CHAPTER 3

Molecular detection and analysis of Y. pestis Abstract The polymerase chain reaction (PCR) has been used to detect Y. pestis in infected fleas, soil samples, and clinical samples for rapid diagnosis of plague. PCR-gradient density electrophoresis (PCR-GDE) has allowed the distinction between the three pathogenic Yersinia species utilizing the rpoB gene. The application of the PCR to dental samples has allowed the confirmation of Y. pestis as the cause of death in archaeological samples dating to 600 AD. In addition, real-time PCR (Rti-PCR) methodology has also been developed for detection of Y. pestis. Target sequences have included unique chromosomal sequences specific for Y. pestis in addition to genes specific for Y. pestis including pla, caf1, Pst. The use of variablenumber tandem repeats (VNTRs) has allowed the mutation rate of Y. pestis to be determined in addition to distinguishing different strains of Y. pestis and the three major biovars. PCR primers have also been developed for confirming the presence of each of the three major virulence plasmids in Y. pestis.

Conventional PCR Campbell et al. (1993) developed a 4-hr. nested PCR assay for detection of Y. pestis. The outer primers YP1/YP2 (Table 1) amplified a 928-bp sequence of the plasminogen activator pla gene of Y. pestis harbored on the 9.7-kb plasmid. The inner nested primers YP1a/YP2a (Table 1) amplified a 458-bp sequence. Among 43 strains of Y. pestis from humans, rats, and fleas 39 (91%) yielded the predicted 928-bp amplicon with the outer primers YP1 and YP2. The nested PCR yielded the predicted 458-bp amplicon from all 43 isolates of Y. pestis. No amplicons were obtained from Y. enteritidis and Y. pseudotuberculosis. Hinnebusch and Schwan (1993) developed a PCR assay to directly detect Y. pestis in infected fleas. Infection of fleas was accomplished by first inoculating one-week old mice intraperitoneally with 103 cells of a virulent strain of Y. pestis. Sick mice were placed in a glass container containing fleas (Xenopsylla cheopsis) derived from a laboratory colony for 48 hrs. Negative controls consisted of fleas allowed to feed on uninfected mice.

158

Robert E. Levin

Individual fleas were macerated in 250 ml of BHI broth which was centrifuge to pellet tissue debri and the supernatant use for the PCR. The PCR primers Yp1/Yp2 (Table1) amplified a 480-bp sequence of the Y. pestis plasminogen activator gene (pla). The sensitivity of detection was the DNA from 10 cells per PCR assay. Norkina et al. (1994) reported on the development of a PCR assay specific for Y. pestis. One pair of primers F1/F2 (Table 1) amplified a 501-bp sequence of the caf1 gene encoding the capsular I antigen harbored on the pFra plasmid. A second pair of primers P1/P2 (Table 1) amplified a 443-bp sequence of the pla gene encoding the plasminogen activator harbored on the pPst plasmid. The limit of sensitivity in pCR reaction mixtures with crude cell lysates was the DNA from 10 – 50 FU following phenol-chloroform extraction. When target cells were mixed with fresh blood of mice, the PCR detection level varied from 400-100 CFU/ml of blood following phenolchloroform extraction. Table 1. PCR primers and DNA probes.

Molecular detection and analysis of Y. pestis

159

The "gold standard" for identifying Y. pestis infected fleas has been the inoculation of mice with pooled flea material. Inoculated mice are then monitored for 21 days and those that die are further analyzed for Y. pestis infection by fluorescent-antibody assay and/or culture. Engalthaler et al. (1999) compared the standard bacteriological assay with the PCR for detection of Y. pestis. A total of 381 field-collected fleas were assayed individually by both PCR and mouse inoculation. The fleas were collected at various locations in Colorado and New Mexico from rodent burrows (270) domestic animals (5), captured animals (70), and animal carcasses (36). The primers Yp1/YP2 (Table 1) amplified a 479-bp sequence of the plasminogen activator gene (pla). Sixty of the 381 flea samples caused death in mice. None of the 321 PCR-negative flea samples caused death. Among the 12 mice that survived inoculation with PCR-positive samples, 10 were later found by serology or culture to have been infected with Y. pestis. This suggests that death of inoculated mice is less reliable than PCR as an indicator of the presence of Y. pestis in flea samples. Among the 270 fleas from rodent burrows, 24 were positive for Y. pestis, 0/5 were positive from domestic animals, 0/70 from captured animals, and 34/36 from animal carcasses. Mouse inoculation assays were found to be comparable to PCR only when surviving as well as dead mice or analyzed for infection. The sensitivity of the assay was found to be the DNA from 10 to 100 CFU of Y. pestis per PCR. The diagnostic value of a PCR assay for confirmation of the diagnosis of plague from bubo aspirates collected from 218 suspected plague patients in a regional hospital of Madagascar was determined by Rahalison et al. (2000). The cultivation of Y. pestis from the bubo samples and the detection of the F1 antigen (Ag) by an ELISA assay were used as reference diagnostic methods. The primers F1/F2 (Table 1) from Norkina et al. (1994) amplified a 501-bp sequence of the caf1 gene harbored on the pFra/pMT1 (96.2-bp) plasmid that encodes the capsular F1 antigen of Y. pestis. Among the 218 patients with suspected plague, 154 were culture positive, 78 were positive with the F1 Ag ELISA and 64 were PCR positive. This translates to the fact that among the total of 218-105 = 113 confirmed positive patients, only 64/113 (57%) were PCR positive. The authors speculated that this relatively low level of detection may have been due to the fact that only 20 μl of bubo sample is diluted with 20 μl of lysing solution and that after centrifugal clarification only 2.5 ml of the sample is used for the PCR. Y. pseudotuberculosis and Y. enterocolitica are divided into serotypes based on differences in their LPS O-side chain (O-antigen) antigenic determinants. Y. pestis has no serotypes because natural mutations in the Oantigen gene cluster prevent the synthesis of O-antigens (Skurnic et al., 2000;

160

Robert E. Levin

Prior et al., 2001). Bogdanovich et al. (2003) utilized the O-antigen gene cluster sequences in Y. pestis and Y. pseudotuberculosis to identify primers Ypf-8608/Ypr-10219 that yielded a 1,612-bp amplicon derived from the wzxwbyJ gene loci (Table 1). All 16 Y. pestis strains were PCR positive regardless of the biovar with these primers, while all 85 Y. pseudotuberculosis strains were negative. A total of 259 bacterial strains were used to asses the specificity of the PCR assay which was 100% for Y. pestis. Rodnedge et al. (2001) identified a unique 41.7-kb chromosomal region specific to Yersinia pestis. The primers 3aF/3aR amplified a 276-pb sequence from Y. pestis in addition to all 8 Y. pestoides (rhamnose positive Y. pestis strains, see chapter 1) strains tested. This 41.7 region was identified on the basis of its absence from Y. pseudotuberculosis and Y. enterocoliticus and yielded negative results with a number of additional genera and species. Cocolin and Comic (2005) developed a molecular detection system for determining the presence of Yersinia in foods. The assay is based on the amplification of a 359-bp PCR product from the RNA polymerase betasubunit gene (rpoB) and subsequent analysis of the resulting amplicons by density gradient gel electrophoresis (DGGE). The primers rpoB1698f/rposB20114r were used in conjunction with a GC clamp added to primer rpoB1698f to improve the sensitivity of detection of mutations by DGGE. The DGGE banding profiles were determined to be species specific. Y. enterocolitica, Y. intermedia, Y. frederiskenii, and Y. kristensenii yielded different band migrations in the gels. The assay was applied to only these 4 of the 11 recognized species of Yersinia. In addition, Y, enterocolitica serotypes O:3, O:5, and O:9 (the only ones examined) were distinguishable. When the PCR-DGGE protocol was applied to 27 raw vegetable samples, a good correlation was obtained when the results of traditional culture methods and the PCR-DGGE methodology were compared. Interestingly, no PCR product could be obtained following enrichment cultivation of the raw vegetables. The PCR-DGGE methodology was applied to colonies obtained following initial enrichment overnight at 20 oC in YPCE medium followed by streaking onto CIN agar and incubating for 8 24 hrs. at 37 oC. Among th 27 raw vegetable samples, 3 were confirmed by PCR-DGGE and sequencing of the resulting amplicons to be Y. enterica. The PCR limit of detection was 102 – 103 CFU. PCR-DGGE bands were also produced by various enterobacteria, with band locations differing from Yersinia species controls. Among the 27 raw vegetable samples, 21 were positive for Yersinia spp. on ICN agar and only 14 were confirmed by PCRDGGE and sequencing of amplicons resulting from CIN colonies. The most frequent Yersinia species from the raw vegetables was Y. frederiksenii which

Molecular detection and analysis of Y. pestis

161

is nonpathogenic. Neither Y. pseudotuberculosis nor Y. pestis were included in the this study.

Real-time (RTi-PCR) Higgins et al. (1998) developed a Rti-PCR assay for detection and quantification of Y. pestis in infected fleas, orophangyeal swabs from aerosol infected monkeys, and from the blood of infected monkeys. The primers Yp pla IS1/Yp pla IS2 (Table 1) amplified a 344-bp sequence of the Y. pestis pla gene. A dual labeled probe MSI002 (Table 1) was labeled a the 5'-end with 6-carboxyfluorescein (FAM) as the fluorescent reporter and at the 3'-end with the quenching dye 6-carboxytetramethylrodamine (TAMRA). The level of detection sensitivity was 2.1 x 105 copies of the pla target gene or 1.6 pg of total cellular DNA. Iqbal et al. (2000) reported on the development of a PCR assay specific for amplification of a DNA sequence of the pesticin (Pst) gene from Y. pestis encoded on the pPCP1 plasmid. The assay utilized primers Forward/Reverse (Table 1) and a probe labeled at the 5’-end with FAM and at the 3’-end with TMRA. The intensity of final fluorescence after 40 thermal cycles was determined with a fluorometer. The level of detection sensitivity was the DNA from three cells. The amplicon size was not stated. Loïez et al. (2003) reported on the development of a Rti-PCR assay for detection of Y. pestis in sputum. Sputum specimens from 20 non plague individuals were inoculated with 10o to 104 CFU/ml. The primers pla-F/pla-R (Table 1) amplified a 69-bp sequence of the Y. pestis plasminogen activator (pla) gene. A minor groove binding probe labeled at the 5'-end with 6carboxyflourescein (FAM) (Table 1) was utilized as the fluorogenic reporter molecule. The assay was found to be 100% specific for Y. pestis. In the absence of inhibitors, sensitivity of detection of 102 CFU/ml of sputum was obtained. When inhibitors were present, detection of Y. pestis DNA required at least 104 CFU/ml. Woron et al. (2006) developed a multiplex, 4-target Rti-PCR assay for Y. pestis. The primers plaf/plar amplified a 270-bp sequence of the pla gene encoding the plasminogen activator and harbored on the pPCP1 plasmid (Table 1). The primers caf1f/caf1r amplified a 176-bp sequence of the caf1 gene, harbored on the pFra plasmid, encoding the F1 capsular antigen (Table 1). The primers CD1f/CD1r amplified a 245-bp sequence of the pCD1 plasmid (Table 1). The primers entF3f/entF3r amplified a 122-bp sequence of the chromosomal entF3 gene (Table 1). This multiplex assay therefore targeted each of the three plasmids of Y. pestis strains in addition to a chromosomal target sequence. Dual labeled probes with utilized black hole

162

Robert E. Levin

quenchers were used to label the probes at the 3'-end and different fluorophores were used to label the 5'- probe ends (Table 1). The sensitivity of detection was less than 85 CFU per RTi-PCR reaction with 100% specificity for Y. pestis. Reihm et al. (2011) evaluated three Rti-PCR assays in two different assay formats, a 5'-nuclease dual labeled probe assays and single fluorophore labeled hybridization probe assays for Y. pestis. Lymph node aspirates from 149 patients yielding 149 samples from Madagascar clinically diagnosed with bubonic plague were used. The primers YPpla S/YPpla R amplified a 232-bp sequence of the pla gene harbored on the pPCP1 plasmid (Table 1). Primers YPcaf S /YPcaf R amplified a 240-bp sequence of he caf1 gene on plasmid pMT1 (Table 1). Primers YPtox U/YPtox R amplified a 168-bp sequence of the ymt gene on plasmid pMT1 (Table 1). Primers Lambda F/Lambda R amplified a 278-bp sequence of the bacteriophage lambda DNA as an internal amplification control. Plasmids are subject to spontaneous loss or alteration of their regulatory operons. It is therefore essential to employ PT-PCR assays for Y. pestis that target genes on more than one plasmid and the bacterial chromosome. Tomaso et al. (2003) developed a real-time PCR multiplex system for specific detection of Y. pestis. A total of six PCR primer pairs were used with accompanying labeled probes: (1) the 16S chromosomal rRNA gene, (2) the yopT gene encoding a cysteine protease residing on the pCD1 plasmid, (3) the caf1 gene encoding the F1 capsular antigen, (4) the plasminogen activator gene pla residing on the pCP1 plasmid, (5) the murine toxin gene ymt also residing on the pCP1 plasmid, and (6) and sequence of the lambda phage genomic DNA as an internal control. Primers and probes for the last three genes and for lambda phage DNA are given in Table 1. Rti-PCR assays for 16S rRNA resulted in a 161-bp amplicon from 25 Y. pestis strains and also from a variety of other Yersinia species but from none of the other 33 bacterial species examined. A 330-bp amplicon derived from the yopT gene located on the pCD1 plasmid was detected with 17 Y. pestis and 22 Y. pseudotuberculosis strains possessing the pCD1 plasmid but did not yield amplicons from any of the other bacterial species. Amplicons were observed for caf1, ymt, and pla from all Y. pestis strains except for plasmid deficient strains. No amplicons were obtained with the primers targeting these three genes from the DNA of any other bacteria species examined. The Rti-PCR multiplex assays with primers for lambda DNA as an internal amplification control did not influence the amplification of Y. pestis targeted sequences. Rti-PCR multiplex assays included 150 pg of lambda DNA selected to yield a Ct value of 30. Multiplex combinations of more than two Y. pestis-specific primer pairs failed to yield amplicons for

Molecular detection and analysis of Y. pestis

163

caf1 and ymt and no fluorescence above the background was observed, presumably due to competitive PCRs. It is possible that by increasing the amount of DNA polymerase and nucleotide tri-phosphates that the adverse effects of competitive PCR could be minimized or eliminated.

DNA characterization of Y. pestis isolates Among 16 restriction nucleases tested, Lucier nd Brubaker (1992) found that only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Y. pestis of sufficient length to allow pulsed-field gel electrophoresis (PFGE). Single dimension PFGE after treatment with SpeI yielded 41 bands, three of which were shown by two-dimensional PFGE using SpeI-ApaI to have co-migrated. From these bands, a refined estimate of 4,397.9 + 134.6 kb was determined for the size of the genomic DNA. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. Girard et al. (2004) determined the rate of mutagenesis of a Y. pestis strain that represented ~21,000 generations of invitro growth. This was accomplished by serially transferring each of 96 clonal linages, derived from a single colony for 10 passages followed by determining genetic diversity among the 96 terminal colonies by using 43 VNTR loci (MLVA). An overall mutation rate of 1.3 x 10-3 mutations/generation for the 43 VNTR markers was obtained. It was estimated that 1,000 generations are required to detect one mutation in a strain of Y. pestis. Strains of Y. pestis contain multiple copies of three insertion sequences (IS100, IS285, and IS1541) scattered over the genome. The genomic instability generated by these insertion sequences (IS) creates polymorphism among the hybridizing restriction fragments Restriction Fragment Length Polymorphism (RFLP) which can be used to subtype this relatively clonal species. Analysis of 61 Y. pestis isolates of worldwide origin indicated that no satisfactory strain clustering was observed with each of these three ISRFLP used individually In contrast, the combination of the three IS-RFLP data (3IS-RFLP) resulted in both an efficient strain discrimination and a robust clustering of the isolates according to their biovar and geographic origin. This geographic clustering was observed even with the Orientalis group although these strains have had only a short period of time (one century) to diverge from the original clone that spread globally from Hong Cong in 1894 during the third pandemic. The best resolution of DNA bands following PCR amplification of the resulting restricted sequences was obtained when the genomic DNAs were first digested with EcoR1 for IS100RFLP and HindII for IS285- and IS1541-RFLP.

164

Robert E. Levin

In 1994, plague reappeared in India in the Beed district of the Maharasta state and Surat City in Guzarat state resulting in the flight of nearly 5,000,000 Surat residents, and the death of 53 individuals. The plague outbreaks of 1994 were associated with unusually heavy flea nuisance, rat deaths and patients with bubonic and pneumonic plague symptoms. Geographical factors, such as earthquakes and unprecedented floods that occurred in these locals are believed to have played a major role in the movement of the disease from wild rodent epizootics to initiate the human plague outbreaks. Kingston et al. (2009) subjected a total of 18 Y. pestis strains from the 1994 plague outbreaks in Maharastra and Gojarat (11 from pneumonic patients and 7 from rodents), 8 Y. pestis strains recovered from liver and spleen of local rodents and 3 Y. pestis strains from pneumonic patients from 2002 plague outbreaks for a total of 29 Y. pestis isolates. Typing methods used were: ribotyping, multiple locus VNTR analysis (MCVA), IS element RFLP analysis, DFR analysis, ERIC-PCR, ERIC-BOX-PCR, and SNP analysis. MLVA genotyping revealed strains of 2 Orientalis, 1 Medievalis, and 1 Antiqua genotypes. The Orientalis Y. pestis strains recovered from rodents and patients from the 1994 plague outbreaks exhibited similar MLVA, ERICPCR, and ERIC-BOX-PCR profiles and these were closely related to the Orientalis strains recovered from rodents trapped on the Deccan plateau. These results indicated Orientalis, Antiqua, and Medievalis biovars to be well established in the Indian subcontinent. ERIC-PCR analysis delineated genotypes similar to those defined by MLVA. Adair et al. (2000) sequenced the entire V antigen gene (lcrV) from 22 strains of Y. pestis. Results indicated that the lcrV gene exhibited very little nucleotide variation among these strains. With the exception of two strains, all sequences were identical. In contrast to the lack of lcrv gene diversity, a tetranucleotide repeat sequence (CAAA)N in the genome of Y. pestis was identified. This variable-number tandem repeat (VNTR) region had nine alleles varying from 2 to 13 tandem repeats which exhibited great diversity among 35 diverse isolates of Y. pestis. The insertion sequence 100 (IS100) is found at least singly on each of the three plasmids of Y. pestis and as multiple copies within the chromosome (Portnoy and Falkow, 1981). Motin et al. (2002) developed a PCR-based genotyping system for detecting variation of the insertion locations of IS100 within the Y. pestis genome. This system was then used to characterize a collection of Y. pestis isolates of different biovars from different geographic global regions. Using sequences from the typically glycerol-negative biovar Orientalis strain CO92, a set of 27-locus-specific primers was designed to amplify fragments of IS100 and its neighboring genes. Geographically diverse members of the Orientalis biovar formed a homogeneous group with identical

Molecular detection and analysis of Y. pestis

165

genotype with the exception of strains isolate from Indochina. In contrast strains belonging to the glycerol-positive biovar Antiqua yielded a variety of genotype banding profiles. Moreover, strains of the biovar Medialis (also glycerol-positive) clustered together with the Antiqua isolates originated from Southern Asia, suggesting close Phylogenetic relationships. Interestingly, a Manchurian biovar Antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar Orientalis isolates. Analysis of the glycerol degradation pathway suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the Orientalis phenotype. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD homologous gene found only in biovar Orientalis strains. The authors postulated that the Nicholisk 51 strain represents a variant of the Orientalis biovar with restored ability to ferment glycerol, suggesting genetic exchange between different Yersinia strains in nature. Lowell et al. (2005) documented the valuable utility of variable-number tandem repeats (VNTRs) as markers for epidemiological investigations of naturally occurring plague and the forensic analysis of potential bioterrorism events. A total of 13 paired sets of Y. pestis isolates were collected during epidemic investigations conducted in New Mexico, Arizona, and Colorado from the 1980's to 2002. Nine sets of isolates were collected during plague case investigations in which isolates were obtained from both human patients and associated environmental samples from mammalian hosts and fleas. Four sets were from fleas or mammalian hosts collected at the same time at different paired locations. A subset of 17 VNTR markers was selected from the 43 VNTR markers previously described for Y. pestis (Adair et al., 2000; Girard et al., 2004; Keim et al., 2000). From the data of Girard et al. (2004) it was calculated that with 17 VNTR markers, that 2,000 generations of growth are required to detect two mutations and that this number of transmission cycles would probably occur during an epizootic period or during one or two seasons of ongoing enzootic transmission in a plague focus area. The VNTR analysis of the human and environmental Y. pestis isolates clearly demonstrated the value of this technique for the identification of likely sources of infection and sites of exposure for human plaque cases. The authors indicated that coupled with case histories and other epidemiological information, MLVA should also be useful for differentiating naturally occurring cases of plague from those occurring from an intentional Y. pestis release.

Molecular detection of Y. pestis McDonough et al., (1998) developed a colony blot DNA hybridization assay for detection of Y. pestis. The DNA probe consisted of a 900-bp

166

Robert E. Levin

restricted sequence of the pPCP1 plasmid present only in Y. pestis strains was labeled with 32P-dCTP by nick translation. The 900-bp sequence was derived from restriction of the pPCP1 plasmid with BamHI and HindII which cut at single sites. The resulting fragments were resolved by agarose gel electrophoresis, with the specific 900-bp fragment eluted and then labeled. The labeled probe was found to be highly specific for strains of Y. pestis carrying the pPCP1 plasmid. Stewart et al. (208) described the development of a quadruplex real timePCR (Rti-PCR) assay using dual labeled probes that exclusively identified Y. pestis using a unique target sequence of the yihN chromosomal gene of 128-bp . In addition, portions of gene sequences carried on each of the three Y. pestis plasmids were also amplified (Table 1). DNA sequences of 194-, 87-, and 144-bp were amplified from the caf1 gene carried on the pFra plasmid, from the lcrV gene carried on the PCD1 plasmid, and the pla gene carried on the pPCP1 plasmid respectively. The quadruplex Rti-PCR assay was validated as 100% specific using a collection of 192 Y. pestis isolates and 52 near-neighbor isolates. The study found that only 72% of natural plague isolates from the states of New Mexico and Utah harbored all three virulence plasmids.

Detection of Y. Pestis in archaeological skeletons The polymerase chain reaction (PCR) and immunological dipstick assays The durability of dental pulp, together with its natural sterility led Drancourt et al. (1998) to hypothesize that it would be a lasting refuge for Y. pestis as a result of lethal systemic infection in plague stricken individuals. DNA extracts were obtained from the dental pulp of 12 teeth extracted from skeletons excavated from 16 and 18 century French graves of persons thought to have died of plague. The primers rpoB-F/rpoB-R (Table 3) amplified a 133bp sequence of the rpoB gene that encodes the RNA polymerase B-subunit of Y. pestis. Primers pla-F/pla-R amplified a 300-bp sequence of the pla gene that encodes the plasminogen activator factor. A single 40 cycle PCR with the rpoB primers failed to yield an amplified product. However, after the product of the first PCR were incorporated into a second similar 40 cycle PCR, four of four samples from ancient presumptively plague teeth were PCR positive. Sequence analysis of the amplicons confirmed the Y. pestis origin of the targeted DNA sequence. Two of four teeth from one burial site and four of eight teeth collected from a geographically distant burial site yielded positive PCR products with the pla primers after a single 40 cycle PCR. Sequence analysis again confirmed the origin of the DNA target sequence to be Y. pestis.

Molecular detection and analysis of Y. pestis

167

Raoult et al. (2000) reported on the extraction of DNA from the pulp of teeth derived from a 14 century grave in France that contained a child and two adults suspected of dying from plague. "Suicide" PCR was applied to the extracts, in which the primers are used only once. When an amplicon of the expected size is generated, it is then sequenced to confirm its identity. A first set of primers INV.D/INV.R (Table 2) targeted an IS200-like element within the env gene of Y. Pestis. A second set of primers YP11D/YP1OR (Table 3) amplified a 148-bp sequence of the pla gene of Y. pestis. A third set of primers YP12D/YP11D (Table 3) amplified a 149-bp sequence of the pla gene of Y. pestis and were used for "suicide PCR". The first set of primers failed to yield amplicons from any DNA extracts from the ancient dental pulp samples. However, amplicons of the expected 148-bp size with the pla primers were obtained from 1/4 child teeth. With the "suicide primers", 19/19 adult teeth yielded the expected 149-bp-amplicon. The sequence of the 148-bp amplicon from the child's tooth shared 100% identity with that of the modern Y. pestis pla sequence in GenBank. A single point mutation (T to C) was present in the amplicons from the adult teeth. The authors emphasized that positive controls should never be used during the molecular detection of an ancient pathogen so as to prevent laboratory contamination of the PCR. The "suicide PCR" was developed to further ensure the absence of amplicon contamination in the laboratory, by using PCR primers designed to hybridize to targets outside genomic regions previously amplified in the laboratory. Since the "suicide primers" are used only once, no previous contamination with expected amplicons can occur. Pusch et al. (2004) compared an immunological dipstick assay for detection of the Y. pestis capsular F1 antigen and a PCR assay for detection of a 93-bp sequence of the caf1 gene that encodes the Y. pestis F1 antigen from archaeological samples. The samples were derived from 12 individuals who succumbed from the black death in the 17th century and were buried with lime on the grounds of a German church. The remains of 12 individuals from the same church grounds from the same time period who were buried without lime were used as nonplague controls. The dipstick assay was found to be superior to the PCR assay. Whereas only 2 of 12 samples from the lime remains were PCR positive, 10 were positive with the dipstick assay. All 12 control samples were negative with both assays. Wiechmann and Grupe (2005) reported on a double inhumation of two female skeletons dating to the last half of the 6th century recovered from a grave site in upper Bavaria and suspected of having expired from plague. Both were female, an adult and juvenile (presumably a mother and child). Extracts from ground teeth were subjected to the PCR using primers YP12D/YP11R (Table 3) from Raoult et al. (2000) to amplify a 149-bp

168

Robert E. Levin

sequence from the pla gene. A second pair of primers YP11D/YP10R (Table 3) from Raoult et al. (2000) was used to amplify a 148-bp sequence of the pla gene in a "suicide PCR". Amplicons of the expected 148-bp were obtained from the tooth extracts of 1/2 teeth from the adult skeleton and 1/4 teeth from the juvenile. Sequencing of the resulting amplicons indicated that they shared 100% identity with that of a modern Y. pestis pla gene in GeneBank with the exception of one amplification product having a single G/T base substitution. The suicide primers yielded the expected 148-bp amplicon from one of the teeth of the juvenile skeleton. The skeletons of six individuals were exhumed from two burial sites in central France dating from the 16th – 18th centuries. The last great plague outbreak in his area of France was from 1629 – 1631. Thick layers of lime had been added to the coffins. During the second plague pandemic, lime was known to be heavily applied to the bodies of buried plague victims. Based on the supposition that the skeletal remains were from plague victims, Beanucci et al. (2009) applied a rapid dipstick assay for detection of the F1 envelope glycoprotein (F1 antigen) of P. pestis from extracts of dental pulp and found all six skeletons positive for Y. pestis. Kacki et al. (2011) reported on the exhumation of nine skeletons in three common graves dated to the 14th century in a province of southern France suggestive of plague. Seven of the nine skeletons tested positive for the presence of the F1 antigen of P. pestis using a dipstick assay applied to bone extracts. Although the F1 antigen-based dipstick assay has met with significant success when applied to archaeological samples, the F1-antigen has certain limitations in immunological assays. The F1-antign is indeed unique to Y. pestis, and its expression is upregulated at 37 oC during the mammalian phase of its infection cycle. The genes encoding the F1-antigen are found on the pMT1 plasmid and therefore immunoassays based on the detection of the F1-antigen from Y. pestis derived from environmental samples and in flea vectors would be limited because the level of expression of the F1-antigen is notably reduced when Y. pestis grows at temperatures below 37 oC. In addition, certain virulent strains fail to produce the F1antigen. The F1 antigen is therefore not an ideal target for immunological detection of Y. pestis which has led to alternative antigens such as the LPS antigen being utilized since the genes encoding its synthesis are chromosomal Prior and Titball, (2002). Haensch et al. (2010) identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central, and southern Europe that were associated archaeologically with the second pandemic (AD 347 – AD 1353) known as the “Black Death” and subsequent resurgencies. DNA analysis was performed with dental pulp or bone samples

Molecular detection and analysis of Y. pestis

169

from 76 human skeletons excavated from putative plague pits England, France, Germany, Italy, and the Netherlands. These plague pits were dated to the 14th and 17th centuries. PCR amplification was used to detect the presence of the Y. pestis-specific pla gene that is located on the multicopy pPCP1 plasmid. Amplicons of appropriate size were repeatedly obtained from ten individuals from France, England and the Netherlands. These results were taken as confirmation that Y. pestis caused the Black death and later epidemics on the entire European continent and over the course of four centuries. In addition, a Y. pestis dipstick immuno-assay specific for the F1 antigen confirmed the presence of the F1 antigen from individuals at the three sites in addition to those from Italy and Germany that were PCR negative. On the basis of 16 - 17 single nucleotide polymorphisms plus the absence of a deletion in the glpD gene encoding glycerol phosphate dehydrogenase, DNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves that are most probably extinct. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are considered ancestral to modern isolates of the Y. pestis biovars Orientalis and Medievalis. Genotyping by single nucleotide polymorphisms (SNPs) revealed that the strains of Y. pestis causing these mass deaths were unrelated to either Medievalis or Orientalis biovars. Among 7 PCR positive individuals from the Netherlands, France, and England, none of the glpD sequences contained the characteristic 93-bp deletion associated with biovar Orientalis, in contrast to prior results (Drancourt et al., 2007). Similarly, the stop codon characteristic of Medievalis strains was absent from the Netherlands and English samples. Therefore, the Y. pestis strains infecting these individuals were concluded as being neither Orientalis nor Medievalis. Assay for 16 single nucleotide polymorphisms indicated that the genotypes of Y. pestis from the archaeological samples from the 14th century were found to be distinct from the Orientalis and Medievalis biovars, and also differed from the modern Antigua biovar. All of these samples were thus distinct from modern Y. pestis from Africa, America, and the Near East, as well as the Pestoides isolates from the former Soviet Union. These results also indicate that at least two related but distinct genotypes of Y. pestis were responsible for the “Black death” and suggest that distinct bacterial populations spread throughout Europe in the 14th century that may now be extinct.

Molecular detection of ciprofloxacin resistance Ciprofloxacin is a fluorinated quinolone antibiotic that blocks DNA replication through inhibition of DNA gyrase activity. Lindler et al. (2001)

170

Robert E. Levin

developed a flourescence resonance energy transfer (FRET)-based PCR assay to detect ciprofloxacin resistant mutants of Y. pestis. Spontaneously resistant mutants of the Y. pestis KIM5 strain were used. Sequencing of gyrA encoded by 65 such mutants revealed that all such isolates contained one of four different point mutations within the quinolone-resistance determining region of gyrA. The FRET-based PCR assay utilized primers LC3/LC4 in conjunction with probe 1/probe 2 (Table 1) to amplify a 261-bp region of gyrA that included all of the detected point mutations with primer sequences complementary to the wild-type Y. pestis. Probe 1 was labeled at the 3'-end with floresceine and probe 2 was labeled at the 5'-end with Red 640. Since other organisms are know to express ciprofloxacin resistance via mutations in gyrB and gyrC, both of these loci were sequenced and compared to that of gyrA for assessment of amplicon specificity. DNA melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates, and resulted in a 4 to 11 oC shift in probe melting temperature.

References 1.

2.

3.

4.

5. 6.

7.

8.

Adair, D, Worsham, P., Hill, K., Klevytska, A., Jackson, P., Friedlander, A., Keim, P. 2000. Diversity in a variable-number tandem repeat from Yersinia pestis. J. Clin. Microbiol. 38:1516-1519. Ayyadurai, S., Houhamdi, L., Lepidi, H., Nappez, C., Raoult, D., Drancourt, M. 2008. Long-term persistence of virulent Yersinia pestis in soil. Microbiol. 154:2865-2871. Bogdanovich, T., Carniel, E., Fukushima, H., Skurnik, M. 2003. Use of Oantigen gene cluster-specific PCRs for th identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis. J. Clin. Microbiol. 41: 5103-5112. Campbell, J., Lowe, J., Walz, S., Ezzell, J. 1993. Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure. J. Clin. Microbiol. 31:748-759. Cocolin, L.,Comi, G. 2005. Use of a culture-endependant molecular method to study the ecology of Yersinia spp. in food. Int. J. Food Microbiol. 105:71-82. Drancourt, M., Aboudharam, G., Signili,M., Dutour, O., Raoult, D. 1998. Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia. Proc. Natl. acad. Sci. USA. 95:12637-12640. Engelthaler, D., gage, K., Montenieri, J., Chu, M., Carter, L. 1999. PCR detection of Yersinia pestis in fleas: comparison with mouse inoculation. J. Clin. Microbiol. 37:1980-1984. Girard, J., Wagner, D., Vogler, A., Keys, C., Allender, C., Drickamer, L., Keim, P. 2004. Differential plague-transmission dynamics detemine Yersinia pestis

Molecular detection and analysis of Y. pestis

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

22.

171

poulation genetic structure on local, regional, and global scales. Proc. Natl. Acad. Sci. USA. 101:8408-8413. Higgins, J., Ezzel, J., Hinnebusch, B., Shipley, M., Henchal, E., Ibrahim, M. 1998. 5'nuclease PCR assay to detect Yersinia pestis. J. Clin. Microbol. 36:2284-2288. Hinnebusch, J., Schwan, T. 1993. New method for plague surveillance using polymerase chain reaction to detect Yersinia pestis in fleas. J. Clin. Microbiol. 31:1511-1514. Iqbal, S., Chambers, J., Good, M., Valdes, J., Brubaker, R. 2000. Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR. Molec. Cell. Probes. 13:109-114. Keim, Pk., Price, L., Klevytska, A., Smith, K., Scupp, J., Okinaka, R., Jackson, P., HughJones, M. 2000. Multiple-locus variable-tandem repeat analysis reveals genetic relationhips within Bacilus anthracis. J. Bacteriol. 182:2928-2936. Kingston, J, Tuteja, U., Kapil, M., Murali, H., Bara, H. 2009. Genotyping of Indian Yersinia pestis strains by MLVA and repetitive DNA sequence based PCRs. Ant. Van Leeuwen. 96:303-312. Kuske, C. Barns, S., Grow, C., Merrill, L., Dunbar, J. 2006. Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes. J. Forensic Sci. 51:548-558. Lindler ,L., Fan, W., Jahan, N. 2001. Detection of ciprofoxacin-resistant Yersinia pestis by fluorogenic PCR using the LightCycler. J. Clin. Microbiol. 39:3649-3655. Lindsay, R., Gamez-Chin, S., McCready, P., Worsham, P., Anderson, G. 2001. Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis. Appl. Environ. Microbiol. 67:3759-3762. Loiez, C., Herwegh, S., Wallet, F., Armand, S., Guinet, F., Courcol, R. 2000. Detection of Yersinia pestis in sputum by real-time PCR. J. Clin. Microbiol. 41:4873-4875. Lowell, J., Wagner, D., Atshabar, B., Antolin, M., Vogler, A., Keimk, P., Chu, M., Gage, K. 2005. Identifying sources of human exposure to plague. J. Clin. Microbiol. 43:650-656. Lucier, T., Brubaker, R. 1992. Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electroprsis. J. Bacteriol. 174:2078-2086. McDonough, K., Schwan, T., Thomas, R., Falkow, S. 1998. Identification of a Yersinia pestis-specific DNA probe with potential for use in plague surveillance. J. Clin. Microbiol. 26:2515-2519. Motin, V., Georgescu, A., Elliott, J., Hu, P., Worsham,P., Ottt, L., Slezak, T., Sokhansanj, B., Regala, W., Brubakr, R., Garcia, E. 2002. Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehyrogenase (glpD). J. Bacteriol. 184:1019-1027. Norkina, O., Kulichenko, A., Gintsburg, L., Tuchkov, V., Popov, Y., Aksenov, M., Dosdov, I. 1994. Development of a diagnostic test for Yersinia pestis by PCR. J. Appl. Bacteriol. 76:240-245.

172

Robert E. Levin

23. Pouillot, F., Derbise, A., Kukkonen, M., Foulon, J., Carniel, E., Simonet, M. 2001. Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid. Microbiol. 151: 3759-3768. 24. Prior, J., Parkil, J., Hitchen, P., Munghall, K., Stevens, K., Morris, H., Reason, A., Oyston, P., Dell, A., Wren, B., Titball, R. 2001. The failure of different strains of Yersinia pestis to produce lipopolysaccharide O-antigens under different growth conditions is due to mutations in the O-antigen gene cluster. FEMS Microbiol. Lett. 197:229-233. 25. Raoult, D., Aboudharam, G., Crubezy, E., Larrouy, G., Ludes, B., Drancourt, M. 2000. Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death. Proc. Natl. Acad. Sci. USA. 97:12800-12803. 26. Rahalison, L, Vololonirina, E., Atsitorahina, M., Chanteau, S. 2000. Diagnosis of bubonic plague by PCR in Madagascar under field conditions. J. Clin Microbiol. 38:260-263. 27. Riehm, J., Rahalison, L., Scholz, H., Thoma, B., Pfeffer, M., Razanakoto, L, Dahouk S., Neubauer, H.,Tomasco, H. 2011. Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague. Molec. Cell. Probes. 25:8-12. 28. Skurnik, M., Peippo, A., Ervela, E. 2000. Characterization of the O-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen gene cluster of Yersinia pestis shows that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotpe O:1b. Mol. Microbiol. 37:316-330. 29. Stewart, A., Satterfield, B., Cohen, M., O’Neill, K., Robison, R. 2008. A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids. J. Med. Microbiol. 57:324-331. 30. Tomaso, H., Reisinger, E., Daouk, S., Frangoulidis, D., Rakin, A., Landt, O., Neubauer, H. 2003. Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridization probes. FEMS Immunol. Med. Microbiol. 38:117-126 31. Torrea, G, Chenal-Francisque, V., Leclercq, A., Carniel, E. 2006. Efficient tracing of global isolates of Yersinia pestis by restriction fragment length polymorphism analysis using three insertion sequences as probes. J. Clin. Microbiol. 44:2084-2092. 32. Woron, A., Nazarian, E., Egan, C., McDonough, A., Cirino, N., Limberger, R., Musser, A. 2006. Development and evaluation of a 4-target multiplex real-time polymerase chain reaction assay for the detection and characterization of Yersinia pestis. Diag. Microbiol. Infect. Dis. 56:261-268.