Multilocus Sequence Typing Website Research Articles

Molecular Epidemiology of Streptococcus equi subsp. zooepidemicus Isolated from Thoroughbred Horses Using Multi Locus Sequence Typing (MLST) in Korea

Background:Multi Locus Sequence Typing (MLST) is a new global molecular typing method used for analyzing the DNA polymorphisms in bacteria. In this study, using MLST, we analyzed the sequence profiles ofStreptococcus (S.) zooepidemicusisolates from the Jeju and Jangsu provinces in South Korea.Objective:This study characterized the molecular epidemiology ofS. zooepidemicusinfection in Thoroughbred horses using MLST with an aim to control and prevent bacterial endometritis in mares.Methods:A total of 79S. zooepidemicusisolates were included in this study. Sequencing of the 7 loci for the MLST analysis was performed as described in the MLST website manual (http://pubmlst.org/szooepidemicus/) with some modifications. For each locus, every unique sequence was assigned a distinct allele number, and each Sequence Type (ST) was defined by a series of 7 integers (the allelic profile) corresponding to the alleles at the 7 loci (arcC,nrdE,proS,spi,tdk,tpi, andyqiL)using the MLST module in the Main Workbench.Results:Among the 79 isolates, 73 different MLST patterns were identified, each corresponding to 1-3 strains. The genetic relationships between the 79 isolates are presented in a dendrogram, and they diverged by up to 11% homology. At 11% homology, three MLST groups, M1, M2, and M3, were identified, and at 26% homology, five subgroups, L1-L5, were observed.We observed various MLST patterns in the strains isolated from Jeju and Jangsu. In addition, by estimating the epidemiological relationships among the strains isolated from Jangsu in 2007 and Jeju in 2009, which had similar MLST patterns, we determined that some strains from Jangsu may have been transported to Jeju.Conclusion:MLST can be used as a framework for determining the epidemiological relationships that form the genetic basis of host and infection site selection.

A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in 2010.

In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations. Vibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations.

English

Multilocus sequence typing (MLST) has been used to characterise methicillin-resistant Staphylococcus aureus (MRSA) isolates into sequence types (STs) and together with SCCmec typing, form the clonal nomenclature for MRSA. MLST was conducted as per the standard protocol on ten out of 236 isolates collected previously from January to December 2009 representing four different SCCmec types. Relationship analysis was performed with eBURST via the MLST website. Four unlinked ‘singleton’ STs were detected: ST30, ST239, ST772 and ST1178. Together with SCCmec typing, five MRSA clones were identified: ST30-IV, ST239-II, ST239-III, ST772-V and ST1178-IV. Clones ST239-III and ST30-IV are already established in Malaysian hospitals and in the local community, respectively. ST772-V is an emerging clone reported previously to have a propensity to displace pre-existing predominant clones. A clone involving the predominant ST in Malaysia (ST239) with SCCmec type II is the first of its kind to be identified. MRSA clones in our centre are very diverse and clone surveillance with large sample sizes should be undertaken as collaborative efforts between local institutions to maximise detection coverage.

Borrelia burgdorferi sensu stricto and Borrelia afzelii: Population structure and differential pathogenicity

MultiLocus sequence typing (MLST) is considered a powerful method to unveil relationships within bacterial populations and it constitutes an economical and fast alternative to whole genome sequencing. We used this method to understand whether there are differences in human pathogenicity within and between different Borrelia burgdorferi sensu lato species. Therefore, 136 strains from human patients or ticks from Europe were included in MLST analyses. The scheme employed used eight chromosomally located housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA). We investigated Borrelia afzelii, one of the predominant species in Europe, and B. burgdorferi sensu stricto (s.s.), because it allowed comparative analysis to strains from the USA. We typed 113 patient isolates as well as 23 tick isolates. For further comparative purposes an additional 746 strains from Europe and the USA were included from the MLST website http://borrelia.mlst.net. We observed an overlap of the B. burgdorferi s.s. populations from Europe and the USA isolated from human patients while there was no overlap of the populations found in tick vectors. Further results indicate that B. afzelii was significantly less associated with disseminated infection than B. burgdorferi s.s. and that B. burgdorferi s.s. from Europe caused neuroborreliosis to a significantly greater extent than B. afzelii or B. burgdorferi s.s. in the USA. Our data suggest that there may be an evolutionary basis of differential interspecies pathogenicity in Borrelia. This was not evident within Borrelia species: we found the same sequence types in patients with disseminated or localized symptoms when the number of strains was sufficiently high. We hypothesize that the finding that B. burgdorferi s.s. in Europe is much more associated with neuroborreliosis than in the USA maybe linked to factor(s) related to the human host, the tick vector or the bacterium itself (e.g. plasmid content and structure).

Types of eae genes in non-O157 Shiga toxin-producing Escherichia coli strains

Objective To analyze the subtypes of eae genes in various non-O157 Shiga toxin-producing Escherichia coli (STEC)strains isolated in China. Methods The complete nucleotide sequences of 10 eae genes were amplified by PCR and sequenced. The BLASTn software was used to analyze the sequences for eae gene subtyping. A phylogenetic tree was constructed based on the 10 eae gene sequences together with the gene sequences of 30 different subtypes in GenBank and those of STEC strains of 7 prevalent serotypes (O157∶H7, O26∶H11, O103∶H2, O111∶H8, O145∶H28, O45∶H2 and O121∶H19) using MEGA 5.0. Multilocus sequence typing (MLST) was performed on the 10 STEC strains with reference to the Escherichia coli (E.coli) MLST website (http: //mlst.warwick.ac.uk/mlst/dbs/Ecoli) for the typing of multiple loci. A minimum spanning tree (MST) was constructed using the BioNumerics software to investigate the phylogenetic relationships between the 10 eae gene-positive STEC strains in this study and hemolytic uremic syndrome-associated enterohemorrhagic E. coli (HUSEC) strains as well as all human STEC strains of O157, O26, O45, O103, O111, O121 and O145 serotypes submitted to the E. coli MLST website database. Results The complete nucleotide sequences of eae genes in 10 non-O157 STEC strains were 2.8 kb in length and belonged to 3 known subtypes. The predominant subtype was β1, accounting for 60% of the 10 STEC strains (6/10), followed by θ and γ1 subtypes with two strains in each type. The eae gene sequences in certain strains were identical to those of the prevalent serotypes. Seven sequence types (STs) were identified from the 10 STEC strains carrying eae gene. Conclusion The eae genes harbored by the non-O157 STEC strains isolated from different specimens in China were diverse and had close phylogenetic relationships with the highly pathogenic and prevalent STEC strains. This study implied that the STEC strains harboring eae gene had high pathogenic potential. Key words: Non-O157 Shiga toxin-producing Escherichia coli; eae gene; Multilocus sequence typing; Subtying

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA.

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.

Multilocus sequence typing of DNA from faecal specimens for the analysis of intra-familial transmission of Helicobacter pylori

This study used multilocus sequence typing (MLST) of total DNA extracted from faecal specimens to genotype Helicobacter pylori to analyse intra-familial transmission. Faecal DNA was extracted and amplified by nested PCR. The products were analysed by direct sequencing and the allele type was determined using an MLST website. Mother-to-child transmission was suspected in at least two of three families, and father-to-child transmission was suspected in one family.

A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

BackgroundThe available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species.Methodology and FindingsWe modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated.ConclusionThe new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.

Identification and proposal of a potentially new clonal complex that is a common cause of MenB disease in central and eastern Canada

This study examined serogroup B meningococci (MenB) from invasive meningococcal disease (IMD) cases in the provinces of Québec and Ontario in the last decade by multilocus sequence typing (MLST) to determine their sequence types (STs) and clonal complexes (CCs). Forty isolates from individual MenB IMD cases were found to belong to 8 related STs, with ST-336 being the founding ST and the other 7 STs being single locus variants of ST-336. Eleven isolates belonged to ST-336, 23 belonged to ST-5571, and the other 6 were represented individually by a single different ST. All but 1 of these 40 isolates have the PorA variable-region type of P1.22,14,36. Interrogation of the Neisseria MLST web site with the present finding did not put any of the 8 related STs into known CCs. Since these 8 related STs were common causes of IMD, with ST-5571 being the most frequently identified ST in Ontario and ST-336 the third most common ST identified in Québec, we propose that ST-336 and its related STs is a potentially new meningococcal clonal complex that is endemic in the Canadian provinces of Québec and Ontario, and they constitute a common cause of IMD.

Importation of KPC-2-producing Escherichia coli from India

Sir, The production of carbapenem-hydrolysing b-lactamases is increasingly reported in Enterobacteriaceae. Among the different types of carbapenemases, the emergence of the Ambler class A KPC-type b-lactamases is of great concern, since those enzymes hydrolyse all b-lactams with the exception of cephamycins. Enterobacterial isolates producing KPC-type b-lactamases were reported in many areas in the USA and subsequently worldwide. The rapid dissemination of KPC enzymes among different enterobacterial species is related to the localization of blaKPC genes on transferable broad host range plasmids and their association with a transposon. This dissemination has also been linked with a ‘successful’ international clone of KPC-producing Klebsiella pneumoniae of sequence type (ST) 258. Early in 2011, a middle-aged patient was transferred from a hospital in Mumbai, India, to the hospital of Dinan, France. The patient suffered from pleurisy due to Streptococcus pneumoniae for which he had received a combination of imipenem, vancomycin and piperacillin/tazobactam in India. Upon admission, a rectal swab revealed the presence of a multidrug-resistant Escherichia coli (designated strain GRU) with reduced susceptibility to carbapenems. No secondary local transmission occurred at the Dinan hospital following the rapid implementation of strict infection control measures. The antibiogram determined by the disc diffusion method and MICs determined by Etest (AB bioMerieux, Solna, Sweden) and interpreted according to the CLSI guidelines revealed that E. coli strain GRU was resistant to all penicillins and expandedspectrum cephalosporins, to ertapenem (MIC .32 mg/L) and to meropenem (MIC 8 mg/L) and was of intermediate susceptibility to imipenem (MIC 1.5 mg/L). The isolate was susceptible to tetracycline and fosfomycin, and MICs of tigecycline and colistin were 1 and 0.5 mg/L, respectively. However, it was resistant at a high level to all fluoroquinolones (MICs .256 mg/L). Molecular investigations performed as described previously identified the blaKPC-2 gene. Isolate GRU also harboured the blaTEM-1 and blaOXA-1 genes. Plasmid location of the blaKPC-2 gene was confirmed by electroporation of a plasmid DNA preparation obtained by the Kieser method into E. coli TOP10 with selection on Trypticase soy plates containing ampicillin (100 mg/L). Molecular and phenotypic analysis of the E. coli transformant confirmed that blaKPC-2 was located on an 20 kb plasmid. The blaKPC-2-positive plasmid was non-typeable using PCR-based replicon typing. No other antibiotic resistance marker was co-transferred. PCR mapping performed as described showed that the blaKPC-2 gene was part of the Tn4401 transposon. It is noteworthy that E. coli GRU additionally harboured a gene encoding the 16S rRNA methylase ArmA, conferring high-level resistance to all aminoglycosides (MICs of gentamicin, netilmicin, kanamycin and tobramycin .256 mg/L). Interestingly, KPC-2and ArmAproducing Enterobacter cloacae and K. pneumoniae isolates have been reported in China and Poland. Multilocus sequence typing (MLST) performed according to the protocol described on the E. coli MLST web site (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/EColi.html) showed that E. coli GRU belonged to ST101, recently reported to be the most frequent NDM-1-producing E. coli clone in the UK and Pakistan. That study reported a KPC-producing E. coli originating from India. It remains to be determined to what extent the spread of KPC-type enzymes will contribute to the problem of carbapenem resistance in India, which currently is commonly regarded as reflecting the dissemination of the NDM-1 carbapenemase.

First Report of Streptococcus pneumoniae Serotype 6D in South America

Streptococcus pneumoniae includes the two serotypes 6A and 6B as well as two recently discovered serotypes, 6C and 6D, in which the wciNα gene is replaced by wciNβ within the cps locus. Serotype 6D occurrence in Asia (2, 4), the Fiji islands (5) and Europe (7, 9) has recently been reported. To our knowledge, we describe here the first description of serotype 6D isolates identified within the Americas. Using the CDC serogroup 6 serotyping scheme (8), we identified 155 serogroup 6 isolates within a larger collection (n = 693) taken from nasopharyngeal carriage in children <2 years old in Peru (2007 to 2009; 541 strains) (unpublished data) and invasive pneumococcal disease (IPD) surveillance in children in Lima hospitals (2006 to 2009; 152 strains) (reference 10 and unpublished data). Among the 155 serogroup 6 isolates, 26, 105, and 22 isolates were identified as serotypes, 6A, 6B, and 6C, respectively (Table 1). We tentatively identified two 6D isolates on the basis of positive reactions with factor sera for serotype 6B (factor 6c) and serotype 6C (factor 6d) and a negative reaction with factor serum for serotype 6A (factor 6b). We verified the serologic results for all 155 serogroup 6 isolates using a recently developed PCR scheme that efficiently resolves all four serogroup 6 serotypes (6). This scheme discriminates between serotypes 6A/6C and 6B/6D using wciP allele-specific reactions. The presence or absence of wciNβ determined by a second reaction subsequently allowed resolution into all four serotypes (Table 1). Table 1. Quellung and PCR results for 155 serogroup 6 isolates The two 6D isolates, both from carriage, shared the new multilocus sequence typing (MLST) profile ST6148 (ST6148, 8-8-263-12-6-104-14) and were both fully susceptible to a variety of antimicrobial agents by broth microdilution. ST6148 is unrelated to previously described 6D genotypes on the MLST website (http://spneumoniae.mlst.net/, last accessed on 5 January 2011). It is, however, highly related to serotype 6A and 6B isolates both from carriage and invasive disease that were characterized from this same study, differing only at the recP allele. These data, together with data published elsewhere, show identical or related multilocus sequence types shared between serotype 6C or 6D strains containing wciNβ and 6A or 6B strains that contain wciNα (3, 4, 7). This suggests that the two different wciN genes are frequently horizontally transferred between different serogroup 6 strains in nature to effect intraserogroup 6 capsular switch events. On the basis of these genotype observations it also circumstantially appears likely that in some cases wciP is cotransferred with wciN, which could effect, for example, a serotype 6A to serotype 6D capsular switch. In summary, we believe that this is the first reported detection of serotype 6D among pneumococcal isolates recovered in South America or elsewhere in the western hemisphere, despite the fact that our U.S.-based surveillance has been vigilant for its appearance using serologic and PCR-based approaches for 6D detection (3, 8). The work presented here validates serotype 6D as the 92nd serotype identifiable by using CDC pneumococcal typing sera (Table 1). Serotype 6C was not effectively targeted by the pneumococcal 7-valent conjugate vaccine according to recent IPD surveillance data (3); however, there are no existing comparable data for serotype 6D. It is hoped that the recently implemented 13-valent conjugate vaccine (1) will effectively target all serogroup 6 pneumococcal diseases.

The Changing Epidemiology of Streptococcus pneumoniae Serotype 19A Clonal Complexes

(See the article by Beall et al, on pages 1360-8.) Streptococcus pneumoniae is a leading cause of pneumonia throughout the world and a major cause of serious invasive disease, especially in the very young and the elderly. The major virulence factor of S. pneumoniae is its polysaccharide capsule, which can be expressed as 91 different capsular serotypes [1, 2].Seven serotypes (those of high prevalence in children prior to 2000; serotype [sero]4, sero6B, sero9V, sero14, sero18C, sero19F and sero23F) constitute a very effective protein conjugate vaccine, PCV7, which has been in use throughout the world, including the United States, since 2000. While the vaccine serotypes contained in PCV7 have declined in prevalence over the last decade, other serotypes, most notably sero19A, have become more prevalent throughout North America and Europe [3–8]. To add cause for concern, many of the sero19A strains circulating are also resistant to multiple antimicrobial agents, making them difficult to treat. Six years after the introduction of PCV7, sero19A is the most common invasive pneumococcal serotype in the United States, with a substantial proportion of its strains being antimicrobial resistant [9]. Although the serotype designation is important for understanding pneumococcal epidemiology, to gain a deeper perspective of the pneumococci circulating globally, a technique termed multilocus sequence typing (MLST) is used. MLST is a nucleotide sequence–based approach to characterizing bacterial strains [10]. It involves the DNA sequencing of internal fragments of 7 housekeeping genes from the pneumococcal genome. The sequences are compared with previously identified sequences (alleles) and assigned an allele number for each of the 7 loci. The 7 number combinations denote an allelic profile of the strain that is then assigned a sequence type (ST) designation. Sequence types are grouped into “clonal complexes.” To be part of clonal complex, members must have typically 5 or 6 loci that match. Interestingly and importantly, isolates with different serotypes can be members of the same clonal complex. Detailed information regarding S. pneumoniae MLST analysis is provided on the pneumococcal MLST website, hosted by Imperial College, London (http://spneumoniae.mlst.net/). The information provided by MLST is global in scope but more in-depth than the serotype designation. In this issue of the Journal, Beall and colleagues describe the recent epidemiology of S. pneumoniae sero19A in the United States from isolates collected through the Centers for Disease Control and Prevention (CDC) Active Bacterial Core (ABC) surveillance program. Their detailed MLST analysis of sero19A isolates has shown that while the prevalence of sero19A strains seems to have stabilized since 2005, the numbers of isolates within specific sero19A clonal complexes have fluctuated significantly. These changes have resulted in declines in clonal complex prevalence, increases in clonal complex prevalence, or appearance of new sero19A clonal complexes. Currently, 3 main clonal complexes dominate the invasive pneumococcal disease sero19A landscape in the United States. These have been designated clonal complex (CC)19919A, CC320/27119A, and CC69519A. These 3 clonal complexes collectively comprise approximately 87% of the sero19A isolates collected from 2005–2007 [11]. Brief descriptions of each of these clonal complexes with respect to prevalence over recent years is given below.

Neisseria meningitidis Serogroup X Sequence Type 767 in Turkey

Neisseria meningitidis is a common inhabitant of the nasopharyngeal tracts of healthy humans and is a significant cause of invasive infections such as meningitis in young children and adolescents worldwide (14). N. meningitidis has been classified into 13 serogroups on the basis of antigenic variation of the capsule, but only serogroups A, B, C, Y, and W-135 have commonly caused invasive infections (8). In Turkey, W-135 has been the most frequently reported serogroup since an international outbreak was reported following the annual Hajj seasons in Saudi Arabia in 2000 and 2001 (10). During the years 2003 to 2008, 17 serogroup W-135 strains (from eight meningitis cases) were isolated from Turkish recruits vaccinated with A+C polysaccharide meningococcal vaccine (10-12). We describe here the first meningococcal meningitis case caused by a serogroup X N. meningitidis strain in Turkey. The patient was a 22-year-old male soldier, working in a military unit, who was admitted to the emergency department with complaints of a 1-week history of fever (39°C), cough, and vomiting on 10 January 2010. There was little petechiate rash on his chest and a lower leg. The laboratory evaluation at that time revealed a leukocyte count of 3,700/mm3, a hemoglobin level of 13.4 g/dl, and an erythrocyte sedimentation rate of 5 mm/h. The patient was then admitted to the infectious disease service with a diagnosis of meningococcal meningitis based on the clinical symptoms and results of confirmatory laboratory tests, including blood culture and cerebrospinal fluid analysis. He was given ceftriaxone (4 g/day) for 10 days and then discharged from the hospital in good condition. Oropharyngeal swab specimens were obtained from the patient's close military personnel contacts, and one person was found to be positive for an N. meningitidis serogroup X strain. He was given a single dose of 500 mg ciprofloxacin immediately. Clinical and oropharyngeal swab specimens were inoculated immediately onto BBL-modified Thayer-Martin medium plates (MTM II; Becton Dickinson Microbiology Systems) and Thayer-Martin medium with VCAT selective supplement (SR10B; Oxoid, Hampshire, England). After incubation, suspect colonies were identified by Gram staining and oxidase reactivity testing and the identification was confirmed by the API NH system (bioMerieux) (4). An antibiotic susceptibility test was performed by the Etest method (AB BIODISK, Solna, Sweden) for ciprofloxacin, ceftriaxone, cefotaxime, penicillin, rifampin, and chloramphenicol (3). The strains were susceptible to the antibiotics tested, except penicillin, with a MIC of 0.75 μg/ml. Serogrouping of the meningococcal isolates was performed by a slide agglutination technique as recommended by Difco Laboratories (Detroit, MI). porA variant region sequencing was performed using the standard primers and identified by querying the respective databases hosted at http://neisseria.org. Multilocus sequence typing (MLST) was performed using the standard primers listed at the Neisseria MLST website (http://pubmlst.org/neisseria/). The isolates had the strain designation X: P1.5-1,10-1: sequence type 767 (ST-767) (cc167). ST-767 meningococci have been found previously among N. meningitidis serogroup A and Y strains in Europe and Africa between 1999 and 2008 (http://pubmlst.org/neisseria/). PFGE typing of NheI-digested DNA was performed by a modification of a previously described method (12). The clinical and carrier isolates had the same genotypic pattern. N. meningitidis serogroup X strains were first described in the 1960s and have been isolated from a few cases of invasive meningococcal diseases in North America, Europe, Australia, and China (2, 6, 7). Outbreaks of N. meningitidis serogroup X strains have been reported in Niger (1), western Kenya (13), and northern Ghana (5). N. meningitidis serogroup X strains were reported to be very efficient in colonization among military recruits in the United Kingdom (9). Before May 2009, eight invasive meningococcal disease cases caused by serogroup W-135 were reported in Turkish soldiers who had been vaccinated with A+C polysaccharide meningococcal vaccine; four of these were fatal. Since that time, a quadrivalent meningococcal polysaccharide vaccine (against A/C/Y/W-135) has been successfully introduced into the Turkish recruit expanded immunization program. We describe here the first meningococcal meningitis case caused by a serogroup X strain in our recruits since the introduction of the quadrivalent vaccine. It was reported that repeated mass vaccination in many African countries might have contributed to colonization by and meningococcal diseases due to serogroup X strains and might result in a changed profile of meningococcal disease (5). We suggest that comprehensive conjugate vaccines including X polysaccharides should be developed. In addition, these data highlight the need for further epidemiological surveillance to carefully monitor the pattern of incidence of meningococcal diseases caused by serogroup X strains and to inform future public health strategies.

Neisseria meningitidis with reduced susceptibility to quinolones in Singapore

Sir, Ciprofloxacin is commonly prescribed as a single-dose chemoprophylactic agent for close contacts of patients with meningococcal infection. Neisseria meningitidis with reduced susceptibility to fluoroquinolones has recently emerged, with sporadic cases reported in France, Australia, Argentina, Spain, Hong Kong and Israel. Recently an outbreak of serogroup A emerged in India, and a cluster of three cases of serogroup B in the USA. To our knowledge, failure of ciprofloxacin prophylaxis due to quinolone resistance has not been reported To date, reduced susceptibility to fluoroquinolones in meningococci has remained rare, with 10 unrelated strains found in an analysis of 5300 isolates in Spain, and only 3 from .5000 isolates in France. Most mutations have arisen in GyrA. However, mutations have also been detected in GyrB and ParE, although their significance remains uncertain. In addition, in vitro changes have been induced in ParC and resistance to quinolones has also been reported through an efflux mechanism. – 4 Following the isolation of N. meningitidis with reduced susceptibility to ciprofloxacin from a 36-year-old man with meningitis in 2008, all stored viable clinical isolates from Singapore’s three largest hospitals (Tan Tock Seng, Singapore General and National University hospitals) were screened for reduced susceptibility to nalidixic acid and rifampicin by disc diffusion, using the CLSI guidelines. Ciprofloxacin MICs were determined by agar plate dilution for isolates with reduced susceptibility to nalidixic acid, using the Australian Gonococcal Surveillance Programme (AGSP) guidelines. MICs obtained with this method give identical results for identical GyrA changes in Neisseria gonorrhoeae and N. meningitidis. All isolates for which the MIC is .0.03 mg/L either have changes in the quinolone resistance determining region (QRDR) or an increased efflux of quinolones, while those for which the MIC is ,0.03 mg/L do not. In the absence of clinical correlation data, an MIC of .0.03 mg/L is taken to indicate reduced susceptibility by both the AGSP and CLSI methods. Multilocus sequence typing (MLST) was carried out using protocols available on the Neisseria MLST website. To characterize the mechanisms of resistance to ciprofloxacin, we sequenced the QRDRs of GyrA, GyrB, ParC and ParE of isolates with a ciprofloxacin MIC of .0.03 mg/L. Penicillin MICs were determined with Etests (AB BIODISK, Solna, Sweden) for all isolates, using Mueller–Hinton agar with 5% sheep blood and interpreted according to CLSI criteria for agar dilution. Of 75 isolates collected between 2000 and 2008, two (3%) were resistant to nalidixic acid, a Group C isolate from 2008 and a Group B isolate from 2002. Both isolates had a raised ciprofloxacin MIC of 0.25 mg/L. All 75 isolates were susceptible to rifampicin. There were 13 isolates (17%) intermediate and 2 (3%) resistant to penicillin (MICs of 0.38 and 0.47 mg/L). MLST confirmed that the two isolates with reduced quinolone susceptibility were unrelated. The 2008 isolate was identified as sequence type (ST)-4821. The 2002 isolate was a new ST with alleles 4, 10, 76, 71, 13, 2, 75 although the aroE allele had one nucleotide different from allele 76 (C to T at position 39). The 2008 ST-4821 isolate had the most common GyrA substitution of threonine to isoleucine at position 91. The 2002 isolate had three mutations detected in GyrA: Thr91!Val; Asn103!Asp; and Ala105!Ser. To our knowledge, the Thr91!Val substitution has not been previously described in N. meningitidis. Neither of the isolates had changes in GyrB, ParC or ParE. Similarities in the mechanism of quinolone resistance in meningococci and gonococci, have prompted concerns that resistance will climb to mirror that of gonococci. Resistance to quinolones among N. gonorrhoeae is now so common that use of ciprofloxacin for the treatment of gonorrhoea is no longer recommended by the US Centers for Disease Control and Prevention. In Singapore, ciprofloxacin resistance among gonococci reached an alarming 61.8% in 2006 (74% in 2008; A. L. Tan, unpublished data). Ciprofloxacin is a prescription-only medicine in Singapore, but is in widespread use. An outbreak of meningococcal meningitis with reduced susceptibility to quinolones has already been seen in India in 2005, a country with liberal use of ciprofloxacin. Although our data are from small numbers, as meningococcal infections are uncommon in Singapore (usually ,10 cases per year), the 3% rate of quinolone resistance in this study is remarkably high when compared with other reports. Surveillance for reduced susceptibility and judicious quinolone use is forever increasing in importance but our local resistance data for the related N. gonorrhoeae may suggest the future is already written.

Population structure of Streptococcus oralis

Streptococcus oralis is a member of the normal human oral microbiota, capable of opportunistic pathogenicity; like related oral streptococci, it exhibits appreciable phenotypic and genetic variation. A multilocus sequence typing (MLST) scheme for S. oralis was developed and the resultant data analysed to examine the population structure of the species. Analysis of 113 isolates, confirmed as belonging to the S. oralis/mitis group by 16S rRNA gene sequencing, characterized the population as highly diverse and undergoing inter- and intra-species recombination with a probable clonal complex structure. ClonalFrame analysis of these S. oralis isolates along with examples of Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae grouped the named species into distinct, coherent populations and did not support the clustering of S. pseudopneumoniae with S. mitis as reported previously using distance-based methods. Analysis of the individual loci suggested that this discrepancy was due to the possible hybrid nature of S. pseudopneumoniae. The data are available on the public MLST website (http://pubmlst.org/soralis/).

Serogroup ANeisseria meningitidiswith Reduced Susceptibility to Ciprofloxacin

Serogroup A<i>Neisseria meningitidis</i>with Reduced Susceptibility to Ciprofloxacin

Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa Isolates from the ocean.

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.

Analysis of Phenotypic Variants of the Serogroup C ET-15 Clone of Neisseria meningitidis by Pulsed-Field Gel Electrophoresis

In Canada, two waves of hyperendemic meningococcal diseases were documented in the last 20 years. Both were due to a unique clone of serogroup C Neisseria meningitidis designated by multilocus enzyme electrophoresis as ET-15 (1). When this clone first appeared, it was characterized by the antigenic formula C:2a:P1.5,2 (1). In 2001, antigenic variants of this ET-15 clone characterized as C:2a:P1.7,1 (7) or C:2a:P1.5 (4) emerged to cause outbreaks (8). In this study, we examined whether pulsed-field gel electrophoresis (PFGE) can be used as a discriminatory tool to differentiate between the antigenic variants C:2a:P1.7,1, C:2a:P1.5, and C:2a:P1.5,2 of the ET-15 clone of N. meningitidis. Eleven serogroup C ET-15 N. meningitidis isolates from invasive meningococcal disease cases were selected for this study. Ten of the 11 serogroup C isolates were identified as serotype 2a. DNA sequencing of the porB gene of the nontypeable isolate identified it as a serotype 2a mutant containing a previously described mutational hot spot (2). There were four different combinations of serosubtype antigens observed for these 11 isolates: 5 with the P1.5 antigen, 3 with the P1.7,1 antigens, 2 with the P1.5,2 antigens, and 1 with the P1.2 antigen. The porA genes of these strains were sequenced, and their PorA variable regions (VRs) are summarized in Fig. ​Fig.11. FIG. 1. Eleven PFGE profiles of SpeI-digested DNA (using Dice coefficient with a position tolerance of 1.5% and optimization of 2.0% on BioNumerics 3.5 software) representing the three Neisseria meningitidis serogroup C ET-15 antigenic variants ... Multilocus sequence typing was performed according to the established method by Maiden et al. (3) and isolates were assigned sequence types according to the Neisseria Multi Locus Sequence Typing website (http://pubmlst.org/neisseria/). An additional region in the fumC gene was amplified to determine the presence of a G-to-A point mutation at position 360, characteristic of ET-15 strains (11). All 11 isolates contained this particular point mutation and were therefore classified as ET-15 strains. PFGE analysis of the 11 isolates that represented the three antigenic variants, C:2a:P1.5,2, C:2a:P1.7,1, and C:2a:P1.5, was performed as described by Tyler and Tsang (9). Restriction enzyme digestion of genomic DNA with NheI (data not shown) and SpeI indicated that serogroup C ET-15 variants had overall similarity and were difficult to distinguish based on the banding patterns they exhibited (Fig. ​(Fig.1).1). Using SpeI, PFGE pattern I was unique to isolates with the serosubtype P1.5 antigen. However, pattern II was common to all three ET-15 antigenic variants, while pattern III was common to C:2a:P1.5,2 and C:2a:P1.7,1 isolates, and pattern IV was common to C:2a:P1.5,2 and C:2a:P1.5 isolates. Despite the widespread acceptance of the PFGE method (4, 10), there is an apparent lack of correlation between an isolate's DNA fingerprint and its antigenic profile. This serves to illustrate a potential limitation of PFGE in the analysis of N. meningitidis strains for molecular epidemiology studies of invasive meningococcal disease. Nevertheless, for localized outbreak analysis, PFGE is still a very useful tool to identify strains linked to a common source (5, 6). In summary, a number of typing tools, including both phenotypic and genotypic methods, should be used in combination with carefully documented epidemiological information for surveillance and analysis of meningococcal disease.

Evaluation of custom-made DNA microarrays for multilocus sequence typing of Neisseria meningitidis

Multilocus sequence typing (MLST) has become the gold standard for typing of a variety of bacterial and fungal micro-organisms. Others recently reported the successful use of the tiling DNA array technology to sequence-type Staphylococcus aureus. We now evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of Neisseria meningitidis. The rationale behind this approach was to minimize the number of microarray probes by exploiting the comprehensive knowledge of polymorphisms combined in the Neisseria MLST website. Initial experiments using model oligonucleotides of 28–32 base-pairs in length revealed that the hybridization protocols used were highly specific. However, despite of several optimization steps, the rate of misidentification of oligonucleotides remained >1.8% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. We assume that the high density of polymorphic sites and the extensive GC-content variations at N. meningitidis MLST loci hinder the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design.

The 1998 Senegal epidemic of meningitis was due to the clonal expansion of A:4:P1.9, clone III-1, sequence type 5 Neisseria meningitidis strains.

Between January and April 1998, a meningitis outbreak due to serogroup A meningococcus took place in Senegal. The outbreak began in Gandiaye, 165 km to the east of Dakar, and progressed towards the towns of Gossas, Niakkhar, Guinguineo, Fatik, Foundiougne, Dioffior, Sokone, Kaolack, and Nioro. At the same time, the outbreak reached regions of Kaffrine, Koungheul, and Tambacounda in the east of Senegal. A total of 1,350 cases and 200 deaths were reported. The WHO Collaborating Center in Marseilles received 24 strains for analysis. All were serogroup A Neisseria meningitidis, type 4 and subtype P1.9. Multilocus enzyme electrophoresis, performed by Institut Pasteur Paris, showed that the strains belonged to clone III-1. DNA restriction fragments generated by endonuclease BglII and analyzed by pulsed-field gel electrophoresis showed 24 indistinguishable fingerprint patterns similar to those of meningococcus strains isolated from African outbreaks since 1988. Three strains were studied by multilocus sequence typing (MLST) with seven loci. The comparison between sequences and existing alleles on the MLST website () allowed us to assign these strains to sequence type 5 (ST5), as their sequences were identical to the consensus at seven loci. All 24 strains were susceptible to penicillin, amoxicillin, chloramphenicol, and rifampin. Subgroup III is finishing its spread towards west of the meningitis belt of Africa. To our knowledge, this is the first time subgroup III, and more precisely ST5, strains are reported as being responsible for a meningitis outbreak in Senegal.