Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multiple Chlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D-, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. The incA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D((s)) strains. Three nucleotide changes were discovered in the D((s)) incA gene, leading to two amino acid changes within the predicted D((s)) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.     

Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.     

Distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae isolates collected in 1995 to 1998 and 2001 to 2006 at a university hospital in South Korea and identification of conjugative plasmids mediating dissemination of 16S rRNA methylase

The distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae collected between 1995 and 1998 and between 2001 and 2006 at a university hospital in South Korea was examined, and conjugative plasmids carrying the 16S rRNA methylase genes were characterized by PCR-based replicon typing and by determination of their antimicrobial resistance pattern. Among the 7,127 isolates, 463 isolates showed a high level of resistance to amikacin, and 218 of the 463 isolates transferred amikacin resistance by conjugation. Among the 218 isolates, armA was detected in 153 isolates (88 Klebsiella pneumoniae, 28 Escherichia coli, 19 Enterobacter cloacae, and 6 Serratia marcescens isolates and 12 isolates of other organisms), and rmtB was detected in 51 isolates (32 K. pneumoniae isolates, 18 E. coli isolates, and 1 Citrobacter freundii isolate). The first appearance of armA was in 1997. The armA gene was carried by conjugative plasmids of replicon groups IncL/M, IncFIIAs, IncF, IncA/C, IncHI2, and Inc(unidentified) in 38, 20, 7, 9, 4, and 75 strains, respectively. The rmtB gene was carried by conjugative plasmids of groups IncA/C, IncF, and IncI1-Iγ in 43 strains, 7 strains, and 1 strain, respectively. Transconjugants that received the IncL/M plasmid carrying armA or the IncA/C plasmid carrying rmtB showed an additional resistance to cefotaxime. Transconjugants that received the IncFIIA plasmid or Inc(unidentified) plasmid carrying the armA gene showed an additional resistance to cefoxitin and a high MIC₅₀ (0.25 mg/liter) of ciprofloxacin. In conclusion, this study demonstrated that the dissemination of 16S rRNA methylase genes among the Enterobacteriaceae is mediated by conjugative plasmids of various incompatibility groups that confer resistance to multiple drugs, including aminoglycosides, extended-spectrum β-lactams, and/or quinolones.     

Multiple insertional events, restricted by the genetic background, have led to acquisition of pathogenicity island IIJ96-like domains among Escherichia coli strains of different clinical origins

We investigated the dissemination of pathogenicity island (PAI) II(J96)-like elements (hra, hly, cnf1, and pap) among 455 Escherichia coli isolates from children and adults with urinary tract infection (UTI), neonates with meningitis or colonized healthy neonates, and 74 reference strains by means of PCR phylogenetic grouping, ribotyping, and PCR analysis of virulence genes. Colocalization of these genes was documented by pulsed-field gel electrophoresis followed by Southern hybridization and long-range PCR (LRPCR) between the hra and the papG alleles. Site-specific insertion of the PAI was determined by LRPCR between hra and tRNA flanking sequences. hra, hly, and cnf1 were found in 113 isolates and consistently colocalized, constituting the backbone of PAI II(J96)-like domains. The prevalence of PAI II(J96)-like domains was significantly higher among UTI isolates than among neonatal meningitis and commensal isolates. These domains were restricted to a few ribotypes of group B2. In contrast to the consistent colocalization of hra, hly, and cnf1, the pap operon was varied: 12% of strains exhibited an allelic exchange of the papG class III allele (papGIII) for the papG class II allele (papGII) (only UTI isolates), and the pap operon was deleted in 23% of strains. No strains harbored papGIII outside the PAI, which appears to be the only source of this allele. PAI II(J96)-like domains were inserted in the vicinities of three different tRNAs--pheU (54%), leuX (29%), and pheV (15%)--depending on the genetic backgrounds and origins of the isolates. Multiple insertional events restricted by the genetic background have thus led to PAI II(J96) acquisition. Specific genetic backgrounds and insertion sites may have played a role in additional recombination processes for E. coli adaptation to different ecological niches.     

Relationship between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis.

Previous studies in our laboratory using a combination of polymerase chain reaction and restriction fragment length polymorphism have identified at least five different genotypes of infectious laryngotracheitis virus (ILTV). However, the virulence of these classes of ILTV was not investigated. In this study, five groups (16 birds each) of 3-week-old specific pathogen free chickens were inoculated via the intratracheal route with 10(3) median embryo infected dose of five different strains of ILTV. Three further groups of chickens were inoculated similarly with the vaccine strains SA2 and A20 or with sterile phosphate-buffered saline (PBS) for comparison. Four days post-inoculation, clinical signs were monitored for scoring, and eight chickens from each group were subsequently euthanized, weighed and subjected to pathological and histopathological examinations. The remaining birds were monitored for clinical signs and mortality until 21 days post-inoculation. All groups inoculated with ILTV strains showed moderate to severe clinical signs 4 days after inoculation. The strain Q1-96 caused only minimal breathing symptoms with a median score that was not significantly different to that of the group inoculated with PBS, but was significantly different to those of the groups inoculated with other ILTV strains. The strain Q1-96 caused severe photophobia and conjunctivitis with a median score that was significantly higher than those of all other groups except for the group inoculated with the strain N3-04. All ILTV strains caused a significant reduction in weight gain when compared with the group inoculated with PBS. The strain Q1-96 caused an average weigh loss of 14% that was significantly higher than those of other ILTV strains. The strains S2-04 and Q1-96 induced only minor microscopic tracheal lesions while all the other ILTV strains, including the vaccine strains A20 and SA2, induced moderate to severe microscopic tracheal lesions. Median scores for microscopic tracheal lesions were well correlated with the number of viral genomes detected in trachea. The results revealed that there is considerable variation among ILTV strains in their tropism for trachea or conjunctiva. In addition it was revealed that ILTV strains with high affinity for conjunctiva can severely affect weigh gain. The ILTV numbers and microscopic lesions in trachea were not found to be reliable indicators of virulence since they are not necessarily correlated with mortality rate in ILT.     

Chlamydia trachomatis IncA Is Localized to the Inclusion Membrane and Is Recognized by Antisera from Infected Humans and Primates

Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.     

Molecular characterization of the VP4, VP6, VP7, and NSP4 genes of lapine rotaviruses identified in italy: emergence of a novel VP4 genotype

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].     

Sequence analysis of the VP7 gene of human rotaviruses G2 and G4 isolated in Japan,China, Thailand,and Vietnam during 2001–2003

Sequence and phylogenetic analyses of the rotavirus VP7 gene were performed on 52 human G2 and G4 strains isolated in Japan, China, Thailand, and Vietnam during 2001–2003. All genotype G2 strains included in the study clustered into lineage II of the phylogenetic tree, together with the majority of global G2 strains detected since 1995. The amino acid substitution at position 96 from aspartic acid to asparagine was noted among the emerging or re‐emerging G2 rotavirus strains in Japan, Thailand, and Vietnam during 2002–2003. Genotype G4 strains detected in Vietnam grouped into lineage Ia of the phylogenetic tree, whereas Japanese G4 strains clustered in lineage Ic which included emerging G4 strains from Argentina, Italy, Paraguay, and Uruguay. It is noteworthy that an insertion of asparagine was found at position 76 in all the Japanese strains and that its presence might be involved in the emergence of G4 rotavirus in Japan during 2002–2003. J. Med. Virol. 82: 878–885, 2010. © 2010 Wiley‐Liss, Inc.     

Properties of strains of Staphylococcus aureus in the 94, 96 complex.

Strains of Staphylococcus aureus that are lysed by typing phages 94 or 96, or by both phages, are usually resistant to lysis by other basic-set typing phages. They are, however, sensitive to several experimental phages and show a number of different lytic patterns when tested against these phages. These differences in susceptibility are due, in part, to immunity imposed by temperate phages carried by the different strains. Resistance to lysis by other basic-set phages was not due to prophage immunity, but to at least one restriction and modification system in such strains. Restrictionless mutants were isolated from one strain in several experiments. These showed an increased sensitivity to many basic-set phages. However, all of these mutants retained the ability to modify the phages to the characteristic "94, 96" specificity. Strains in the 94, 96 complex showed a remarkable homogeneity in biological traits. The majority were non-pigmented, and produced lipase, fibrinolysin, alpha and delta haemolysins and enterotoxin B. This homogeneity may well be a reflexion of the restriction and modification systems in these strains, that prevent the acquisition of genetic material from strains outside the complex. A new lytic group V is proposed for members of the 94, 96 complex.     

武汉市新分离2株乙型脑炎病毒E基因分型及序列分析

目的 对2009年武汉市新分离的2株乙型脑炎（简称乙脑）病毒进行基因分型和序列分析,了解本地乙脑病毒株的分子生物学特性。方法 将2009年从三带喙库蚊中分离的两株乙型脑炎病毒用RT-PCR法扩增E基因,将其进行测序,并用DNAstar and MegAlign软件与其他基因型代表株进行比对。结果 16组样品检出两株阳性（WHJX9-09、WHJX10-09）,这两株阳性均属于GI型。两株新分离JEV之间的核苷酸和氨基酸同源性分别为98.9％和100％。同目前在武汉市使用的疫苗株SA-14-14-2相比,核苷酸同源性分别为87.4％、87.9％,氨基酸同源性为96.9％。共有15个氨基酸发生变异分布在3个不同结构域,中和位点没有变异但是神经毒力位点仍然存在。结论 武汉市本地新分离乙脑病毒基因型为GI型,不同于1988年在武汉检出的GⅢ型的基因型,和疫苗株SA-14-14-2相比,其神经毒力并没有减弱,但疫苗产生的抗体对新出现的GI型乙脑病毒仍有中和作用。因此提高乙脑疫苗的接种率并配合防蚊灭蚊措施对控制乙脑疫情依然至关重要。同时有必要对本市蚊虫及乙脑患者进行长期的病原学监测工作,为乙脑预测预警体系的建立提供科学依据。     

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.     

Genetic variability and prevalence of Bartonella henselae in cats in Berlin, Germany, and analysis of its genetic relatedness to a strain from Berlin that is pathogenic for humans

Nineteen Bartonella henselae strains and one Bartonella clarridgeiae strain were isolated from blood samples of 97 pet cats and 96 stray cats from Berlin, Germany, indicating prevalence rates of 1 and 18.7%, respectively, for B. henselae and 0 and 1%, respectively, for B. clarridgeiae. Eighteen of 19 B. henselae isolates corresponded to 16S rRNA type II. Pulsed-field gel electrophoresis (PFGE) analysis revealed seven different PFGE types among the feline B. henselae strains. Interestingly, all feline isolates displayed low genetic relatedness to B. henselae strain Berlin-1, which is pathogenic for humans.     

Detection of beta-lactamases in the Bacteroides fragilis group

One hundred and thirty three strains of Bacteroides fragilis group isolated from clinical specimen were tested for beta-lactamase activity against five beta-lactam antibiotics, ampicillin, cefaloridin, cefuroxime, cefotaxime and cefoxitin. The Minimal Inhibitory Concentration of antibiotics was determined using the agar dilution method. Beta-lactamase production was detected by a microbiological method in 128 of the 133 (96%) strains. The detected beta-lactamases had a broad-spectrum activity, hydrolyzing both penicillins and cephalosporins (101 strains). Some strains had a wide activity against beta-lactam antibiotics, including cefoxitin (7 strains); among these strains, 3 were found hydrolyzing imipenem.     

Presence of F-like OriT base-pair sequence on the virulence plasmids of Salmonella serovars Gallinarum,Enteritidis, and Typhimurium,but absent in those of Choleraesuis and Dublin

The 90 kb virulence plasmids of Salmonella biosers Gallinarum and Pullorum (expressed as serovar Gallinarum) are non-conjugative. They were, however, found to be readily mobilized by IncFI and IncFIV plasmids, but not by conjugative IncA, IncB = C, IncH, IncL and IncM R plasmids. No virulence plasmids of serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium were mobilized by any of these conjugative plasmids. The 90 kb virulence plasmid of Gallinarum was shown to hybridize to the Tra genes region of IncFI and IncFIV plasmids, suggesting it contains some F Tra genes region. It also hybridized with 75% homology to the virulence plasmid of Typhimurium, and, in decreasing order, to those of Dublin, Enteritidis, and Choleraesuis. A probe made of 319 basepairs of the F OriT region hybridized with approximately 45% homology to the virulence plasmid of Gallinarum, 63% to that of Typhimurium and 50% to that of Enteritidis. The probe, however, failed to hybridize to the plasmid DNAs of IncA, IncB = C, IncH, IncL and IncM, and to the virulence plasmids of Choleraesuis and Dublin.     

Pap,papG and prsG DNA sequences in Escherichia coli from the fecal flora and the urinary tract

The pap gene clusters encode P fimbriae and fimbriae-associated G adhesins. DNA sequence analysis has resolved three G adhesin variants (papG_J96, papG_IA2 and prs G_J96) that differ in receptor specificity and therefore in binding to epithelial cells. In this study, DNA probes specific for the pap gene cluster or the papG_J96, pap G_IA2 and prsG_J96 adhesin sequences were used to examine 74 fecal and 204 urinary Escherichia coli isolates (67 from acute pyelonephritis, 71 from acute cystitis and 66 from asymptomatic bacteriuria). In accordance with previous studies, a higher frequency of pap⁺ strains was found in the urinary strains (71%) than in the fecal (20%) E. coli isolates. The papG_IA2, and prs G_J96 sequences were more frequent among urinary (42% pap G⁺_IA2, 23% prs G⁺_J96) than among fecal (18% pap G⁺_IA2, 5% prs G⁺_J96) isolates. None of the isolates hybridized with the papG_J96 probe. Pap⁺ strains accounted for 82% of the pyelonephritis, 69% of the cystitis and 61% of the asymptomatic bacteriuria strains. The papG_IA2 genotype dominated in acute pyelonephritis strains (72% pap G⁺_IA2, 16% prs G⁺_J96). The prsG_J96 genotype was most frequent in cystitis strains (25% pap G⁺_IA2, 37% prs G⁺_J96). The asymptomatic bacteriuria strains formed an intermediate group (30% papG⁺_IA2, 14% prs G⁺_J96). Most of the pap G⁺_IA2 strains expressed P fimbriae which agglutinated human erythrocytes, sheep erythrocytes and Galα1-4Galβ latex beads. The prsG⁺_J96 strains varied in agglutination of human and sheep erythrocytes and Galα1-4Galβ-latex beads. The results demonstrated that the papG_IA2 and prs G_J96 adhesin DNA sequences differ in disease association.     

The phylogenetic position of Chlamydia strains isolated from monkeys and humans with Chlamydial pathology in the family Chiamydiaceae. Genotypic and phenotypic properties of this pathogen

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S - 23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the GenBank electronic database has been demonstrated. Differences in the 16S - 23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and non-plasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B- B, Ba, D, Da, E, L1, L2, L2a; intermediate group - F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life and development cycle disorder and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration.     

2011年北京市5岁以下婴幼儿A组轮状病毒腹泻监测

目的了解北京市5岁以下婴幼儿轮状病毒腹泻患者的流行特点。方法2011年1月至12月在北京市3家医院肠道门诊收集输液两次以上5岁以下腹泻患者病例604例,采集粪便标本,应用酶联免疫吸附试验检测轮状病毒抗原,阳性标本用半巢式反转录PCR进行基因分型。结果604例标本中96例轮状病毒抗原阳性,阳性率为15．89％。秋冬季为A组轮状病毒腹泻的发病高峰,其中11月份A组轮状病毒阳性率最高（55．56％,30／54）。轮状病毒腹泻主要发生于3—23月龄患者（93．76％,90／96）。对轮状病毒抗原阳性标本进行G／P分型,G分型以G3＋G9型混合感染占第一位（37．50％,36／96）,其它型别主要有G3型（28．12％,27／96）,G1型（11．46％,11／96）,G9型（9．38％,9／96）,G2型（8．33％,8／96）。P分型以P8型为主要优势株（86．46％,83／604）,其次为P4型（10．42％,10／96）。最常见的G／P基因型组合为G3＋G9／[P8]（36．46％,35／96）,其次为G3／[P8]（26．04％,25／96）,GI／[P8]（11．46％,11／96）,G9／[P8]（9．38％,9／96）,C2／[P4]（8．33％,8／96）。结论A组轮状病毒是北京市婴幼儿腹泻的主要病原之一,2011年主要流行株基因型组合为G3＋G9／P[8]。     

High diversity of Panton–Valentine leukocidin-positive, methicillin-susceptible isolates of Staphylococcus aureus and implications for the evolution of community-associated methicillin-resistant S. aureus

In total, 100 Staphylococcus aureus isolates from diverse cases of skin and soft-tissue infection at a university hospital in Saxony, Germany, were characterised using diagnostic microarrays. Virulence factors, including Panton-Valentine leukocidin (PVL), were detected and the isolates were assigned to clonal groups. Thirty isolates were positive for the genes encoding PVL. Only three PVL-positive methicillin-resistant S. aureus (MRSA) isolates were found, two of which belonged to European clone ST80-MRSA IV and one to USA300 strain ST8-MRSA IV. The remaining methicillin-susceptible PVL-positive isolates belonged to a variety of different multilocus sequence types. The predominant strains were agrI/ST22, agrII/CC5, agrIII/CC30 and agrIV/ST121. In order to check for possible bias caused by regional or local outbreak strains, an additional 18 methicillin-susceptible, PVL-positive isolates from the UK were tested. Approximately two-thirds of the UK isolates belonged to types that also comprised approximately two-thirds of the isolates from Saxony. Some methicillin-susceptible PVL-positive isolates (agrI/ST152, agrIII/ST80 and agrIII/ST96) closely resembled known epidemic community-acquired MRSA (CaMRSA) strains. These findings indicate that the current rise in PVL-positive CaMRSA could be caused by the dissemination of novel SCCmec elements among pre-existing PVL-positive strains, rather than by the spread of PVL phages among MRSA strains.