Sequence Type Complex Research Articles

Genetic Diversity and Virulence Profiles of Escherichia coli Isolates Causing Spontaneous Bacterial Peritonitis and Bacteremia in Patients with Cirrhosis

Among patients with cirrhosis, infections caused by Escherichia coli organisms that translocate from the gut are a frequent and severe complication. One hundred ten E. coli isolates from 110 cirrhotic patients with spontaneous bacterial peritonitis and/or spontaneous bacteremia were characterized for their phylogenetic group and virulence genotype (34 extraintestinal virulence factor genes). Genetic relatedness was investigated by enterobacterial repetitive intergenic consensus sequence type 2 (ERIC-2) PCR typing and multilocus sequence typing. Phylogenetic groups A, B1, B2, and D accounted for 24%, 4%, 48%, and 24% of the population, respectively. Overall, 68 distinct ERIC-2 profiles were encountered. Eleven clonal groups, represented by multiple isolates (2 to 11) from the same sequence type (ST) or sequence type complex, were identified. These clonal groups accounted for 54 (49%) isolates overall. Membership in one of these clonal groups was more frequent among B2 isolates than non-B2 isolates (67% versus 32%, P < 0.001). The most frequent sequence types were ST95 (n = 13) and ST73 (n = 8), followed by the ST14 and ST10 complexes (n = 7). ST131 and ST69 were represented by three isolates each. Clonal group-associated isolates exhibited a greater prevalence of 11 virulence genes, including pap elements, than the other isolates. However, no association between clonal groups and host factors, type of infection, or mortality was observed. In conclusion, E. coli isolates causing spontaneous bacterial peritonitis and bacteremia in cirrhotic patients are genetically diverse. However, approximately half of the isolates belong to familiar clonal groups and exhibit extensive virulence profiles that may be associated with greater invasive potential.

The genetic structure of Neisseria meningitidis populations in Cuba before and after the introduction of a serogroup BC vaccine

We investigated the population genetics in collections of meningococci sampled in Cuba during the period 1983–2005, thereby covering a period before and after the introduction of an antimeningococcal B–C vaccine. A total of 163 case isolates and 210 isolates from healthy carriers were characterized by multilocus sequence typing (MLST) and sequence determination of porA, porB and fetA genes. A total of 56 sequence types (STs) including 28 new STs were identified among these isolates. The analysis of surface antigens revealed variants 3-1 and 3-8 to be prevalent for porB; variant F5-1 was the most common FetA epitope, and variants 19 and 15 corresponded to the prevalent variable regions 1 (VR1) and VR2 PorA epitopes, respectively. The strongest associations between specific surface protein variants and clonal complexes were detected in lineages ST-32 and ST-53. All ST-32 complex isolates possessed porB3 alleles, and the most frequent antigen combination among ST-32 complex isolates was P1.19,15;F5-1. Variants PorB3-64 at PorB and P1.30 at PorA VR2, in combination with the PorA VR1 variants P1.12-1, P1.7 and P1.7-2 as well as the FetA variants F1-2 and F1-7, dominated the ST-53 complex organisms. Furthermore, we observed a statistically significant association between the most frequent porA, porB and fetA alleles and strain invasiveness. Finally, this study showed that the application of VA-MENGOC-BC®, the Cuban antimeningococcal vaccine, reduced the number and frequency of the hypervirulent Clonal Complexes ST-32 and ST-41/44, and also impacted on other lineages. The vaccine also affected the genetic composition of the carrier-associated meningococcal isolates. The number of carrier isolates belonging to hypervirulent lineages decreased significantly after vaccination, and ST-53, a sequence type common in carriers, became the predominant ST.

Occurrence of ST23 Complex Phylogroup A Escherichia coli Isolates Producing Extended-Spectrum AmpC β-Lactamase in a French Hospital

Extended-spectrum AmpC beta-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.

Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway

Clinical isolates of Escherichia coli with reduced susceptibility to oxyimino-cephalosporins and not susceptible to clavulanic acid synergy (n = 402), collected from Norwegian diagnostic laboratories in 2003–2007, were examined for the presence of plasmid-mediated AmpC β-lactamases (PABLs). Antimicrobial susceptibility testing was performed for β-lactam and non-β-lactam antibiotics using Etest and Vitek2, respectively. The AmpC phenotype was confirmed using the boronic acid test. PABL-producing isolates were detected using ampC multiplex-PCR and examined by blaAmpC sequencing, characterization of the blaAmpC genetic environment, phylogenetic grouping, Xbal- pulsed-field gel electrophoresis (PFGE), multi-locus sequence typed (MLST), plasmid profiling and PCR-based replicon typing. For the PABL-positive isolates (n = 38), carrying blaCMY-2 (n = 35), blaCMY-7 (n = 1) and blaDHA-1 (n = 2), from out- (n = 23) and in-patients (n = 15), moderate-high MICs of β-lactams, except cefepime and carbapenems, were determined. All isolates were resistant to trimethoprim-sulphamethoxazole. Multidrug resistance was detected in 58% of the isolates. The genes blaCMY-2 and blaCMY-7 were linked to ISEcp1 upstream in 32 cases and in one case, respectively, and blaDHA-1 was linked to qacEΔ1-sull upstream and downstream in one case. Twenty isolates were of phylogenetic groups B2 or D. Thirty-three XbaI-PFGE types, including three clusters, were observed. Twenty-five sequence types (ST) were identified, of which ST complexes (STC) 38 (n = 7), STC 448 (n = 5) and ST131 (n = 4) were dominant. Plasmid profiling revealed 1–4 plasmids (50–250 kb) per isolate and 11 different replicons in 37/38 isolates; blaCMY-2 was carried on transferable multiple-replicon plasmids, predominantly of Inc groups I1 (n = 12), FII (n = 10) and A/C (n = 7). Chromosomal integration was observed for blaCMY-2 in ten strains. CMY-2 is the dominant PABL type in Norway and is associated with ISEcp1 and transferable, multiple-replicon IncI1, IncA/C, or IncFII plasmids in nationwide strains of STC 448, STC 38 and ST131.

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.

Genetic and virulence-phenotype characterization of serotypes 2 and 9 of Streptococcus suis swine isolates.

The aim of this study was to analyze the genetic characteristics and virulence phenotypes of Streptococcus suis, specifically, in clinical isolates of serotypes 2 and 9 (n = 195), obtained from diverse geographical areas across Spain. Pulsed-field gel electrophoresis (PFGE) typing identified 97 genetic profiles, 68% of which were represented by single isolates, indicative of a substantial genetic diversity among the S. suis isolates analyzed. Five PFGE profiles accounted for 33.3% of the isolates and were isolated from 38% of the herds in nine different provinces, indicative of the bacterium's widespread distribution in the Spanish swine population. Representative isolates of the most prevalent PFGE profiles of both serotypes were subjected to multilocus sequence typing (MLST) analysis. The results indicated that serotypes 2 and 9 have distinct genetic backgrounds. Serotype 2 isolates belong to the ST1 complex, a highly successful clone that has spread over most European countries. In accordance with isolates of this complex, most serotype 2 isolates also expressed the phenotype MRP(+)EF(+)SLY(+). Serotype 9 isolates belong to the ST61 complex, which is distantly related to the widespread European ST87 clone. Also, in contrast to most isolates of the European ST87 clone, which express the large variant MRP*, the majority of serotype 9 isolates (97.9%) did not express the protein.

Extended-spectrum β-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2

In this study, we investigated the population structure of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Spain and determined possible associations between specific multilocus sequence typing (MLST) types and ESBL types. Ninety-two ESBL-producing E. coli isolates from 11 Spanish hospitals were studied. The predominant ESBLs in this collection were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%). Phylogenetic groups and MLST types were studied. Thirty-seven isolates (40.2%) belonged to phylogroup A, 26 (28.3%) to group B1, 13 (14.1%) to group B2 and 16 (17.4%) to group D. Fifty-six sequence types (STs) were identified, but ST131 (eight isolates) and ST167 (five isolates) were the most prevalent. The most common ST complexes were ST10 (13 isolates; 14.3%) and ST23 (10 isolates; 11%). Escherichia coli ST131 carried six different ESBLs (CTX-M-1, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15 and SHV-12), E. coli ST10 complex carried five ESBLs and E. coli ST23 complex carried four ESBLs. A great diversity of MLST types was observed among Spanish ESBL-producing E. coli isolates.

Semi-automated rep-PCR for rapid differentiation of major clonal groups of Escherichia coli meningitis strains

DiversiLab, a semi-automated repetitive-sequence-based PCR (rep-PCR) device, is a highly integrated platform designed for rapid bacterial genotyping. Here, we evaluated the capacity of the DiversiLab system to determine the genetic relatedness of Escherichia coli neonatal meningitis (ECNM) strains and to identify clonal groups. We analyzed 80 isolates representative of the diversity of ECNM strains in Europe and North America and 52 E. coli reference (ECOR) strains belonging to phylogenetic groups A, D, and B2. All the strains had previously been characterized by means of multilocus sequence typing (MLST). The DiversiLab dendrogram clustered all but 8 of the strains according to their phylogenetic groups. After defining a rep-PCR type complex (RPTc) based on an average similarity threshold of 95% between rep-PCR types, we observed excellent agreement between RPTc and sequence type complexes (STc) in groups D and B2. In group A, rep-PCR typing was more discriminative than MLST, dividing the 25 ECOR group A strains into 19 RPTc, compared to only 10 STc. In the highly virulent clonal group B2 1, mainly composed of O1, O2, O18, and O45:K1 strains, the DiversiLab system individualized a particular subgroup of O2:K1 strains. In addition, among O18:K1 strains the system identified a particular genetic background associated with pathogenicity island II J96-like domains. Thus, the DiversiLab system is a rapid and powerful tool for identifying and discriminating clonal groups among ECNM strains.

Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis ( srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A–L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.

Streptococcus suisMeningitis without History of Animal Contact, Italy

<i>Streptococcus suis</i>Meningitis without History of Animal Contact, Italy

High prevalence of Neisseria meningitidis hypervirulent lineages and emergence of W135:P1.5,2:ST-11 clone in Southern Brazil

The aim of this study was to characterize Neisseria meningitidis strains causing invasive disease in Rio Grande do Sul (RS), during 2003-2005, monitoring the occurrence of hypervirulent lineages, as well as to determine the diversity of PorA VR types for the corresponding isolates and clinical specimens. Isolates and clinical specimens were characterized by MLST and PorA VR typing. This study demonstrated high prevalence of some hypervirulent lineages and emergence of new ones, including the emergence of lineages W135:P1.5,2:ST-11 complex, and C:P1.22,14-6:ST-103 complex. These lineages are probably responsible for the increasing incidence of serogroups C and W135, despite the overall decrease in serogroup B cases during the period. The most prevalent complex was serogroup B ST-32/ET-5 complex. The most prevalent PorA types found for serogroup B were P1.19,15, P1.7,16, and P1.18-1,3, representing a different distribution of PorA types compared to other states of Brazil. This study highlights the importance of monitoring each population, even within the same country. The different distribution of PorA VR types in RS has implications in vaccine design and efficacy. Detailed and accurate meningococcal characterization is an important element in studies of meningococcal epidemiology, population biology, and evolution and provides information for the design of control strategies.

Association between mortality of Escherichia coli meningitis in young infants and non-virulent clonal groups of strains

To identify factors associated with Escherichia coli meningitis (ECM) mortality in infants aged <3 months, the clinical, biological and bacterial characteristics of isolates from 99 cases of ECM were compared, including the phylogenetic group, multilocus sequence type, O serogroup and sequence O type (a combination of sequence type complex (STc) and O serogroup) and virulence genotype. All 99 isolates were susceptible to the initial antimicrobial treatment. The mortality rate (14%) was not influenced by term or post-natal age. Hypotension or seizures were the sole clinical predictive factors for fatal outcome (p <0.01), and abnormal initial trans-fontanellar ultrasound was associated with death (p 0.03). Seventy-seven isolates belonged to the common sequence O types (STc29(O1), STc29(O18), STc29(O45), STc301(O7), STc304(O16), STc697(O83), STc700(O1)) causing neonatal meningitis. None of the phylogenetic groups and none of the virulence determinants were distributed differently between survivors and non-survivors, except that the aerobactin gene (iucC) was less frequent in lethal isolates (94% vs. 71%, p 0.02). Isolates belonging to rare sequence O types were more likely to be lethal (OR 4.3, p 0.01), although they induced a lower level of bacteraemia than common sequence O types such as STc29(O18) and STc29(O45) in a neonatal rat model. These results suggest that unidentified human genetic risk-factors may be more important than strain virulence in predicting ECM mortality.

Genetic characteristics of serogroup A meningococci circulating in China, 1956–2005

Neisseria meningitidis serogroup A accounted for 95% of cases of meningococcal disease in China during the last century. To understand the circulation of these organisms in China over a 50-year period, 275 serogroup A meningococcal isolates collected between 1956 and 2005 were characterised by multilocus sequence typing (MLST) and PorA typing. In total, 44 sequence types (STs), belonging to five hyperinvasive lineages, and ten singletons were identified in this collection. The ST-5 complex and the ST-1 complex represented 52.8% (86/163) and 44.2% (72/163), respectively, of isolates from cases of infection and, overall, 93.1% (256/275) of all isolates. Three prevalent clones (ST-5, P1.5-2,10; ST-3, P1.7-1,10; and ST-5, P1.20,9) were involved in four national epidemics in 1959, 1967, 1977 and 1984. ST-5 was replaced by ST-7 in the late 1980s, such that ST-7 isolates with P1.20,9 represented >86% of isolates from cases of infection after 2000. The data also revealed that the collection contained 19 PorA VR types, of which P1.7-1,10 and P1.20,9 were the predominant types in the ST-1 and ST-5 common lineages, respectively. Three other hyperinvasive lineages (ST-11 complex, ST-32 complex and ST-4821 complex) were isolated only from carriers. It was concluded that serogroup A meningococci of the ST-5 complex and the ST-1 complex were responsible for most cases of meningococcal disease in China during the past 50 years.

Allelic variation and prevalence of serum opacity factor among the Streptococcus suis population

Serum opacity factor of Streptococcus suis (OFS) has recently been identified as a virulence determinant of an S. suis strain. In this study, we investigated the prevalence and variations of the ofs gene among 108 S. suis isolates from diseased and healthy pigs, and human patients. PCR screening and sequencing analysis showed that besides the ofs gene reported already (designated type 1), there were three allelic variants of ofs (designated types 2 to 4). Type-1 and type-2 ofs genes were expected to encode functional OFS, and SDS extracts of the isolates with type-1 ofs and type-2 ofs opacified horse serum. Culture supernatants of the isolates with type-2 ofs also showed strong serum opacification activity. In contrast, type-3 ofs was interrupted by a point mutation and type-4 ofs was disrupted by either insertion of an IS element or genetic rearrangement, and therefore the SDS extracts and culture supernatants of the isolates with type-3 ofs and type-4 ofs did not show serum opacification activity. Regardless of their origins, approximately 30 % of the isolates possessed functional OFSs, although type-2 ofs was found only in three isolates from healthy pigs. Multilocus sequence typing analysis showed that most of the isolates with type-1 ofs belonged to the sequence type (ST)1 complex, and most of the isolates with type-3 ofs and type-4 ofs belonged to the ST27 complex. The isolates with type-2 ofs were not assigned to a major ST complex. These results suggest that type-1 OFS contributes to the virulence of a limited number of S. suis isolates, i.e. those of the ST1 complex type, whereas other S. suis may not possess this category of virulence factor; the importance of type-2 OFS is obscure.

Impact of Meningococcal Serogroup C Conjugate Vaccines on Carriage and Herd Immunity

In 1999, meningococcal serogroup C conjugate (MCC) vaccines were introduced in the United Kingdom for those under 19 years of age. The impact of this intervention on asymptomatic carriage of meningococci was investigated to establish whether serogroup replacement or protection by herd immunity occurred. Multicenter surveys of carriage were conducted during vaccine introduction and on 2 successive years, resulting in a total of 48,309 samples, from which 8599 meningococci were isolated and characterized by genotyping and phenotyping. A reduction in serogroup C carriage (rate ratio, 0.19) was observed that lasted at least 2 years with no evidence of serogroup replacement. Vaccine efficacy against carriage was 75%, and vaccination had a disproportionate impact on the carriage of sequence type (ST)-11 complex serogroup C meningococci that (rate ratio, 0.06); these meningococci also exhibited high rates of capsule expression. The impact of vaccination with MCC vaccine on the prevalence of carriage of group C meningococci was consistent with herd immunity. The high impact on the carriage of ST-11 complex serogroup C could be attributed to high levels of capsule expression. High vaccine efficacy against disease in young children, who were not protected long-term by the schedule initially used, is attributed to the high vaccine efficacy against carriage in older age groups.

In Humans, Thailand

To the Editor: Streptococcus suis is an important zoonotic pathogen for swine and humans. Among 33 serotypes, serotype 2 is more frequently isolated from diseased pigs than other serotypes (1). However, not all serotype 2 strains are virulent, and degree of virulence varies among strains (2). Previous studies have reported several S. suis putative virulence factors, including the polysaccharide capsule, the muramidase-released protein, the extracellular factor, and suilysin (3–5). Some of these factors have been used as virulence-associated markers, and the association of the factors of S. suis isolates with virulence or clinical background has been suggested in Europe (2,5). However, because many virulent isolates lacking these factors have also been isolated from clinical cases in Canada (6), they cannot be used as virulence markers in North America. Recent analysis of S. suis isolates by multilocus sequence typing (MLST) suggested the association of some clonal groups with particular clinical manifestations. That is, most invasive isolates belonged to the sequence type (ST) 1 complex, while the ST27 and ST87 complexes were found to include a higher proportion of lung isolates (7). Although S. suis has been prevalent worldwide, the geographic location of the isolates used so far was mainly Europe, North America, and East Asia (7–9). Moreover, the clonal association with virulence of S. suis has been discussed mainly on the basis of clinical and experimental data in swine (7). In this report, to broaden understanding of the population structure of S. suis as a zoonotic agent, we characterize 20 S. suis isolates (Table) recovered from humans in Thailand in 1998–2002. Table Epidemiologic data of Streptococcus suis isolates from patients in Thailand, 1998–2002* Serotyping by coagglutination tests showed that 19 of the 20 isolates belonged to serotype 2, while the remaining 1 (MNCM07) was serotype 14. MLST analysis resolved the 20 isolates into 8 STs (Table). By using eBURST (http://eburst.mlst.net), we assigned 4 isolates (MNCM01, MNCM06, MNCM07, and MNCM16) from 1 case of endocarditis and 3 cases of meningitis to the ST1 complex. The remaining isolates were assigned to the ST27 complex with a less-stringent group definition (Table), although ST101 (MNCM21) and ST104 (MNCM50) shared only 2 alleles with ST27 and were incorporated into this complex by a chaining effect. Regarding the clinical cases from which the ST27 complex isolates were recovered, the patients had meningitis, endocarditis, septicemia, septic shock, diarrhea, and respiratory involvement. The 2 ST complexes both contained isolates from deceased patients (Table). All the isolates assigned to the ST1 complex were positive for the suilysin gene sly, the extracellular factor gene epf or its variant, and the muramidase-released protein gene mrp or its variant. With the exception of MNCM21 and MNCM50, which had only sly, all isolates classified into the ST27 complex were negative for sly and epf but positive for mrp or its variant. These results showed the congruence between STs and the virulence-associated gene profiles and further support the usefulness of MLST for epidemiologic studies of S. suis. Of the 3 major clonal complexes identified so far in S. suis (ST1, ST27, and ST87), the ST1 complex particularly attracts considerable public attention as a clonal group that may have the potential for a higher degree of virulence than the others (7), and most (96%) of the human isolates investigated so far, including ST7 isolates, which caused the largest outbreak in China, belong to the ST1 complex (7–9). In this study, although no ST7 isolate was found, 4 isolates were assigned to the ST1 complex. This further confirmed the gravity of the ST1 complex not only for swine industries but also for public health. In contrast to the ST1 complex, only 4 human clinical isolates have so far been reported to belong to the ST27 complex. Three of the 4 are isolates from Canada that belong to ST25 (7). The remaining 1 is from Japan and assigned to ST28 (8). Unlike in previous reports, 80% of the human clinical isolates (16 isolates) characterized in this study were assigned to the ST27 complex. Although previous studies suggested that members of the ST27 complex may have lower potential to cause invasive diseases in swine (7), all the isolates were isolated from blood or cerebrospinal fluid of the patients, suggesting a high degree of invasiveness (Table). Because it is unknown whether the ST27 complex is also dominant among isolates from diseased pigs in Thailand, future surveillance will be necessary to know the situation in pigs. However, our data indicate that the ST27 complex is another clonal group that should be assessed for its importance for human infection. Because mrp, epf, and sly are not appropriate as virulence markers for the ST27 complex members, development of novel virulence markers will be needed for efficient discrimination of S. suis strains virulent for humans.

Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 complex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak.

Characterization of ST-4821 complex, a unique Neisseria meningitidis clone

Ten outbreaks of a new serogroup C meningococcal disease emerged during 2003–2005 in China. The multilocus sequence typing results indicated that unique sequence type 4821 clone meningococci were responsible for these outbreaks. Herein, we determined the entire genomic DNA sequence of serogroup C isolate 053442, which belongs to ST-4821. Comparison of 053442 gene contents with other meningococcal genomes shows that they have similar characteristics, including thousands of repetitive elements and simple sequence repeats, numerous phase-variable genes, and similar virulence-related factors. However, many strain-specific regions were found in each genome. We also present the results of a genomic comparison of 28 ST-4821 complex isolates that were isolated from different serogroups using comparative genomic hybridization analysis. Genome comparison between the newly emerged hyperinvasive isolates belonging to different serogroups will further our understanding of their respective pathogenetic mechanisms.

Characterization of serogroup A Neisseria meningitidis from invasive meningococcal disease cases in Canada between 1979 and 2006: Epidemiological links to returning travellers.

Serogroup A Neisseria meningitidis has repeatedly caused epidemics of invasive meningococcal disease (IMD) in developing nations since the 1960s. The present study is the first detailed study of serogroup A bacteria isolated in Canada. Thirty-four serogroup A meningococcal isolates collected from individuals with IMD in Canada between 1979 and 2006 were characterized by serology and multilocus sequence typing of seven housekeeping enzyme genes and genes encoding three outer membrane protein antigens. Isolates were assigned to either the sequence type (ST)-1 or the ST-5 clonal complex. Clones within the ST-1 complex were recovered between 1979 and 1992, while clones of the ST-5 complex were isolated between 1987 and 2006; respectively, they accounted for 70.6% and 29.4% of all isolates studied. Isolates of the ST-1 complex were characterized by serosubtype antigen P1.3 or P1.3,6 with PorB allele 60 (serotype 4) and FetA sequence F5-1, while isolates of the ST-5 complex were characterized by serosubtype antigen P1.9 with PorB allele 47 (also serotype 4) and FetA sequence F3-1. The Canadian serogroup A IMD isolates likely originated in travellers returning from hyperendemic or epidemic areas of the globe where serogroup A bacteria circulate. Although the Canadian cases of serogroup A IMD were caused by clones known to have caused epidemics in developing countries, disease incidence remained low in Canada.

The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence

The restriction endonuclease Type II R.NmeDI from Neisseria meningitidis 2120 (serogroup C, ST-11 complex) was characterized. The cloned nmeDIR gene was expressed in Escherichia coli cells, and the endonucleolytic and restriction activities of R.NmeDI were then observed in vitro and in vivo. The nmeDIR gene consists of 1056 bp coding 351 aa protein with a calculated molecular weight of M(r) = 39 000 ± 1000 Da. The R.NmeDI enzyme was purified to apparent homogeneity following overexpression, using metal affinity chromatography. This enzyme recognizes a palindrome sequence and cleaves double-stranded DNA upstream and downstream of its recognition sequence (12/7) RCCGGY (7/12) (R = A/G, Y = C/T) cutting out a 25-bp fragment. R.NmeDI cleaves in two steps. The enzyme cleaves the first strand randomly on either side of the recognition sequence generating an intermediate, and the second cleavage occurs more slowly and results in the production of a final reaction product. The R.NmeDI endonuclease requires two recognition sequences for effective cleavage. The tetramer is an active form of the R.NmeDI enzyme.