High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis

Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology.     

Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes

An outbreak of lymphogranuloma venereum (LGV) infections has recently been reported from The Netherlands and other European countries. The Swedish surveillance system has identified three LGV cases since 2004, all with clinically suspected infection in men who have sex with men (MSM). In order to assess the prevalence of LGV in a high-risk group of MSM and include clinically atypical cases, retrospective analysis of 197 Chlamydia trachomatis-infected men was performed. Sequencing of the ompA gene showed a different serotype distribution compared to recent Swedish studies in heterosexual populations. The most common types were G (45%), D (27%), and J (26%), whereas the normally predominant type E accounted for only 4% of the chlamydia cases. Furthermore, certain ompA genotype variants of the dominant serotypes were highly prevalent among MSM, and the reason for this is discussed. No additional case of LGV was detected by retrospective analysis of the high-risk MSM population. This indicates that, thus far, LGV in Sweden is only a result of sporadic import from infected MSM clusters abroad.     

Serotype 14 variants of the France 9V(-3) clone from Baltimore, Maryland, can be differentiated by the cpsB gene

European serotype 14 variants of the France 9V(-3) clone, which have arisen through recombination events involving the penicillin binding protein 1a (pbp1a) gene, have cpsB sequences distinct from those of the 9V(-3) clone. Serotype 14 variants of the 9V(-3) clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible Streptococcus pneumoniae strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), pbp1a PCR restriction profiles, and cpsB and pbp1a sequences. The cpsB genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V(-3) clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V(-3) clone nor to the R6 strain from positions 1498 to 1710 of the pbp1a gene. All serotype 14 strains had cpsB sequences that differed by or=16% (78 to 83 of 476 bp) divergent from that of the 9V(-3) clone. Allowing for a 2-bp difference in the cpsB sequence resulted in the highest correlation between the cpsB gene and serotype. Overall, 95% (84 of 88) of the strains were classified correctly by serotype with the cpsB sequence. The distal recombination site of the Baltimore serotype 14 variants of the 9V(-3) clone was not identical to that of the European serotype 14 variants. The cpsB gene was serotype specific regardless of whether capsular switching occurred. Although the correlation between serotype and the cpsB sequence was high, the overall diversity of the cpsB gene within a serotype likely will limit the role of this gene in a sequence-based serotyping method.     

青海同德藏族人群感染HBV基因型、血清型调查

目的 了解青海同德藏族人群乙型肝炎病毒(HBV)基因型、血清型的分布状况.方法 用巢式PCR扩增青海同德藏族人群表面抗原(HBsAg)阳性者的HBV S、C基因,测定其序列,判定其基因型和血清型.结果 源于自然人群的311份血清标本中,同时获得S和C基因序列者271份,其中C型基因10例,占3.7%,C/D重组型261例,占96.3%;血清ayw2亚型259例,占95.6%,adr亚型10例,占3.7%,adw2亚型2例,占0.7%.结论 青海同德藏族人群感染HBV以C/D重组型为主,血清型以ayw2为主.     

Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <10(2) CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.     

Invasive neonatal GBS infections from an area-based surveillance study in Italy

During an area-based study, 75 group B streptococcus (GBS) strains isolated both from early-onset disease (EOD, 37 strains) and from late-onset disease (LOD, 38 strains) were analysed for serotype, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing profiles, protein markers and antibiotic resistance. Serotype III, possessing the rib gene, was the most frequent (54 strains, 72%) and responsible for 89.5% and 54% of LOD and EOD, respectively. Forty-six serotype III strains belonged to the same PFGE type and clonal complex 17, already described as an over-represented clone in neonatal invasive GBS infections. Other serotypes were Ia (9.3%), II (6.7%), Ib (5.3%), V (5.3%) and IV (1.3%). Seventeen PFGE groups were identified comprising strains with related sequence types; conversely, strains displaying the same sequence type could belong to different PFGE groups. When both neonate and maternal strains from vaginorectal swabs and/or milk were available (eight cases), they were indistinguishable. Resistance to erythromycin (12%) was associated with a constitutive resistance to clindamycin in five cases (four carrying the erm(B) gene and one both the erm(B) and mef(E) genes) and with an inducible clindamycin resistance in two cases (one possessing the erm(A) gene, the other the erm(T) gene). Two isolates displayed the M phenotype (mef(E) gene). All strains but five were resistant to tetracycline, mostly mediated by the tet(M) gene (97.1%). The study underlined the importance of an active surveillance system for the elucidation of a GBS population structure causing neonatal infections and allowed the detection of rare antibiotic resistance determinants [erm(T)].     

Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.     

Genetic variation of NSP1 and NSP4 genes among serotype G9 rotaviruses causing hospitalization of children in Melbourne, Australia, 1997-2002

Serotype G9 rotaviruses have emerged as one of the leading causes of gastroenteritis in children worldwide. We examined 29 representative G9 rotavirus isolates from a 6-year collection (1997-2002) and determined the level of variation in genes encoding non-structural proteins, NSP1 and NSP4. Northern hybridization analysis with a whole genome probe derived from the prototype G9 strain, F45, revealed that the NSP1 gene (gene 5) of two isolates (R1 and R14) did not exhibit significant homology. Complementary DNA probes of R1 and R14 genes 5 were used in Northern blot hybridization and indicated the presence of at least two gene 5 alleles among Melbourne G9 rotaviruses. Nucleotide sequence analysis revealed that isolates carrying the R14 gene 5 shared 94-98% sequence identities with one another, while sequence identity to R1 was 78%. Surprisingly, R1 displayed 96% nucleotide identity with the prototype serotype G1 strain, Wa. The detection of different alleles of NSP1 genes prompted us to investigate the level of variation in another non-structural protein, NSP4, a multifunctional protein and the first viral-encoded enterotoxin. Phylogenetic analysis indicated that while all isolates clustered into one group containing the Wa NSP4 allele (genotype 1), isolate R1 was most closely related to Wa. This study reveals new information about the diversity of non-structural proteins of G9 rotaviruses.     

Genomic characterization of rotavirus strains obtained from hospitalized children with diarrhoea in Bangladesh

Faecal samples were obtained from 111 children hospitalized with acute watery gastroenteritis in a children's hospital in Bangladesh from January to December, 1989. Rotavirus was detected as the aetiologic agent in 35 (32%) of these patients. The electrophoretic pattern of ds-RNA extracted from the rotaviruses showed 11 different migration patterns (six short and five long), as well as some short and long mixed profiles. All four major G serotypes were identified by amplification of a segment of the gene for VP7 using the polymerase chain reaction. A specific serotype could be assigned in 32 (91%) cases. Serotypes G1-G4 accounted for 11%, 37%, 11%, and 23% of isolates respectively. Three (9%) mixed serotypes were identified by this method and were found to be mixtures of serotypes G1 and G4, G2 and G4, and G3 and G4. © 1994 Wiley-Liss, Inc.     

Dominance of serotype Ia among group B Streptococci causing invasive infections in nonpregnant adults in Portugal

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.     

Analysis of Trichosporon isolates obtained from the houses of patients with summer-type hypersensitivity pneumonitis

Summer-type hypersensitivity pneumonitis (SHP) is type III or IV allergies developed by repeated inhalation of arthroconidia of Trichosporon species. We identified 105 strains obtained from the homes of 36 SHP patients by analysis of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes; in addition, we analyzed the IGS genotypes of the strains. Serologically, Trichosporon species are classified as serotype I, II, III, or I-III. Of the 105 strains, 43 (41.1%), 53 (50.5%), and 9 (8.6%) strains were isolated as serotypes I, II, and III, respectively. Serotype I, II, and III strains were recovered from 19 (52.8%), 29 (80.6%), and 4 (11.1%) of the 36 houses of SHP patients, respectively. No serotype I-III strains were isolated from the houses. Of 43 serotype I strains, 42 (97.7%) were identified as Trichosporon dermatis, and the remaining one was T. terricola. Of 53 serotype II strains, 37 (69.8%) were identified as T. asahii, and the remaining serotype II isolates were T. aquatile (1.9%), T. coremiiforme (7.5%), T. faecale (1.9%), T. japonicum (15.1%), and T. ovoides (3.8%). There were nine serotype III strains comprised of T. montevideense (77.8%) and T. domesticum (22.2%). Intraspecies diversity was found only in T. asahii. This microorganism also causes opportunistic infections (trichosporonosis); seven genotypes of its IGS 1 region have been identified. While the strains of T. asahii obtained from Japanese patients with trichosporonosis were genotype I, the strains from the houses of SHP patients were genotype III. Based on our analysis, we conclude that the strains that play the most significant roles in the development of SHP are T. dermatis, T. asahii genotype 3, and T. montevideense, representing serotypes I, II, and III, respectively.     

Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus.

The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.     

乙肝疫苗长期免疫地区低龄人群乙型肝炎病毒基因序列与突变现状

目的 通过乙型肝炎流行病学调查和病毒基因检测,了解乙肝疫苗长期免疫地区低年龄人群的基因序列与突变特征.方法 从乙肝监测点收集乙型肝炎病毒表面抗原阳性血清,取其中年龄小于16岁者,扩增包括preS和S基因在内的基因序列片段,共1100碱基,序列测定后与标准基因型别比较,确定病毒基因及血清型别,确定a抗原决定簇的氨基酸替代突变发生率,选取一株病毒进行全基因扩增、克隆和序列测定.结果 共检测样本35例,33例血清扩增出乙肝病毒基因序列,其中30例乙型肝炎病毒基因型为B型,占90.9%,3例乙型肝炎病毒基因型为C型,占9.0%,1例血清型为ayw,3例为adr,其余29例为adw,共有5例在a抗原决定簇发生氨基酸替代突变,发生率约为15.2%.其中5856号血清扩增乙型肝炎病毒全基因为B型,血清型为adw,共3215个碱基.结论 该地区乙型肝炎基因型主要为B型,血清型为adw,人群中a抗原决定簇某些位点已发生与疫苗免疫逃逸相关的突变.     

Antigenic and molecular characterization of isolates of the Italy 02 infectious bronchitis virus genotype.

As part of an epidemiological surveillance of infectious bronchitis virus (IBV) in Spain, four Spanish field isolates showed high S1 spike sequence similarities with an IBV sequence from the GenBank database named Italy 02. Given that little was known about this new emergent IBV strain we have characterized the four isolates by sequencing the entire S1 part of the spike protein gene and have compared them with many reference IBV serotypes. In addition, cross-virus neutralization assays were conducted with the main IBV serotypes present in Europe. The four Spanish field strains and the Italy 02 S1 sequence from the NCBI database were established as a new genotype that showed maximum amino acid identities with the 4/91 serotype (81.7% to 83.7%), the D274 group that included D207, D274 and D3896 strains (79.8% to 81.7%), and the B1648 serotype (79.3% to 80%). Furthermore, on the basis of these results, it was demonstrated that the Italy 02 genotype had been circulating in Spain since as early as 1997. Based on the average ratio of synonymous:non-synonymous (dS/dN) amino acid substitutions within Italy 02 sequences, no positive selection pressures were related with changes observed in the S1 gene. Moreover, phylogenetic analysis of the S1 gene suggested that the Italy 02 genotype has undergone a recombination event. Virus neutralization assays demonstrated that little antigenic relatedness (less than 35%) exists between Italy 02 and some of the reference IBV serotypes, and indicated that Italy 02 is likely to be a new serotype.     

Serotype 3 is a common serotype causing invasive pneumococcal disease in children less than 5?years old, as identified by real-time PCR

Serotype 3 is one of the most often detected pneumococcal serotypes in adults and it is associated with serious disease. In contrast, the isolation of serotype 3 by bacterial culture is unusual in children with invasive pneumococcal disease (IPD). The purpose of this study was to learn the serotype distribution of IPD, including culture-negative episodes, by using molecular methods in normal sterile samples. We studied all children<5 years of age with IPD admitted to two paediatric hospitals in Catalonia, Spain, from 2007 to 2009. A sequential real-time polymerase chain reaction (PCR) approach was added to routine methods for the detection and serotyping of pneumococcal infection. Among 257 episodes (219 pneumonia, 27 meningitis, six bacteraemia and five others), 33.5% were identified by culture and the rest, 66.5%, were detected exclusively by real-time PCR. The most common serotypes detected by culture were serotypes 1 (26.7%) and 19A (25.6%), and by real-time PCR, serotypes 1 (19.8%) and 3 (18.1%). Theoretical coverage rates by the PCV7, PCV10 and PCV13 vaccines were 10.5, 52.3 and 87.2%, respectively, for those episodes identified by culture, compared to 5.3, 31.6 and 60.2% for those identified only by real-time PCR. Multiplex real-time PCR has been shown to be useful for surveillance studies of IPD. Serotype 3 is underdiagnosed by culture and is important in paediatric IPD.     

Comparison of the roles of calcineurin in physiology and virulence in serotype D and serotype A strains of Cryptococcus neoformans

The calcineurin gene was cloned and disrupted in serotype D strains of Cryptococcus neoformans. Serotype A and serotype D calcineurin mutants were inviable at 37 degrees C and avirulent in mice, whereas only serotype A mutants were cation stress sensitive. Thus, calcineurin plays conserved and divergent roles in serotype A and serotype D strains.     

Alterations of the SDHD gene locus in midgut carcinoids,Merkel cell carcinomas,pheochromocytomas, and abdominal paragangliomas

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor-suppressor gene SDHD (succinate-ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas.     

Analysis of group B streptococcal isolates from infants and pregnant women in Portugal revealing two lineages with enhanced invasiveness

The populations of group B streptococcus (GBS) associated with vaginal carriage in pregnant women and invasive neonatal infections in Portugal were compared. GBS isolates were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) profiling, and multilocus sequence typing (MLST). Serotypes III and V accounted for 44% of all colonization isolates (n = 269), whereas serotypes III and Ia amounted to 69% of all invasive isolates (n = 64). Whereas serotype Ia was associated with early-onset disease (EOD), serotype III was associated with late-onset disease (LOD). Characterization by PFGE and MLST identified very diverse populations in carriage and invasive disease. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and the infrequent ST24. In contrast, serotype III was found in a large number of PFGE clusters and STs, but a single PFGE cluster defined by ST17 was found to be associated with invasive disease. Although serotype III was associated only with LOD, ST17 showed an enhanced capacity to cause both EOD and LOD. Our data reinforce the evidence for enhanced invasiveness of ST17 and identify a lineage expressing serotype Ia capsule and represented by ST23 and ST24 as having enhanced potential to cause EOD.