Search

Objective: To evaluate the immunogenicity of the meningococcal penicillin-binding protein 2 (PBP2) and its potential as a vaccine candidate. Methods: The immunogenicity of meningococcal PBP2 was investigated using acute and convalescent sera from patients who recovered from meningococcal disease. Sera were tested against purified recombinant PBP2s corresponding to meningococcal isolates of different genetic lineages, of different serogroups and with various susceptibility levels to penicillin G. Mice were vaccinated with recombinant PBP2 and challenged with Neisseria meningitidis. A purified anti-PBP2 rabbit IgG was also used for passive protection experiments in mice. Results: Convalescent patients' sera recognized PBP2s from different strains, showing that this protein is immunogenic in meningococcal disease. Vaccination with purified recombinant PBP2 and purified anti-PBP2 rabbit IgG antibody conferred protection against experimental meningococcaemia in mice. Conclusion: These data argue for considering meningococcal PBP2 as a vaccine candidate.

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.

The clinical presentations of meningococcal diseases other than meningitis or meningococcemia may lead to erroneous diagnosis. Although several reports have described unusual meningococcal diseases, the Neisseria meningitidis strains involved in these forms have been poorly characterized. In this study, meningococcal arthritis and pericarditis were confirmed by isolation of N. meningitidis and/or detection of meningococcal DNA in synovial or pericardial fluid, respectively, and meningococcal pneumonia was detected by isolation of N. meningitidis from blood. From 1999 through 2002, meningococcal disease was bacteriologically confirmed in 26 cases of arthritis, 6 cases of pericarditis, and 33 cases of pneumonia by the National Reference Center for the Meningococci in Paris. We found a statistically significant association between strains of serogroup W135, mostly of the clonal complex ET-37, and arthritis. Pneumonia was most frequently diagnosed in patients aged >70 years, and 54.5% of the strains belonged to serogroup W135, although these strains had heterogeneous phenotypes. Bacteremia is a key step in the pathophysiology of meningococcal disease and precedes any form of invasive infection.

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.

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 IS 100 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.

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.

Yersinia pestis is the causative agent of plague, a zoonotic disease transmitted to humans through flea bites and typically characterized by the appearance of a tender and swollen lymph node, the bubo. Human-to-human transmission can occur, through either the bite of fleas (bubonic plague) or respiratory droplets, causing an overwhelming infection called pneumonic plague. The last plague pandemic began in Hong Kong in 1894 and spread throughout the world, establishing many endemic foci. Antibiotics and enforcement of public health measures significantly decreased the morbidity and mortality associated with the disease but did not allow its eradication. In fact, plague . . .

Neisseria meningitidis belong to the genus Neisseria of the family Neisseriaceae. Two species are pathogenic for humans: Neisseria gonorrhoeae, N. meningitidis. These bacteria have the typical shape of Gram-negative diplococci. They are strict aerobes, non motile, oxydase and catalase positive, acidifying a limited number of sugars. Meningococcal strains that are isolated from invasive infections are usually easily identified. However, strains that are isolated from respiratory pathway may show atypical profiles. Beside their bacteriological determination and antibiotic susceptibility testing, N. meningitidis strains are phenotyped serologically (serogroup, serotype, serosubtype). Serogroup is based on the antigenic nature of the meningococcal capsule. It is crucial to determine serogroup to select the appropriate vaccines to prevent secondary cases as well as for epidemiological surveillance of this potentially epidemic bacterium.

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.

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.

International audience; Induction of type-IIA secreted phospholipase A2 (sPLA2-IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA2-IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili-defective mutant exhibited significantly lower capacity to stimulate sPLA2-IIA synthesis than the wild-type strain. Moreover, pili isolated from a LOS-defective strain induced sPLA2-IIA expression and nuclear factor kappa B (NF-kappaB) activation. These data suggest that pili are potent inducers of sPLA2-IIA expression by AM, through a NF-kappaB-dependent process.

International audience; We developed a model of sequential influenza A virus (IAV)-Neisseria meningitidis serogroup C (Nm) infection in BALB/c mice. Mice infected intranasally with a sublethal IAV dose (260 pfu) were superinfected intranasally with Nm. Fatal meningococcal pneumonia and bacteremia were observed in IAV-infected mice superinfected with Nm on day 7, but not in those superinfected on day 10. The susceptibility of mice to Nm superinfection was correlated with the peak interferon-gamma production in the lungs and decrease in IAV load. After Nm challenge, both IAV-infected and uninfected control mice produced the inflammatory cytokines interleukin (IL)-1 and IL-6. However, IL-10 was detected in susceptible mice superinfected on day 7 after IAV infection, but not in resistant mice. This model of dual IAV-Nm infection was also used to evaluate the role of bacterial virulence factors in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the crgA gene, negatively regulating expression of the pili and capsule, upon contact with host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified in genomic studies.

Infections due to Neisseria meningitidis are a major public health concern. In France, during 1999-2002, a total of 2167 clinical isolates of N. meningitidis from invasive infections were studied at the National Reference Center for Meningococci (Paris). Serogroup B strains were the most common (58%), followed by serogroup C strains (29%) and serogroup W135 strains (8%). Various phenotypes were observed, reflecting heterogeneity in the meningococcal population. Strains were susceptible to antibiotics currently used for treatment and chemoprophylaxis of meningococcal infections. However, the prevalence of meningococci with reduced susceptibility to penicillin is increasing. Such strains were heterogeneous and accounted for approximately 30% of isolates during this period, warranting continued surveillance of this phenomenon.

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, 16:1ω9cis, 17:0-cyc, and 18:1ω9trans 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 .

Yersinia spp. are gram-negative bacteria belonging to the family Enterobacteriaceae (3). The genus is composed of 11 species which can be divided into three pathogenicity groups. Strains nonpathogenic for humans are found mainly in the environment and sometimes also as transient intestinal saprophytes. These include Yersinia intermedia, Y. frederiksenii, Y. kristensenii, Y. mollaretii, Y. bercovieri, Y. aldovae, Y. rohdei, Y. ruckeri, and Y. enterocolitica biotype 1A (38). Strains with a moderate level of pathogenicity are widespread in cold and temperate countries and are responsible for mild enteric infections in humans (38). They are not lethal for mice at low doses and correspond to Y. enterocolitica strains of biotypes 2 to 5 (14). The group of high-pathogenicity strains is composed of Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica biotype 1B (46), which are all lethal for mice at low doses and cause disseminated infections in humans. This high level of pathogenicity correlates with the presence of a pathogenicity island (PAI) called the high-pathogenicity island (HPI) in Yersinia, as it differentiates low- and high-pathogenicity strains (10). PAIs are large pieces of chromosomal DNA that carry virulence genes, possibly acquired by phage-mediated horizontal transfer (24). The HPI of Y. enterocolitica 1B (10) is a chromosomal fragment of 45 kb that carries virulence genes known as the yersiniabactin locus that are involved in iron uptake (10, 39). Yersiniabactin is a siderophore (26) that endows the bacterium with the ability to acquire the iron molecules necessary for its in vivo growth and dissemination. Most of the length of the HPI is occupied by the yersiniabactin locus (Fig. ​(Fig.1),1), which comprises the genes irp1 to irp5, involved in nonribosomal biosynthesis of the siderophore (22, 39), and the outer membrane siderophore receptor gene fyuA (27, 42). The G+C content of these genes is higher than the chromosomal background. The yersiniabactin locus is highly conserved among Y. enterocolitica 1B strains (10) and in the two other high-pathogenicity species, Y. pseudotuberculosis (7) and Y. pestis (2, 8, 20). The region located on the right side of this locus, between irp2 and the right-hand boundary of the Y. enterocolitica HPI (Fig. ​(Fig.1),1), has not yet been sequenced. However, based on hybridization experiments with Y. pestis probes (8), it is most likely that this region carries genes recently described as composing another part of the yersiniabactin locus in Y. pestis (17). The right-hand border of the Y. enterocolitica HPI is defined by an asn-tRNA gene (10). In contrast, the ≈15-kb region of the Y. enterocolitica HPI extending on the left side of the yersiniabactin locus (Fig. ​(Fig.1)1) is not well conserved among various isolates of this species (10) and is different from the regions found on the HPIs of the two other high-pathogenicity species (7, 8). The only genes identified until now on this 15-kb region correspond to a cluster of three repeated sequences (IS1400 [10], IS1328 [41], and RS3 [10]) (Fig. ​(Fig.1).1). The left-hand boundary of the island has not been precisely identified. FIG. 1 Genetic organization of the HPI of Y. enterocolitica Ye8081. The HPI is represented by the gray line, and the identified genes are represented by arrows. Dashed lines below the diagram represent probes, named on the basis of their size in kilobases and ... The HPI of Y. enterocolitica 1B thus shows nearly all of the criteria of a typical PAI as defined by Hacker and colleagues (24): (i) it is a large chromosomal DNA fragment, (ii) it carries virulence genes essential for the expression of the high-virulence phenotype, (iii) it harbors several repeated sequences, (iv) it is bordered on one side by a tRNA gene, and (v) its G+C content is different from that of the rest of the chromosome. However, while PAIs are often described as mobile elements, and while the HPIs of Y. pestis (18, 19) and Y. pseudotuberculosis (7) are able to undergo spontaneous deletion from the host chromosome, the Y. enterocolitica HPI seems to be stable. Our previous attempts to identify spontaneous HPI deletion mutants among natural isolates of Y. enterocolitica 1B or following repeated subcultures of high-pathogenicity strain Ye8081 were unsuccessful (16). To date, no precise excision of this island has been reported. The aim of this study was to confirm whether the HPI of Y. enterocolitica was a stable feature of this organism’s genome and, if it was, determine the species-specific mechanism of stabilization.

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 distanceor = 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.

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.

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.

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.

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)

Previously, a group of 40 Yersinia enterocolitica-like strains that were isolated from water and fish were called group X2. These strains produced acid from L-rhamnose, did not ferment sorbose, cellobiose, melibiose, or raffinose, and rarely fermented sucrose (5% in 48 h. 10% in 7 days). This pattern of reactions separated group X2 strains from Yersinia enterocolitica, Yersinia intermedia, Yersinia frederiksenii, and Yersinia kristensenii. Positive reactions for acetoin production (Voges-Proskauer test), ornithine decarbox-ylase, and lack of acid production from melibiose distinguished group X2 strains from both Yersinia pseudotuberculosis and Yersinia pestis. Group X2 strains exhibited variable reactions only in tests for citrate utilization, hydrolysis of Tween 80, and acid production from maltose. Genetically, group X2 strains formed a single deoxyribonucleic acid hybridization group with an average level of relatedness of 86% or more (86% as determined by the S1 method at 60°C or by the hydroxyapatite method at 75°C and 92% as determined by the hydroxyapatite method at 60°C). The level of divergence among related sequences in 60°C reactions was 0.5%, as determined by the hydroxyapatite method. The relatedness of group X2 strains to other Yersinia species was 42 to 73% in 60°C reactions. The divergence in these reactions was 11.0 to 15.5%, and the relatedness to other yersiniae in 75°C reactions was 21 to 38%. Group X2 strains were 11 to 32% related to 67 species of the Enterobacteriaceae that belonged to genera other than Yersinia. On the basis of these biochemical and genetic data, we believe that group X2 represents a single new species in the genus Yersinia. The name Yersinia aldovae sp. nov. is proposed for this species. The type strain of Y. aldovae is strain CNY 6005 (= CDC 669-83 = ATCC 35236).

We have previously shown that under iron limitation, different Yersinia species synthesize new polypeptides. Two of them, the high-molecular-weight proteins (HMWPs), are expressed only by the highly pathogenic strains. In the present study, the HMWPs from Y. enterocolitica serovar O:8 were purified by gel filtration, and specific antibodies were obtained. Using these antibodies, we show that the two polypeptides were synthesized de novo during iron starvation and that they were found essentially in the bacterial outer membrane fractions, although the majority of the molecules were not exposed on the cell surface. We also demonstrate that the two proteins had common epitopes and that the HMWPs of the high-virulence-phenotype species Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serovar O:8 (a strain different from the one used to purify the proteins) are antigenically related. The less pathogenic and nonpathogenic strains did not exhibit cross-reacting material, suggesting that these strains do not synthesize even an altered form of the HMWPs.

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.