Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis.

Plasmid-mediated high-level resistance to multiple antibiotics was reported in a clinical isolate of Yersinia pestis in Madagascar in 1997. We describe a second Y. pestis strain with high-level resistance to streptomycin, isolated from a human case of bubonic plague in Madagascar. The resistance determinants were carried by a self-transferable plasmid that could conjugate at high frequencies to other Y. pestis isolates. The plasmid and the host bacterium were different from those previously associated with multiple-drug resistance, indicating that acquisition of resistance plasmids is occurring in this bacterial species. Emergence of resistance to streptomycin in Y. pestis represents a critical public health problem since this antibiotic is used as the first-line treatment against plague in many countries.

High homogeneity of the Yersinia pestis fatty acid composition.

The cellular fatty acid compositions of 29 strains of Yersinia pestis representing the global diversity of this species have been analyzed by gas-liquid chromatography to investigate the extent of fatty acid polymorphism in this microorganism. After culture standardization, all Y. pestis strains studied displayed some major fatty acids, namely, the 12:0, 14:0, 3-OH-14:0, 16:0, 16:1omega9cis, 17:0-cyc, and 18:1omega9trans compounds. The fatty acid composition of the various isolates studied was extremely homogeneous (average Bousfield's coefficient, 0.94) and the subtle variations observed did not correlate with epidemiological and genetic characteristics of the strains. Y. pestis major fatty acid compounds were analogous to those found in other Yersinia species. However, when the ratios for the 12:0/16:0 and 14:0/16:0 fatty acids were plotted together, the genus Yersinia could be separated into three clusters corresponding to (i) nonpathogenic strains and species of Yersinia, (ii) pathogenic Yersinia enterocolitica isolates, and (iii) Yersinia pseudotuberculosis and Y. pestis strains. The grouping of the two latter species into the same cluster was also demonstrated by their high Bousfield's coefficients (average, 0.89). Therefore, our results indicate that the fatty acid composition of Y. pestis is highly homogeneous and very close to that of Y. pseudotuberculosis.

Failure of oily chloramphenicol depot injection to treat plague in a murine model.

Effective low-cost single-dose therapy would be invaluable in treating human plague. The efficacy of single- or two-dose injections of oily chloramphenicol (OCm) was compared with that of standard multiple injections of reference drugs (streptomycin or chloramphenicol) in a murine plague model. A single injection of OCm was ineffective. Two doses cleared bacteraemia and limited bacterial growth in the mouse spleen but were less effective in reducing mortality than standard therapy. However, because of the marked pharmacokinetic differences between mice and humans, the failure of depot injection of OCm in murine plague treatment is not indicative of its ineffectiveness in human plague.

Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis.

Plague, one of the most devastating diseases of human history, is caused by Yersinia pestis. In this study, we analyzed the population genetic structure of Y. pestis and the two other pathogenic Yersinia species, Y. pseudotuberculosis and Y. enterocolitica. Fragments of five housekeeping genes and a gene involved in the synthesis of lipopolysaccharide were sequenced from 36 strains representing the global diversity of Y. pestis and from 12-13 strains from each of the other species. No sequence diversity was found in any Y. pestis gene, and these alleles were identical or nearly identical to alleles from Y. pseudotuberculosis. Thus, Y. pestis is a clone that evolved from Y. pseudotuberculosis 1,500-20,000 years ago, shortly before the first known pandemics of human plague. Three biovars (Antiqua, Medievalis, and Orientalis) have been distinguished by microbiologists within the Y. pestis clone. These biovars form distinct branches of a phylogenetic tree based on restriction fragment length polymorphisms of the locations of the IS100 insertion element. These data are consistent with previous inferences that Antiqua caused a plague pandemic in the sixth century, Medievalis caused the Black Death and subsequent epidemics during the second pandemic wave, and Orientalis caused the current plague pandemic.

The high-pathogenicity island of Yersinia enterocolitica Ye8081 undergoes low-frequency deletion but not precise excision, suggesting recent stabilization in the genome.

Highly pathogenic strains of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are characterized by the possession of a pathogenicity island designated the high-pathogenicity island (HPI). This 35- to 45-kb island carries an iron uptake system named the yersiniabactin locus. While the HPIs of Y. pestis and Y. pseudotuberculosis are subject to high-frequency spontaneous deletion from the chromosome, we were initially unable to obtain HPI-deleted Y. enterocolitica 1B isolates. In the present study, using a positive selection strategy, we identified three HPI-deleted mutants of Y. enterocolitica strain Ye8081. In these three independent clones, the chromosomal deletion was not limited to the HPI but encompassed a larger DNA fragment of approximately 140 kb. Loss of this fragment, which occurred at a frequency of approximately 5 x 10(-7), resulted in the disappearance of several phenotypic traits, such as growth in a minimal medium, hydrolysis of o-nitrophenyl-beta-D-thiogalactopyranoside, Tween esterase activity, and motility, and in a decreased virulence for mice. However, no precise excision of the Ye8081 HPI was observed. To gain more insight into the molecular basis for this phenomenon, the putative machinery of HPI excision in Y. enterocolitica was analyzed and compared to that in Y. pseudotuberculosis. We show that the probable reasons for failure of precise excision of the HPI of Y. enterocolitica Ye8081 are (i) the interruption of the P4-like integrase gene located close to its right-hand boundary by a premature stop codon and (ii) lack of conservation of 17-bp att-like sequences at both extremities of the HPI. These mutations may represent a process of HPI stabilization in the species Y. enterocolitica.

Phenotypic and genotypic characterization of virulent yersinia enterocolitica strains unable to ferment sucrose.

Several atypical sucrose-negative Yersinia strains, isolated from clinical samples and sometimes associated with symptoms, proved to have full virulence potential in in vitro and in vivo testings. DNA-relatedness studies revealed that they were authentic Yersinia enterocolitica strains. Therefore, atypical sucrose-negative Yersinia isolates should be analyzed for their virulence potential.

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes.

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis. In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis, has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis. A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance > or = 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica, the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica, is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.

Recent emergence of new variants of Yersinia pestis in Madagascar.

Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain.

[Serodiagnosis of human pathogenic Yersinia infections]. / Sérodiagnostic des infections humaines à Yersinia pathogènes.

OBJECTIVES: The test classically used for the serological diagnosis of yersiniosis is based on the technique of agglutination of killed bacteria. The sensitivity of this technique is low and numerous cross reactions are observed. The aim of this work was to develop a new serological test based on the ELISA method and to compare the results obtained by this method and by agglutination. METHODS: The antigens used for the ELISA test were the virulence plasmid-encoded proteins present in all pathogenic Yersinia and specific for this bacterial genera. The background of this technique was determined using 30 normal human sera. Validation of the ELISA method was performed with human sera negative (23) or positive (72) by the agglutination technique. The sensitivity of the ELISA test was compared with that of agglutination using 10 patients' sera which remained negative by the agglutination test despite the isolation of a pathogenic Yersinia. Finally, the ELISA specificity was determined with the sera of patients suffering from tuberculosis (20), typhoid fever (6), suspected or confirmed brucellosis (5) and suspected yersiniosis but having a positive Wright reaction (13). RESULTS: We show here that, in addition to the advantages inherent in the ELISA technique, this test is more sensitive and specific than the agglutination test. Furthermore, the ELISA test can be used for all pathogenic Yersinia, whatever their serotype and biotype. CONCLUSION: The use of the ELISA test should significantly improve the serodiagnosis of yersiniosis.

Comparison of three molecular methods for typing and subtyping pathogenic Yersinia enterocolitica strains.

The efficiency of pulsed-field gel electrophoresis (PFGE), ribotyping and restriction enzyme analysis of the virulence plasmid (REAP) for typing and subtyping strains of Yersinia enterocolitica was compared. All three techniques gave concordant results, and the strains studied could be separated into three distinct clusters: (1) heterogeneous strains of biotype 1A and serotype O5 (1A/O5); (2) one 3/O3 strain and all 2/O9 strains; and (3) all 4/O3, 2/O5 and two 3/O3 strains. Within cluster 3, the 2/O5 and 3/O3 strains were related more closely to each other than to the 4/O3 isolates. With ribotyping, PFGE and phage-typing, the 4/O3 isolates were subdivided into two homogeneous groups, corresponding to strains of phage type IXb and strains of other phage types, respectively. Similarly, ribotyping and PFGE subdivided the 2/O9 strains into two conserved groups (I and II), but REAP gave a different subdivision and identified a new REAP pattern (P3). The three techniques confirmed the clear distinction between the heterogeneous group of non-pathogenic 1A/O5 strains and the well conserved group of pathogenic 2/O5 strains. Additional plasmids were identified in some 3/O3 strains. Combined, the results indicated that REAP (with EcoRI) and ribotyping (with EcoRV) are valuable alternatives to bioserotype determination, and PFGE is the most suitable technique for epidemiological tracing.

Susceptibility to beta-lactam agents of Yersinia enterocolitica biotype 4, serotype O3 isolated in various parts of the world.

Forty-eight human isolates of Yersinia enterocolitica of biotype 4, serotype O3 from various parts of the world were examined for susceptibility to 13 beta-lactam agents. The intracellular beta-lactamases of each of the 48 strains were examined. Isolates from Europe, Asia and Brazil (phage type VIII) or South Africa and Hungary (phage type IXa) produced both enzyme A and enzyme B, whereas isolates from New Zealand and Australia (phage type IXb) lacked the cephalosporinase enzyme B. Among the seven strains isolated in Canada belonging to phage type IXb, three strains expressed only enzyme A (group I) whereas the other four strains produced both enzymes A and B (group II). The high susceptibility to the combination of amoxycillin and clavulanate observed in one subtype was explained by the absence of the cephalosporinase enzyme B. A simple disk diffusion test with this antibiotic combination was shown to be effective in the detection of enzyme B in Y. enterocolitica 4/O3.

Plague pandemics investigated by ribotyping of Yersinia pestis strains.

Yersinia pestis is the causative agent of plague, a disease which has caused the deaths of millions of people and which persists now in endemic foci. The rRNA gene restriction patterns (i.e., ribotypes) of 70 strains of Y. pestis, isolated on the five continents over a period of 72 years, were determined by hybridization with a 16S-23S rRNA probe from Escherichia coli. The combination of the EcoRI and EcoRV patterns resulted in the elucidation of 16 ribotypes. Two of them (B and O) characterized 65.7% of the strains studied, while the 14 other ribotypes were found in no more than three strains each. A relationship was established between biovars and ribotypes: strains of biovar Orientalis were of ribotypes A to G, those of biovar Antiqua were of ribotypes F to O, and those of biovar Medievalis were of ribotypes O and P. Great heterogeneity in rRNA restriction patterns was found among strains isolated in Africa; this heterogeneity was less pronounced among Asian isolates and was completely absent from the American strains. Pulsed-field gel electrophoresis was performed on the DNAs of some strains, but it appeared that different colonies from the same strain displayed different pulsed-field gel electrophoresis patterns and therefore that this technique was not suitable for comparison of Y. pestis isolates. In contrast, the ribotypes of individual colonies within a given strain were stable and were not modified after five passages in vivo. A clear correlation between the history of the three plague pandemics and the ribotypes of the strains could be established.

Assessment of a fluoroquinolone, three beta-lactams, two aminoglycosides, and a cycline in treatment of murine Yersinia pestis infection.

Amoxicillin, cefotaxime, ceftriaxone, gentamicin, doxycycline, and ofloxacin were active in vitro, like the reference drug streptomycin, against the virulent strain Yersinia pestis 6/69M. The comparative efficacies of these drugs in vivo were evaluated in a standardized and reproducible mouse model of systemic infection. Each antibiotic was injected intravenously once, at 24 h postinfection, and then repeatedly during 48 h. In vivo results were measured by counting the viable bacteria recovered from the whole spleens of mice sacrificed at selected times. All the drugs were manifestly successful; ceftriaxone, ofloxacine, and the reference drug were the most effective. Therefore, gentamicin and doxycycline could be used, depending on the clinical forms of the Y. pestis infection. Further investigations on beta-lactams, especially those used in the present study, could be carried out to confirm or not confirm their activities against Y. pestis. Ofloxacin appeared to be as active and to perform as rapidly as streptomycin in the treatment of murine Y. pestis infection, which is in agreement with the previous successes obtained with the use of fluoroquinolones in the treatment of murine infections caused by other pathogenic yersiniae.

Survey of the irp2 gene among Yersinia pestis strains isolated during several plague outbreaks in northeast Brazil.

The irp2 gene codes for a 190 kDa protein (HMWP2) synthesized when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil was studied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to be positive with the A13 probe, indicating that the locus was lost after subculture in vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcoRV or AvaII, indicating that the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host.

Relationship between loss of pigmentation and deletion of the chromosomal iron-regulated irp2 gene in Yersinia pestis: evidence for separate but related events.

The irp2 gene, coding for a 190-kDa iron-regulated protein (HMWP2), and the hemin storage locus (hms), which determines Yersinia pestis pigmentation, are each located on a large chromosomal fragment which carries virulence genes and deletes spontaneously. To determine whether the two loci are located on one unstable fragment or on two different excisable DNA segments, the pigmentation status and the presence of irp2 in 43 strains of Y. pestis isolated in various parts of the world were examined. Three different types were observed: Pgm+ Irp2+ (39.5%), Pgm- Irp2- (44.2%), and Pgm- Irp2+ (16.3%). No Pgm+ Irp2- strain was found. These three types were also recovered in vitro from the parental strain Saigon 55-12-39 (Pgm+ Irp2+), but again, no Pgm+ Irp2- colony was observed. Pgm- Irp2- derivatives were obtained from a single Pgm- Irp2+ colony, indicating sequential loss of the two traits. The fact that the genomic SpeI restriction patterns obtained by pulsed-field gel electrophoresis were specific for each of the three variants suggested that distinct large-scale chromosomal rearrangements had occurred in the Pgm- Irp2+ and Pgm- Irp2- derivatives. The virulence of Pgm- Irp2+ bacteria in mice was ca. 10(7)-fold lower than that of the Pgm+ Irp2+ strains injected subcutaneously but was not significantly decreased when injected intravenously. In contrast, the Pgm- Irp2- microorganisms were markedly less pathogenic (10(6)-fold) than the Pgm+ Irp2+ strains injected intravenously and were 100 times less virulent than the Pgm- Irp2+ strains injected subcutaneously.

Chromosomal irp2 gene in Yersinia: distribution, expression, deletion and impact on virulence.

Iron starvation induces the synthesis of two high molecular weight proteins (HMWP1 and 2) in Yersinia. The presence of the irp2 gene coding for the HMWP2 was investigated in 170 Yersinia strains. This gene was absent from all avirulent or weakly pathogenic species and was restricted to highly pathogenic strains. One hundred percent of the potentially highly pathogenic Yersinia enterocolitica biotype 1B harbored irp2 but surprisingly, 70.4% of the Yersinia pseudotuberculosis tested lacked the gene. Only serotypes I and III of Y. pseudotuberculosis harbored the locus, however, synthesis of HMWPs was detected uniquely in the former. In Yersinia pestis, overall 55.3% of the strains tested had the gene, with an uneven distribution among Orientalis (65.2%), Antiqua (66.6%) and Medievalis (0%) geographic variants. Except for one Y. pestis strain, the irp2 restriction profiles were identical for all strains of Y. pestis and Y. pseudotuberculosis tested. All Y. enterocolitica 1B displayed the same irp2 pattern, different from that of the other two species. In vitro spontaneous deletion of irp2 was not obtained in Y. enterocolitica 1B but was observed in both Y. pseudotuberculosis and Y. pestis. Repeated subcultures of Y. pestis increased progressively the proportion of irp2-deleted colonies, yielding an almost pure irp2-deleted strain after 16 subcultures. A clear correlation was established between the presence of irp2 and the level of virulence of Y. pseudotuberculosis and Y. pestis.

Molecular cloning, iron-regulation and mutagenesis of the irp2 gene encoding HMWP2, a protein specific for the highly pathogenic Yersinia.

Under iron-starvation, the highly pathogenic Yersinia synthesize several iron-regulated proteins including two high-molecular-weight polypeptides (HMWP1 and HMWP2). From the chromosome of Yersinia enterocolitica serovar O:8 (strain Ye 8081), the genes coding for the HMWP2 (irp2) and its promoter were cloned into plasmid pUC18 (pIR2) and used as a probe. We show here that the irp2 gene is present only in the highly pathogenic strains and that its promoter is iron-regulated in Escherichia coli. After introduction of the pIR2 plasmid into a fur mutant of E. coli, both the iron-starved and the iron-replete bacteria expressed the HMWP2. Repressibility of irp2 by iron was restored by introduction of a plasmid carrying the fur gene. These results demonstrate that the irp2 promoter is controlled by the Fur repressor in E. coli. Mutagenesis of the chromosomal irp2 gene of Yersinia pseudotuberculosis was obtained by homologous recombination with a 1 kb fragment of this gene cloned on the suicide plasmid pJM703.1. Inactivation of irp2 resulted in the non-expression of both HMWPs, while introduction of plasmid pIR2 into the mutant strain led to the synthesis of the HMWP2 only. Therefore, it is probable that the genes coding for the HMWPs constitute an operon where irp2 is upstream of irp1. When comparing the virulence of the wild-type strain and of its irp2 mutant derivative, we found that the 50% lethality (LD50) for mice of the mutant strain was increased, whatever the route of infection, but more markedly when injected parenterally. Accordingly, these data demonstrate that a mutation in the irp2 gene alters the pathogenicity of Y. pseudotuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)