Search

ABSTRACTFood-borne salmonellosis is a major manifestation of gastrointestinal disease in humans across the globe. Accurate and rapid identification methods could positively impact the identification of isolates, enhance outbreak investigation, and aid infection control. The SNaPshot multiplex system is a primer extension-based method that enables multiplexing of single nucleotide polymorphisms (SNPs). Here the method has been developed for the identification of five Salmonellaserotypes, commonly detected in the United Kingdom, based on serotype-specific SNPs identified in the multilocus sequence typing (MLST) database of Salmonella enterica. The SNPs, in genes hemD, thrA, purE, and sucA, acted as surrogate markers for S. entericaserovars Typhimurium, Enteritidis, Virchow, Infantis, and Braenderup. The multiplex primer extension assay (MPEA) was conducted in two separate panels and evaluated using 152 Salmonella entericaisolates that were characterized by MLST. The MPEA was shown to be 100% specific and sensitive, within this collection of isolates. The MPEA is a sensitive and specific method for the identification and detection of Salmonellaserotypes based upon SNPs seen in MLST data. The method can be applied in less than 6 h and has the potential to improve patient care and source tracing. The utility of the assay for identification of Salmonellaserotypes directly from clinical specimens and food samples warrants further investigation.

ABSTRACTA total of 88 uropathogenic Escherichia coliisolates, including 68 isolates from urine and 20 isolates from blood, were characterized by multilocus sequence typing (MLST). MLST has identified an important genetic lineage of E. coli, designated sequence type 131 (ST-131), represented by 52 of these isolates, 51 of which were resistant to extended-spectrum cephalosporins. ST-131 appears to be a drug-resistant uropathogenic strain of E. coliresponsible for causing urinary tract infections and bacteremia and is widely disseminated among both community and hospital patients from different geographical areas in the northwest of England. Application of MLST has helped to define the population biology which may underpin the epidemiology of pathogenic E. colistrains. The portability of MLST allows the accurate monitoring of this antibiotic-resistant uropathogenic strain of E. coliand will enhance surveillance for this important group of organisms.

ABSTRACTInfections associated with Neisseria meningitidisare a major public health problem in England, Wales, and Northern Ireland. Currently, over 40% of cases are confirmed directly from clinical specimens using PCR-based methodologies without an organism being isolated. A nested/seminested multilocus sequence typing (MLST) system was developed at the Health Protection Agency Meningococcal Reference Unit to allow strain characterization beyond the serogroup for cases confirmed by PCR only. This system was evaluated on a panel of 20 meningococcus-positive clinical specimens (3 cerebrospinal fluid and 17 blood samples) from different patients containing various concentrations of meningococcal DNA that had corresponding N. meningitidisisolates. In each case, the sequence type generated from the clinical specimens matched that produced from the corresponding N. meningitidisisolate; the sensitivity of the MLST system was determined to be less than 12 genome copies per PCR. The MLST system was then applied to 15 PCR meningococcus-positive specimens (2 cerebrospinal fluid and 13 blood samples), each from a different patient, involved in three case clusters (two serogroup B and one serogroup W135) for which no corresponding N. meningitidisorganisms had been isolated. In each case, an MLST sequence type was generated, allowing the accurate assignment of individual cases within each of the case clusters. In summary, the adaptation of the N. meningitidisMLST to a sensitive nested/seminested format for strain characterization directly from clinical specimens provides an important tool for surveillance and management of meningococcal infection.

ABSTRACTThe haploid pathogenic yeast Candida glabratais the second most common Candidaspecies isolated from cases of bloodstream infection. The clinical relevance of C. glabratais enhanced by its reduced susceptibility to fluconazole. Despite this, little is known of the epidemiology or population structure of this species. We developed a multilocus sequence typing (MLST) scheme for C. glabrataand used it to fingerprint a geographically diverse collection of 107 clinical isolates and 2 reference strains. Appropriate loci were identified by amplifying and sequencing fragments of the coding regions of 11 C. glabratagenes in 10 unrelated isolates. The 6 most variable loci (FKS, LEU2, NMT1, TRP1, UGP1, and URA3) were sequenced in the collection of 109 isolates. From the 3,345 bp sequenced in each isolate, 81 nucleotide sites were found to be variable. These defined 30 STs among the 109 strains. The technique was validated by comparison with random amplified polymorphic DNA and the complex DNA fingerprinting probes Cg6 and Cg12. MLST identified 5 major clades among the isolates studied. Three of the clades exhibited significant geographical bias. Our data demonstrate for the first time, with such a large geographically diverse strain collection, that distinct genetic clades of C. glabrataprevail in different geographical regions.

ABSTRACTThe genetic diversity of porBgenes from meningococcal isolates characterized as serotype 22 was investigated by gene sequencing. This procedure identified seven distinct porBsequences, demonstrating variation in the PorB protein recognized by the serotype 22 monoclonal antibody. This is consistent with the genetic heterogeneity of serotype 22 meningococci reported previously.