PB1/+ - - - - - - -
Y. pestis KIM 5 - + + + - - +
Y. pestisKuma - - - - - - -
Effect of Media on Congo Red-Uptake in pYV/pCD-
Bearing Y. enterocolitica, Y. pseudotuberculosis, and
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.
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.