A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.
An acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis was diagnosed in 13 patients from the Russian Far East in 2002. We amplified and sequenced four portions of three rickettsial genes from the patients' skin biopsy results and blood samples and showed that the amplified rickettsial genes belong to R. heilongjiangensis, which was recently isolated from Dermacentor sylvarum ticks in nearby regions of China. This rickettsia, belonging to subgroup of R. japonica, was previously suggested to be pathogenic for humans on the basis of serologic findings. We tested serum samples with different rickettsial antigens from 11 patients and confirmed increasing titers of immunoglobulin (Ig) G and IgM to spotted fever group rickettsiae, including R. heilongjiangensis. Clinical and epidemiologic data on these patients show that this disease is similar to other tick-borne rickettsioses.
Staphylococcus aureus is a pathogen and a skin commensal that is today also common in the infant gut flora. We examine the role of S. aureus virulence factors for gut colonization. S. aureus isolated from quantitative stool cultures of 49 Swedish infants followed from birth to 12 months of age were assessed for 30 virulence-associated genes, spa type, and agr allele by serial polymerase chain reaction (PCR) assays. Strains carrying genes encoding collagen-binding protein, and the superantigens S. aureus enterotoxin O/M (SEO/SEM) had higher stool counts than strains lacking these genes, whereas genes for S. aureus enterotoxin A (SEA) were associated with low counts. A cluster of strains belonging to agr allele I and the spa clonal cluster 630 (spa-CC 630) that carried genes encoding SEO/SEM, SEC, collagen-binding protein, and elastin-binding protein were all long-time colonizers. Thus, certain S. aureus virulence factors might promote gut colonization.
The taxonomic position of 12 isolates tentatively assigned to the genus Bifidobacterium on the basis of a limited phenotypic characterization was examined. The isolates were collected between 1978 and 2005 in Belgium, Sweden and Norway, and originated from various human clinical samples, including urine, blood, urethra, oral cavity, tonsil, and abscesses of lung and aortic valve. On the basis of band number and clustering analysis, repetitive DNA element-based PCR fingerprinting using the BOXA1R and (GTG)(5) primers indicated that the clinical isolates represented a taxon probably not belonging to the genus Bifidobacterium. Analysis of 16S rRNA gene sequence similarities revealed that the isolates were most closely affiliated to Parascardovia denticolens LMG 18312(T) (93.0-93.2 %), Scardovia inopinata LMG 18313(T) (92.9-93.1 %) and other members of the Bifidobacteriaceae, indicating that the isolates belong to a novel genus within that family. This observation was further substantiated by the results of partial sequencing of the heat-shock protein 60 gene (hsp60) and determination of the DNA G+C contents (47.3-48.3 mol%). Members of the novel taxon can be phenotypically distinguished from S. inopinata, P. denticolens and Gardnerella vaginalis by the ability to grow on agar under aerobic conditions and on the basis of positive reactions for acid production from L-arabinose, raffinose, salicin and D-xylose. Unambiguous phenotypic differentiation from Aeriscardovia aeriphila and Bifidobacterium species may be difficult, so phenotypic analyses should be complemented by molecular methods. The values for DNA-DNA binding among four members of the novel genus were in the range of 89-100 %, indicating that the strains should be considered as a single novel species of a novel genus, for which the name Alloscardovia omnicolens gen. nov., sp. nov. is proposed. The type strain of Alloscardovia omnicolens is CCUG 31649(T) (=LMG 23792(T)).
Laboratory for Vaccine Preventable Diseases. National Institute of Public Health and the Environment, Anthonie van Leeuwenhoeklaan 9, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.
This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin–antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms—endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).
This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin–antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms—endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).
An analysis of a 5.4-kbp cryptic plasmid detected in the course of whole-genome sequencing of the Yersinia pestis medieval biovar isolated in the Russian Central Caucasian high-mountain plague focus was performed. The identification of the nucleotide sequence of this cryptic plasmid and its structural and functional analysis revealed that it contained eight open reading frames, among which the following genes were identified: the rep gene of a replication protein, the virB6 gene of a type-IV secretion system inner membrane protein, the virB5gene of the type-IV secretion system minor pilin, and a number of genes probably associated with secretion and transport. A general analysis of the pCKF plasmid DNA showed that the adenine content was 28.34%, the cytosine content was 20.5%, the guanine content was 17.87%, and that of thymine was 33.28%, while the total G+C content appeared to be 38.38%. The G+C content of the chromosome of the Y pestis strain C-627 is 47.6%, which indicates that the pCKF plasmid was obtained from a microorganism equally-phylogenetically distant from the Yersinia bacteria andany other bacteria from the Enterobacteriaceae family. A comparison of the amino acid sequences.of hypothetical proteins encoded by pCKF plasmid with analogous proteins encoded by other bacteria was carried out. The possible contribution of the pCKF plasmid to the maintenance of the most ancient known phylogenetic line of Y. pestis medieval biovar, 2.MEDO, was discussed.
Antimicrobial activity of tigecycline and comparative agents against clinical isolates of staphylococci and enterococci from ICUs and general hospital wards at three Swedish university hospitals.
Clinical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoping University, Linkoping, Sweden. carcl@imk.liu.se
The activities of tigecycline and comparative agents on staphylococci and enterococci isolated from patients at general hospital wards (GHWs) and intensive care units (ICUs) at 3 university hospitals in Sweden were investigated. Oxacillin disc diffusion and minimal inhibitory concentration with E-test were used. The presence of mecA, vanA or vanB genes was determined with PCR. Statistically significant higher incidence of clindamycin, fusidic acid, rifampicin and multidrug-resistant CoNS was found at ICUs compared to GHWs. Resistance rates were low among S. aureus. Tigecycline, linezolid and vancomycin were the only agents with high activity against methicillin-resistant S. aureus and multidrug-resistant CoNS. Resistance rates were low among E. faecalis, except for high-level gentamicin-resistant (HLGR) E. faecalis. E. faecium showed high resistance rates to ampicillin, piperacillin/tazobactam and imipenem. The HLGR rates among E. faecium were lower than the rates for E. faecalis. Tigecycline and linezolid were the only drugs with high activity against all enterococci including vancomycin-resistant enterococci. No statistically significant differences in susceptibility rates were found between the ward levels for S. aureus and enterococcal isolates and no statistically significant differences were found between the hospitals.
Helicobacter pylori has been proven to be responsible for causing various gastrointestinal disorders including gastric adenocarcinoma. Several genes of pathogen (the genes of the cag-PAI, vacA, iceA, and babA) either in combination or independently have been reported to significantly increase the risk of ulceration/gastric carcinoma, with the cagA gene having the strongest predictive value. Pursuit to identify new genes which could serve as a marker of overt disease progression, lead to the discovery of hrgA gene.
Fifty-six indigenous strains of H. pylori from subjects with various gastric disorder were screened to assess the status of hrgA gene along with the cagA gene using simple polymerase chain reaction using specific oligonucleotide primers. Post-amplification, amplicons were subjected for sequencing to identify any strain specific variations in sequences from the H. pylori isolated from different disease manifestations. Histopathological analysis was done to ascertain any significant change in the histological scores of subjects infected with cagA+/hrgA+ and cagA-/hrg+ strains.
All the 56 (100%) subjects amplified with the oligonucleotide primers specific to hrgA gene, whereas 81.71% subjects showed the presence of cagA gene. Sequencing of the amplimers showed 99% homology. Histology of the cagA+/hrgA+ and cagA-/hrg+ subjects did not show any significant difference.
hrgA gene of Helicobacter pylori is not a ideal surrogate marker for identifying individuals with higher risk of developing overt gastro-duodenal diseases such as neoplasia of the stomach.
