[Show abstract][Hide abstract] ABSTRACT: As part of an ongoing study of the response of the Streptococcus pneumoniae population to conjugate vaccination, we applied multi-locus sequence typing (MLST) to 291 isolates sampled from nasopharyngeal carriage in Massachusetts children. We found 94 distinct sequence types (STs), including 19 that had not been previously recorded, and a xpt allele containing a large insertion. Comparison with a similar sample collected in 2007 revealed no significant overall difference in the ST composition (p=0.51) suggesting that the population has reached a new equilibrium following the introduction of 7 valent vaccination in 2000. Within serotypes, a large and statistically significant increase (p=0.014 Fisher's Exact test) was noted in the prevalence of the major multiresistant clone ST 320, which is apparently outcompeting ST 199 among serotype 19A strains. This sample will be used as a baseline to study the future evolution of the pneumococcal population in Massachusetts following introduction of vaccines with higher valency.
[Show abstract][Hide abstract] ABSTRACT: We carried out multilocus sequence typing (MLST) on 148 pneumococcal carriage isolates collected from children <24 months old in the Upper River Division, the Gambia. MLST revealed a diverse population. Seventy-six different sequence types (STs) were found, the most common of which were 802 and 919, associated with 23F and 6A serotypes, respectively. Comparison with the MLST database showed that only 11 of the STs found in the present sample had been reported outside Africa. Six STs showed evidence of capsular switching (172, 802, 847, 1730, 1736, and 1737). Serotype switches were confirmed by microarrays that detected capsule genes. Of isolates analyzed by using microarrays, 40/69 (58%) harbored the tetM resistance determinant. A statistical genetic analysis to detect recombination found that 49/144 (34%) isolates showed significant (P<0.05) evidence of admixture, which is greater than that observed in similar samples from the United Kingdom (5%) and Finland (2%). We hypothesize that large amounts of admixture could reflect the high prevalence of multiple carriage in this region, leading to more opportunities for homologous recombination between strains. This could have consequences for the population response to conjugate vaccination.
[Show abstract][Hide abstract] ABSTRACT: Vaccination against 7 serotypes of Streptococcus pneumoniae has led to the near extinction of vaccine serotypes in both disease and asymptomatic carriage. In carriage, vaccine serotypes have been replaced by nonvaccine serotypes.
We used multilocus sequence typing to analyze a sample of 294 isolates of S. pneumoniae carried by Massachusetts children (aged, 3 months-7 years) and examine the results for serotype switching and association with antimicrobial resistance.
Eighty-six distinct sequence types (STs) were found, 10 of which exhibited a serotype other than that which would be expected from previous carriage samples. We interpret this as evidence of past or recent serotype switching. Switched variants include ST 320, which is a common and increasing source of multidrug resistance in this community. Switching events within serogroups were more common than expected by chance (P = 0.043 by a Monte Carlo approach). Using multilocus sequence typing data and eBURST analysis, we also describe clonal dynamics within the important replacement serotypes 19A, 15B/C, 35B, and the recently described 6C.
Some strains generated by serotype switching are increasingly important parts of the carriage population. In the case of 19A, it appears that the majority of increase is due to ST 320, a recently reported switched variant. This may have consequences for the STs causing invasive pneumococcal disease.
[Show abstract][Hide abstract] ABSTRACT: Methods for assigning strains to bacterial species are cumbersome and no longer fit for purpose. The concatenated sequences of multiple house-keeping genes have been shown to be able to define and circumscribe bacterial species as sequence clusters. The advantage of this approach (multilocus sequence analysis; MLSA) is that, for any group of related species, a strain database can be produced and combined with software that allows query strains to be assigned to species via the internet. As an exemplar of this approach, we have studied a group of species, the viridans streptococci, which are very difficult to assign to species using standard taxonomic procedures, and have developed a website that allows species assignment via the internet.
Seven house-keeping gene sequences were obtained from 420 streptococcal strains to produce a viridans group database. The reference tree produced using the concatenated sequences identified sequence clusters which, by examining the position on the tree of the type strain of each viridans group species, could be equated with species clusters. MLSA also identified clusters that may correspond to new species, and previously described species whose status needs to be re-examined. A generic website and software for electronic taxonomy was developed. This site http://www.eMLSA.net allows the sequences of the seven gene fragments of a query strain to be entered and for the species assignment to be returned, according to its position within an assigned species cluster on the reference tree.
The MLSA approach resulted in the identification of well-resolved species clusters within this taxonomically challenging group and, using the software we have developed, allows unknown strains to be assigned to viridans species via the internet. Submission of new strains will provide a growing resource for the taxonomy of viridans group streptococci, allowing the recognition of potential new species and taxonomic anomalies. More generally, as the software at the MLSA website is generic, MLSA schemes and strain databases for other groups of related species can be hosted at this website, providing a portal for microbial electronic taxonomy.
[Show abstract][Hide abstract] ABSTRACT: Pilus components of Streptococcus pneumoniae encoded by rlrA were recently shown to elicit protection in an animal model of infection. Limited data are available on the prevalence of the rlrA operon in pneumococci; therefore, we investigated its distribution and its antigenic variation among disease-causing strains.
The prevalence of rlrA and its association with serotype and genotype were evaluated in a global panel of 424 pneumococci isolates (including the 26 drug-resistant clones described by the Pneumococcal Molecular Epidemiology Network).
The rlrA islet was found in 130 isolates (30.6%) of the defined collection. Sequence alignment of 15 rlrA islets defined the presence of 3 clade types, with an overall homology of 88%-92%. The presence or absence of a pilus-encoding operon correlated with S. pneumoniae genotype (P < .001), as determined by multilocus sequence typing, and not with serotype. Further investigation identified a positive trend of rlrA occurrence among antimicrobial-resistant pneumococci.
On the basis of S. pneumoniae genotype, it is possible to predict the incidence of the rlrA pilus operon in a collection of pneumococcal isolates. This will facilitate the development of a protein vaccine.
The Journal of Infectious Diseases 03/2008; 197(6):888-96. · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gonorrhoea is an important cause of sexual ill health and is concentrated in geographical areas and demographic groups. This study explores the distribution of gonorrhoea across London.
Epidemiological data on all gonorrhoea cases were collected from 13 major genitourinary clinics in London between 1 June and 30 November 2004. Samples were stored centrally and typed using NG-MAST. The postcode of each case's main residence was used to calculate incidence of gonorrhoea by borough using data from the UK 2001 census and a population survey on residence of men who have sex with men (MSM).
2,891 cases were confirmed, 1,822 of which had postcode data, resided in London, and had their strain successfully typed. There was a very high incidence of gonorrhoea in MSM (1,834 per 100,000 population) and heterosexuals of black ethnicity (392 per 100,000). The incidence among heterosexuals was highest in City of London (390 per 100,000, 95% CI 213 to 566), Southwark (308 per 100,000, 95% CI 280 to 336), Hackney (284 per 100,000, 95% CI 254 to 313), and Lambeth (216 per 100,000, 95% CI 194 to 239) and was not associated with measures of social deprivation (correlation coefficient = 0.0008, p = 0.97) but was strongly associated with black ethnicity (correlation coefficient = 0.48, p = 0.01). 45% of cases had one of the 21 major strains; eight of these strains were significantly clustered geographically and persisted for a shorter duration than those that were not clustered. Patients travelled a mean of 7.7 km from their home to the clinic.
High gonorrhoea incidence in London is observed in MSM and heterosexuals of black ethnicity. Endemic strains in both MSM and heterosexuals are diagnosed at multiple clinics. Interventions, including partner notification, must therefore operate between clinics.
[Show abstract][Hide abstract] ABSTRACT: The long-term effects of selective pressure from conjugate pneumococcal vaccine on the serotype distribution and antimicrobial resistance of carriage and invasive isolates of Streptococcus pneumoniae are unknown. Early changes demonstrate a reduction in vaccine serotypes and an increase in nonvaccine serotypes (NVT) among both carriage and invasive isolates. Ongoing surveillance is necessary to identify emerging invasive serotypes and antimicrobial susceptibilities.
Enhanced surveillance of invasive pneumococcal disease in Massachusetts began in October 2001 and remains ongoing. Isolates from children less than 5 are sent to the Massachusetts Department of Public Health and subsequently to the Maxwell Finland laboratory for serotyping and determination of antimicrobial susceptibility. Annual incidence rates for vaccine serotype and NVT disease are calculated using 2000 census data.
NVT caused 72%-91% of invasive pneumococcal disease annually in children less than 5 years of age between 2002 and 2005. Serotype 19A has emerged as the most frequent cause of IPD in Massachusetts. A multidrug-resistant clone (ceftriaxone, amoxicillin, azithromycin and trimethoprim-sulfamethoxazole) (MLST 320) was first identified in Massachusetts in 2005.
Three years after the introduction of pneumococcal conjugate vaccine for universal administration to children less than 2 in Massachusetts, a significant increase in invasive disease due to serotype 19A was observed. Although MLST 199 remains the most frequent sequence type among invasive isolates (of 19A), a multidrug-resistant sequence type, not previously identified in Massachusetts, has become an important cause of invasive disease. Further surveillance of the changing ecology of S. pneumoniae is necessary as a 4-year time period is not sufficient to fully evaluate the impact of PCV of pneumococcal infections.
[Show abstract][Hide abstract] ABSTRACT: In response to the selective pressure of pneumococcal conjugate vaccine, increased asymptomatic carriage of antibiotic-nonsusceptible nonvaccine serotypes (NVTs) has been observed. Possible mechanisms include de novo acquisition of resistance, serotype switching, introduction of new clones, and expansion of existing clones.
To investigate the process of increased antibiotic nonsusceptibility among replacing serotypes, we applied multilocus sequence typing to samples of 126 and 222 pneumococci collected in 2001 and 2004, respectively, from the nasopharynges of children <7 years of age in 16 Massachusetts communities.
We found no evidence of penicillin resistance due to either serotype switching or de novo acquisition. Nonetheless, resistance increased through the expansion of previously recognized clones of NVTs, particularly in serotypes 19A, 15A, and 35B. In 19A, several unrelated clones increased in frequency, whereas, in the other 2 serotypes, single resistant lineages were responsible for the increased prevalence of resistant strains.
The decreased prevalence of antibiotic resistance with the introduction of heptavalent pneumococcal conjugate vaccine is likely to be partially eroded over time as vaccine-included serotypes are replaced by resistant clones of NVTs. The clinical significance of this will depend on the pathogenic potential of replacing clones to cause local (e.g., otitis media) or invasive disease.
The Journal of Infectious Diseases 03/2007; 195(3):347-52. · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular typing of Neisseria gonorrhoeae and contact tracing provide a combined approach for analysis of sexual networks in metropolitan areas, although there are some difficulties in application. Our aim was to examine the application of high-throughput molecular approaches that can identify individuals in linked sexual networks.
We characterised 2045 isolates of N gonorrhoeae from patients presenting at 13 major sexually transmitted infection clinics in London, UK, between June 1 and Nov 30, 2004. All isolates were assigned a sequence type (strain) on the basis of the sequences of internal fragments of two highly polymorphic loci, por and tbpB. These types were matched to demographic and behavioural data obtained at the clinic for each patient. We assessed the congruence in the demographic and behavioural characteristics of individuals infected with the same strain.
We identified 21 prevalent strains in this diverse gonococcal population, each infecting between 20 and 124 individuals. Seven of these strains were predominantly from men who have sex with men; the remaining 14 were predominantly from heterosexual people. No differences were recorded between the strains associated with men who have sex with men in the demographic or behavioural characteristics of infected individuals. By contrast, significant differences in age (p<0.0001), ethnicity (p=0.001), proportion of women (p=0.01), and HIV status (p=0.03) were noted between the 14 prevalent heterosexual-associated strains. Heterosexuals with strains not shared by others in the sample were significantly older (p=0.0005) and more likely to have had sex outside the UK (p<0.0001) than those sharing a strain with at least one other.
The discriminatory high throughput strain characterisation method applied here identified localised transmission networks and suggests little bridging between networks of men who have sex with men and heterosexual networks.
The Lancet 07/2006; 368(9530):139-46. · 39.21 Impact Factor