Multiple-Locus Variable-Number Tandem-Repeat Analysis of Pathogenic Yersinia enterocolitica in China

The predominant bioserotypes of pathogenic Yersinia enterocolitica in China are 2/O: 9 and 3/O: 3; no pathogenic O: 8 strains have been found to date. Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) based on seven loci was able to distinguish 104 genotypes among 218 pathogenic Y. enterocolitica isolates in China and from abroad, showing a high resolution. The major pathogenic serogroups in China, O: 3 and O: 9, were divided into two clusters based on MLVA genotyping. The different distribution of Y. enterocolitica MLVA genotypes maybe due to the recent dissemination of specific clones of 2/O: 9 and 3/O: 3 strains in China. MLVA was a helpful tool for bacterial pathogen surveillance and investigation of pathogenic Y. enterocolitica outbreaks

Lipopolysaccharide O1 Antigen Contributes to the Virulence in Klebsiella pneumoniae Causing Pyogenic Liver Abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K1− O1) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain

PCR-Based Identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the Agent of Rhinoscleroma

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma

Screening for anthrax occurrence in soil of flooded rural areas in Poland after rainfalls in spring 2010

[b]Introduction and objective[/b]. Anthrax spores remain viable and infectious in soil for decades. Flood water can percolate towards the surface the spores buried in soil. Moreover, the flood water might transport spores to areas previously unaffected. After the water recedes the spores located on the surface of the ground can be consumed by grazing animals and cause outbreaks of anthrax. [b]Materials and method[/b]. Soil samples were collected in areas of Poland most affected by floods in 2010 (Lubelskie, Świętokrzyskie, Podkarpackie and Mazowieckie provinces). After heating with the aim to kill vegetative forms of bacteria, the samples were cultured on PLET agar and the resulted colonies were investigated in terms of motility and presence of anthrax specific chromosomal (SG-749, plcR) and plasmid markers (capB, pagA). [b]Results.[/b] In total, 424 spore-forming, aerobically growing isolates were collected from the tested soil samples. Eighty-nine of them were non-motile. All the isolates were negative in PCR for anthrax specific chromosomal and plasmid markers. Conclusions. Spores of [i]B. anthracis[/i] that could be related to risk of anthrax outbreaks were not detected in soil samples tested in this study. The negative results presented may not be proof that Poland is country free of anthrax. The results, however, may suggest a relatively low risk of anthrax outbreaks being triggered in the sampled area

Some Bacillus thuringiensis strains share rpoB nucleotide polymorphisms also present in Bacillus anthracis

Identification of Bacillus anthracis is considerably difficult because of its very high phenotypic and genotypic similarity to other members of the Bacillus cereus group. Differentiation methods based on morphological and phenotypic characteristics are time-consuming, and false results may be obtained for atypical strains. On the other hand molecular discrimination based on the presence of two B. anthracis virulence plasmids, pXO1 and pXO2, is not sufficient because plasmids can be lost or transferred to other bacilli. Therefore, several chromosomal markers have been investigated and applied (1, 8). In 2001 Qi et al. (7) described single nucleotide polymorphisms (SNPs) in the rpoB gene and their usefulness for B. anthracis identification. Since then, several articles describing various molecular methods for rpoB sequence-based detection of B. anthracis have been published (for example see references 2 and 9). We conducted studies of single-strand conformation polymorphisms (SSCPs) of the rpoB gene in a large collection of B. cereus group strains. Surprisingly, we found that the nucleotide sequence of the rpoB gene fragment containing the marker SNPs of two reference strains of Bacillus thuringiensis was identical to that of the homologous region in B. anthracis. Therefore, rpoB gene-based tools could not distinguish these strains from B. anthracis, thus resulting in false-positive anthrax identification

Specific Bacillus anthracis identification by a plcR-targeted restriction site insertion-PCR (RSI-PCR) assay

A RSI-PCR assay was developed for the detection of a Bacillus anthracis-specific nonsense mutation in the plcR gene. The assay specificity was tested using 170 Bacillus spp. strains including 47 strains of B. anthracis. The plcR RSI-PCR distinguished Bacillus cereus group strains closely related to B. anthracis from the anthrax agent. The assay was found to be a robust, simple and cost effective tool for B. anthracis identification. In contrast to previously developed real time PCR-based methods, the RSI-PCR needs basic molecular biology equipment only, and thus may be easily introduced in developing countries, where anthrax is endemic