A Procedure for Monitoring the Presence of the Virulence Plasmid (pYV) in Yersinia pestis Under Culture Conditions

Abstract

The pathogenicity of Yersinia pestis depends on the presence of a virulence plasmid (pYV). The unstable nature of pYV in Y. pestis leads to the eventual outgrowth of pYV-less cells due to its higher growth rate. Thus, it was necessary to develop procedures to monitor the presence of the plasmid during cultivation, storage, and laboratory manipulations. A procedure was developed to monitor the presence of pYV in Y. pestis by using low calcium response and Congo red binding techniques. The selection of pYV in the isolated clones was confirmed by polymerase chain reaction and by expression of pYV-associated phenotypes. Thus, using this procedure, low calcium response-Congo red-positive clones can be isolated for use in the development of growth models of virulent Y. pestis in food.

Introduction

Y ersinia pestis , Yersinia enterocolitica, and Yersinia pseudotuberculosis cause diverse human diseases. Y. enterocolitica and Y. pseudotuberculosis can induce intestinal distress when consumed in contaminated food. Y. pestis, the agent of bubonic plague, can cause oro-pharyngeal plague as a result of the consumption or handling of meat from infected animals (Christie et al., 1980; Arbaji et al., 2005; Bin Saeed et al., 2005). Thus, food may have a role in the dissemination of Y. pestis. All three species have been shown to carry a 70-kb virulence plasmid (pYV also known as pCD1), which is directly involved with virulence (Perry and Fetherston, 1997; Brubaker, 2006; Carniel, 2006; Chen et al., 2010). The pYV-associated temperature-dependent phenotypes, which are expressed at 37°C but not at 28°C, include colony morphology/size (size = 1.13 mm), low calcium response (Lcr, pin point colony, size = 0.36 mm), crystal violet (CV) binding (dark-violet colony), Congo red (CR)-uptake (red pin point colony, size = 0.36 mm), autoagglutination (AA = cells agglutinate), hydrophobicity (HP = forms clumps), and expression of a series of released proteins (Yops) (Lazere and Gemski, 1983; Lachica and Zink, 1984; Skurnik et al., 1984; Bhaduri et al., 1987, 1991; Straley, 1991; Bhaduri, 2001; Robins-Browne, 2001; Weagant et al., 2007; Bhaduri and Sommers, 2008). At the same time, expression of these phenotypes at 37°C results in the loss of pYV (Straley, 1991; Straley et al., 1993; Bhaduri, 2001; Robins-Browne, 2001). These well-characterized pYV-associated virulence determinants are used by researchers to determine plasmid maintenance, for isolation/detection, and as an indication of virulence for various serotypes of pYV-bearing Y. enterocolitica in food (Koeppel et al., 1993; Juríková et al., 1995; Bhaduri and Cottrell, 1997; Bhaduri et al., 1997; Robins-Browne, 2001; Fredriksson-Ahomaa and Korkeala, 2003; Weagant et al., 2007), as well as to determine the presence of pYV in Y. pestis (Bhaduri and Sommers, 2008). The pYV is unstable in all three pathogens and the loss of the plasmid after cultivation or during food processing results in avirulent clones (not lethal to mice; does not cause plague) (Kwaga and Iversen, 1991; Straley, 1991; Bhaduri, 2001; Robins-Browne, 2001; Bhaduri and Sommers, 2008). We observed that laboratory manipulation such as repeated transfer of cultures, extended storage at 4°C, or at −20°C, as well as subculturing Y. pestis at temperatures >30°C lead to the loss of pYV (Tamplin and Bhaduri, unpublished data; Bhaduri and Sommers, 2008; Bhaduri, 2009). Moreover, pYV is more unstable in Y. pestis than in the other species of Yersinia (Bhaduri and Sommers, 2008; Bhaduri, 2009). The loss of pYV leads to the eventual overgrowth by cells lacking pYV and results in the loss of virulence and the concomitant disappearance of associated virulence characteristics (Bhaduri, 2001; Straley et al., 1993; Weagant et al., 2007; Bhaduri and Sommers, 2008). In our previous study on the growth of Y. pestis in ground beef, we found that the cultures lost pYV during preparation of the inoculum in brain heart infusion (BHI) broth (Bhaduri, 2009). We were not able to maintain pYV in cells from the stock cultures using the standard procedures developed previously in our laboratory (Bhaduri, 2001). Thus, the growth model of Y. pestis in this ground beef study does not reflect the actual behavior of pYV-bearing Y. pestis. In our initial studies in ground beef, we observed that the growth rates of pYV-less cells were 0.096 and 0.287 CFU/h at 10°C and 25°C, respectively (Tamplin and Bhaduri, unpublished data), whereas, for pYV-bearing cells, the growth rates were 0.057 and 0.233 CFU/h at 10°C and 25°C, respectively (Bhaduri, 2009). The difference in growth between pYV-positive and pYV-less strains of Y. pestis was more pronounced at lower temperatures. There was no growth of the pYV-bearing strain at 0 and 4°C (Bhaduri, 2009) as compared to the growth rates of pYV-less strains of 0.003 and 0.016 CFU/h for pYV-less cells at 0°C and 4°C, respectively, in ground beef (Bhaduri, 2009; Tamplin and Bhaduri, unpublished data). Therefore, the absence of pYV leads to a faster growth rate and does not represent the true growth rate of the pYV-bearing strain. Hence, it is critical to maintain pYV in Y. pestis to properly study the growth behavior of pYV-bearing Y. pestis in food to develop a realistic growth model of this pathogen. The unstable nature of pYV necessitates an examination of the presence of the pYV and its virulence characteristics during laboratory manipulation and investigations. In this regard, there is no method available to monitor the presence of pYV in Y. pestis cells so that pYV-bearing cells could be used for investigations. Hence, the present study was initiated to develop a procedure to monitor the presence of pYV in Y. pestis cells during storage and culturing by using the Lcr-CR binding techniques (Bhaduri and Sommers, 2008) followed by polymerase chain reaction (PCR) assays and determining the expression of pYV-associated virulence characteristics. Thus, this procedure will allow only the Lcr-CR-positive pYV-bearing clones to be used to develop growth models and related studies in food.

Materials and Methods

Bacteria

Y. pestis KIM5, a derivative of strain KIM (Kurdistan Iran man), which lacks the chromosomally encoded pigmentation (Pgm⁻) locus but harbors pYV (Bearden and Perry, 2008), was used in this study. This strain is conditionally virulent (a conditional mutant is infectious if inoculated intravenously) and it is the only strain available that can be used in a BL2 laboratory facility (Bearden and Perry, 2008). This strain is well characterized, and has been extensively used to study the microbiology and molecular pathogenesis of Y. pestis (Brubaker, 2006; Bearden and Perry, 2008; Bhaduri and Sommers, 2008).

Preparation of media

BHI broth was prepared as recommended by the supplier (Becton-Dickinson, Sparks, MD). Calcium-deficient CR magnesium oxalate agar (CR-MOX) was prepared by adding 20% D-galactose (Sigma Chemical Co., St. Louis MO), 0.25 M sodium oxalate (Sigma Chemical Co.), 0.25 M magnesium chloride (Sigma Chemical Co.), and 1% CR (Sigma Chemical Co.) to tryptic soy agar (Becton-Dickinson), as described previously (Bhaduri et al., 1991). Low-calcium (238 μM Ca²⁺) CR BHI agarose (CR-BHO) was prepared by adding agarose (GibcoBRL, Grand Island, NY) to a final concentration of 1.2% to BHI broth supplemented with 0.1% magnesium chloride (Sigma Chemical Co.), and CR (Sigma Chemical Co.) was added at a concentration of 75 μg/mL as described previously (Bhaduri et al., 1991).

Presence of pYV and virulence characteristics in Lcr and CR uptake positive clones

The phenotypic virulence characteristics of pYV, including Lcr, CR uptake, and CV binding, were determined as described previously (Bhaduri et al., 1991; Bhaduri and Sommers, 2008). In both CR-MOX and CR-BHO, pYV expressed Lcr and CR uptake facilitated differentiation of pYV-bearing cells from pYV-less cells for subsequent isolation of pYV-bearing cells (Bhaduri et al., 1991; Bhaduri and Sommers, 2008). These two media were compared to determine which medium is more suitable for monitoring the presence of pYV under culture conditions. The presence of pYV was also confirmed by a PCR assay targeting a key regulatory gene virF, from pYV, which encodes a transcriptional activator for expression of pYV-encoded outer membrane proteinYop51 (Nakajima et al., 1992; Bhaduri, 2003; Thoerner et al., 2003).

Results and Discussion

Since pYV of Y. pestis is easily lost during laboratory manipulations such as subculturing, the procedures outlined in Flow Chart 1 were developed to monitor the presence of pYV and differentiate pYV-positives clone from pYV-less colonies during laboratory investigations. After receiving the pYV-bearing strain of Y. pestis KIM5 from Dr. Susan Straley (Department of Microbiology and Immunology, University of Kentucky, Lexington, KY), the presence of pYV was immediately confirmed by demonstrating that virulence-associated phenotypes were present and by a PCR assay targeting the pYV-encoded virF (Fig. 1, lane 3) (Bhaduri, 2003; Bhaduri and Sommers, 2008). The strain was then stored at −80°C. Since pYV is readily lost during the first passage of subculturing and laboratory manipulation (Bhaduri and Sommers, 2008; Bhaduri, 2009; Tamplin and Bhaduri, unpublished data), a procedure was developed to ensure that the working culture retained pYV in the cells. At the initial stage, it is essential to culture first on CR-MOX and CR-BHO to isolate pYV-bearing clones from the frozen stock culture. The pYV-positive colonies appeared as red pinpoint colonies (0.36 mm in diameter) showing both Lcr and CR uptake, whereas pYV-less colonies appeared as much larger white or orange colonies (1.37 mm in diameter) (Bhaduri et al., 1991; Bhaduri and Sommers, 2008). The colony morphology and CR uptake can be used to differentiate between pYV-positive clones and pYV-less colonies. The Lcr- and CR-positive clones were further confirmed as pYV positive by the PCR assay (Fig. 1, lane 4 [CR-MOX] and lane 5 [CR-BHO]) and by pYV-associated Lcr, CR uptake, and CV binding phenotypes (see Flow Chart 1). These pYV-bearing clones were inoculated into BHI broth for the preparation of frozen and working stock cultures as described in Flow Chart 1. Before frozen storage and preparation of working stock cultures, the culture prepared in BHI broth at 28°C was tested for the presence of pYV and its virulence-associated phenotypes (Fig. 1, lane 6). The Lcr-CR positive clones on CR-MOX were used as working stock cultures and could be used for 15 days for laboratory studies by storage at −80°C. After that period of storage the red pin point colonies lost pYV (Fig. 1, lane 11). The CR-BHO was also successfully used to ensure the selection of pYV in Y. pestis although CR uptake was not as intense as on CR-MOX. The Lcr-CR positive clones were used as working stock cultures from CR-BHO and could be stored for 30 days at 2°C. The reason for the shorter storage stability of pYV in Y. pestis on CR-MOX may be the use of oxalate, which chelates calcium. The use of oxalate in this medium raises the possibility that other essential ions may be sequestered by oxalate, thereby affecting bacterial behavior (Bhaduri et al., 1991).

FIG. 1.

Confirmation of presence of pYV in the original strain, and CR-positive clones from CR-MOX, CR-BHO, and BHI broth using polymerase chain reaction assay targeting a key regulatory gene virF from pYV. The primer pairs (5'-TCATGGCAGAACAGCAGTCAG-3' and 5'-ACTCATCTTACCATTAAGAAG-3') for detection of the virF gene (430- to 1020-nucleotide region) amplified a 591-bp product from the pYV. The low calcium response-CR⁺ clones showed the presence of 591-bp products from pYV (lanes 2–10 and 12). Lane 1, 50–1000 bp ladder marker; lane 2, negative control with no template, lane 3, original KIM5 strain as positive control; lanes 4 and 5, low calcium response-CR⁺ colonies from the CR-MOX and CR-BHO, respectively (Flow Chart 2; #1); lane 6, BHI broth (Flow Chart 2; 1st passage; #2); lane 7, stock culture on CR-MOX (#3; 1st passage); lane 8, stock culture on CR-BHO (#3; 1st passage); lane 9, BHI broth (#4, 2nd passage from CR-MOX); lane 10, BHI broth (#4, 2nd passage from CR-BHO); lane 11 (#5, 2^nd passage on CR-MOX) showing the absence of 591-bp product, and lane 12 (#5, 2^nd passage on CR-BHO). pYV, virulence plasmid; CR, Congo red; BHI, brain heart infusion; BHO, BHI agarose; MOX, magnesium oxalate agar.

FlOW CHART 1.

Isolation and maintenance of virulence plasmid in Yersinia pestis.

To ensure the validity of this procedure for selecting pYV in Y. pestis cells, we also examined and monitored pYV stability during the subculturing of pYV-bearing cells in BHI broth, CR-MOX, and CR-BHO. Y. pestis from stock cultures stored at 2°C on CR-MOX and CR-BHO and were subcultured as explained in Flow Chart 2. The presence of pYV in Y. pestis cells in each medium and after each passage was monitored and confirmed at every step of culture transfer (#2–5) by the PCR assay for pYV and by expression of pYV-associated phenotypic virulence characteristics, including Lcr, CR uptake, and CV binding. The PCR data for the presence of pYV are shown in Fig. 1. PCR results confirm that primers amplified a 591-bp product from the pYV (virF gene) from each phase of the culture as described above and all PCR-positive clones on CR-MOX and CR-BHO showed their virulent phenotypic characteristics, including Lcr, CR uptake, and CV binding. The presence of virF gene demonstrates the presence of pYV, which confers pYV-associated phenotypes.

FlOW CHART 2.

Confirmation of virulence plasmid in Yersinia pestis.

Conclusions

The procedure developed here provides a method to ensure the selection of pYV in Y. pestis and for studying pYV-bearing Y. pestis without losing pYV during experimental procedures by storing at −80°C for 30 days. Although CR-BHO is a better medium for subculturing of pYV-bearing Y. pestis, the pYV-bearing red pin point colonies are more easily detectable on CR-MOX due to more intense absorption of CR in the cells (Bhaduri and Sommers, 2008). Hence, the use CR-MOX for the preparation of stock cultures and to monitor the selection of pYV is recommended for investigations on the growth of pYV-bearing Y. pestis in food. At present, the development of a growth model for pYV-bearing Y. pestis in ground beef and pork is in progress using the pYV-bearing clones isolated by this procedure.

Disclaimer

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Footnotes

Disclosure Statement

No competing financial interests exist.

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