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PubMed2003

Collaborative consensus for optimized multilocus sequence typing of Candida albicans.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

Candida albicans↗

PubMed2003

Multilocus sequence typing of Candida glabrata reveals geographically enriched clades.

The haploid pathogenic yeast Candida glabrata is the second most common Candida species isolated from cases of bloodstream infection. The clinical relevance of C. glabrata is 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. glabrata and 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. glabrata genes 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. glabrata prevail in different geographical regions.

Antifungal Agents↗

PubMed2003

Development of a multilocus sequence typing method for analysis of Listeria monocytogenes clones.

This study is a first step in the development of multilocus sequence typing (MLST) method for Listeria monocytogenes. Nine housekeeping genes were analyzed in a set of 62 strains isolated from different sources and geographic locations in Spain. These strains were previously characterized by pulsed-field gel electrophoresis (PFGE). Because of low diversity, two loci were discarded from the study. The sequence analysis of the seven remaining genes showed 29 different allelic combinations, with 22 of them represented by only one strain. The results of this sequence analysis were generally consistent with those of PFGE. Because MLST allows the easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be a useful tool for the listeriosis surveillance systems that will allow the identification and distribution of analysis of L. monocytogenes clones in the environment.

Alleles↗

PubMed2003

Evolution of a vancomycin-intermediate Staphylococcus aureus strain in vivo: multiple changes in the antibiotic resistance phenotypes of a single lineage of methicillin-resistant S. aureus under the impact of antibiotics administered for chemotherapy.

A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 micro g/ml for the initial isolate to an MIC of 8 micro g/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates-apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

Anti-Bacterial Agents↗

PubMed2003

Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras, Greece.

Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with the mecA- and Tn554-specific DNA probes were used to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected in 1993 and 1998 to 2000 at the University Hospital of Patras, Patras, Greece. Representatives of each clonal type were analyzed by spaA typing, multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing. The results indicated the existence of two successive international MRSA clones: (i) a clonal type with PFGE type A, sequence type (ST) 30 (ST30), and SCCmec type IV, which was very similar to a clone widely spread in the United Kingdom, Mexico, and Finland, and (ii) a clonal type with PFGE type B, ST239, and SCCmec III, which was related to the Brazilian clone. Both clones seem to be widespread in Greece as well. A novel MRSA clone is also described and is characterized by a new MLST type (ST80) associated with SCCmec type IV and with the presence of Panton-Valentine leukocidin genes.

Bacterial Typing Techniques↗

PubMed2003

Genetic relatedness of levofloxacin-nonsusceptible Streptococcus pneumoniae isolates from North America.

We characterized 32 levofloxacin-nonsusceptible Streptococcus pneumoniae (LNSP) isolates obtained from a broad geographic region of North America over a 5-year period by using capsular serotypes, antimicrobial susceptibility profiles, BOX-PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sixteen international clones identified by the Pneumococcal Molecular Epidemiology Network also were included for comparison. Fifteen serotypes were represented, with serogroups 6, 9, 14, 19, and 23 accounting for 63% of isolates. Among isolates whose quinolone resistance-determining regions were sequenced, all contained gyrA and parC point mutations. Sixty-three percent were penicillin susceptible, and 84% were erythromycin susceptible. BOX-PCR analysis identified 39 different band patterns among 32 LNSP and 16 international clones and grouped 16 isolates, including 2 international clones, into seven unrelated groups of 2 to 4 isolates each. PFGE analysis identified 35 different band patterns among 32 LNSP and 16 international clones and grouped 21 isolates, including 3 international clones, into eight unrelated groups of 2 to 6 isolates each. MLST performed on 10 isolates identified five allelic profiles and separated 9 isolates into four groups of 2 to 3 isolates each. Overall, each typing method indicated that the LNSP were heterogeneous and that resistance to fluoroquinolones was not closely associated with a particular serotype or with coresistance to other antimicrobial classes and suggests that LNSP have likely arisen through independent mutational events as a result of selective pressure. However, seven LNSP were found to be related to three international clones by PFGE.

Anti-Bacterial Agents↗

PubMed2003

Phenotypic and genotypic characterization of two penicillin-susceptible serotype 6B Streptococcus pneumoniae clones circulating in Italy.

Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain(6B)-2. The pbp2b, pbp2x, dhf, and pspA genes of the isolates had identical restriction profiles; and the partial sequence of pspA was identical to that of clone Spain(6B)-2. In all isolates the resistance determinants erm(B) and tet(M) were inserted in a Tn1545-like element; 11 isolates carried cat as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their pspA profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary(19A)-6 and Poland(23F)-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.

Adult↗

PubMed2004

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

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

Bacterial Proteins↗

PubMed2004

Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital.

Three hundred seventy-five consecutive methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered between 1998 and 2002 at the Nuestra Señora de Candelaria University Hospital in Tenerife, Spain, were analyzed by molecular fingerprinting techniques to determine MRSA clonal types and their prevalence over time. After determining antibiotic susceptibility, we used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize MRSA isolates and to establish PFGE types. Additionally, several selected isolates representative of each major PFGE type were tested by multilocus sequence typing (MLST) and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec), generating the corresponding sequence type (ST)-SCCmec types. Results of PFGE, supported by those of MLST and SCCmec typing, allowed us to identify six MRSA clones within the five major PFGE types and document temporal shifts in the prevalence of these MRSA clones from 1998 to 2002. Four of the clones were the pandemic "Iberian" (designated ST247-MRSA-IA), EMRSA-15 (ST22-MRSA-IV), EMRSA-16 (ST36-MRSA-II), and the so-called pediatric (ST5-MRSA-IV) clones, while the other two (ST125-MRSA-IVA and ST146-MRSA-IVA) clones could be derived from the pediatric one. The most striking temporal shift in the dominance of MRSA clones was the replacement of the multidrug-resistant and highly epidemic Iberian clone by the so-called British EMRSA-16 clone during the 5-year surveillance period. Our results are in accordance with previously stated findings showing the worldwide hospital dominance of relatively few pandemic and presumably virulent MRSA clones. We report for the first time the detection in Spain of the British EMRSA-15 and pediatric clones, as well as the abrupt replacement of the Iberian by the EMRSA-16 as the major MRSA clone.

Anti-Bacterial Agents↗

PubMed2004

Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia.

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80slv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.

Anti-Bacterial Agents↗

PubMed2004

Prevalence and molecular analysis of macrolide and fluoroquinolone resistance among isolates of Streptococcus pneumoniae collected during the 2000-2001 PROTEKT US Study.

The PROTEKT US (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin in the United States) surveillance program was established to determine the prevalence and mechanisms of antibacterial resistance among bacterial pathogens from patients with community-acquired respiratory tract infections. In year 1 of the PROTEKT US study, 10,103 isolates of Streptococcus pneumoniae, including 3,133 erythromycin-resistant strains and 81 levofloxacin-resistant strains, were collected from 206 centers. We report on the molecular analyses of these resistant strains. The resistance genotypes among the 3,044 typed macrolide-resistant isolates overall were mef(A) (n = 2,157; 70.9%), erm(B) (n = 530; 17.4%), mef(A) erm(B) (n = 304; 10.0%), and erm(A) subclass erm(TR) (n = 5; 0.2%). Fifty (1.6%) macrolide-resistant isolates were negative for the mef and the erm resistance genes. Seventy-eight (96.3%) of the 81 levofloxacin-resistant isolates analyzed possessed multiple mutations in the gyrA, gyrB, parC, and/or parE quinolone resistance-determining regions. A total of 43 known multilocus sequence typing (MLST) profiles (or single- or double-locus variants) accounted for 75 of 81 isolates. There was no evidence of dissemination of fluoroquinolone-resistant clones within the United States; however, 12 isolates with the same MLST profile were located in one center in Massachusetts. Almost 90% of the erythromycin-resistant isolates and approximately one-third of the levofloxacin-resistant isolates were multidrug resistant.

Anti-Bacterial Agents↗

PubMed2004

Isolates of Burkholderia pseudomallei from Northern Australia are distinct by multilocus sequence typing, but strain types do not correlate with clinical presentation.

Melioidosis is the disease caused by the saprophytic organism Burkholderia pseudomallei. Previous studies have suggested some strain tropism and differential virulence. In this study, we defined strains by multilocus sequence typing (MLST) of isolates taken from the Top End of Australia's Northern Territory and compared the results with those of other strains typed worldwide. We specifically sought clinical and geographical correlates of strain types. Among 87 Australian isolates, 48 sequence types were defined. None of the sequence types in this study has been found elsewhere in the world. Strains were distributed widely throughout the region, and the different presentations of disease, including neurological and prostatic infection, were associated with many different strains. There was excellent congruence between pulsed-field gel electrophoresis and MLST, and the two typing methods had a similar level of strain discrimination. The work suggests that host and environmental factors may be more important in determining disease presentation than infecting strain type. It is possible that the distinct but diverse strain types found in this study reflect Australia's geographical isolation over many millions of years.

Bacterial Proteins↗

PubMed2004

Genetic analysis of diverse disease-causing pneumococci indicates high levels of diversity within serotypes and capsule switching.

We have used multilocus sequence typing (MLST) and serotyping to build a phylogenetic framework for pneumococcal disease isolates in Scotland that provides a snapshot of the relationships between capsular type and genotype. The results show that while the MLST type correlates with the serotype, isolates within a serotype can belong to a number of individual clonal complexes or sequence types (STs). We also show that isolates of the same ST can express different capsular polysaccharides, i.e., display capsular switching, and that this phenomenon is observed both for capsular types commonly isolated from patients with invasive disease and for serogroups less commonly isolated from patients with invasive disease but which may commonly be carried asymptomatically in the human nasopharynx.

Adolescent↗

PubMed2004

mecA Locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone.

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 272G polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

Bacterial Proteins↗

PubMed2004

Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types.

A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sodA). The number of alleles ranged from five (dutA and ddl) to eleven (recA). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages). However, A(-) B(+) variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A(-) B(+) variant isolates. C. difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.

Alleles↗

PubMed2004

Identification of Campylobacter jejuni multilocus sequence type ST-21 clonal complex by single-nucleotide polymorphism analysis.

Conserved single-nucleotide polymorphisms (SNPs) which characterize the allelic profile of the major epidemiological lineage ST-21 were identified from the alleles within the current Campylobacter jejuni multilocus sequence typing (MLST) database. Allelic discrimination assays were designed for the detection of SNPs, enabling rapid strain profiling for clonal complex ST-21. This method is suitable for epidemiological investigations and is complementary to full MLST.

Alleles↗

PubMed2005

Distribution of major genotypes among methicillin-resistant Staphylococcus aureus clones in Asian countries.

To investigate the evolutionary pattern and genotypic characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains in the Asian region, 74 MRSA strains isolated from 12 Asian countries were analyzed by multilocus sequence typing (MLST) and SCCmec typing. Overall, a total of 16 genotypes based on sequence type and SCCmec types were identified among MRSA strains from Asian countries. Data revealed two major genotypes of MRSA strains in Asia, with unique geographic distributions. By MLST analysis, all strains from Korea and Japan except one belonged to clonal complex 5 (CC5) while most MRSA isolates from other Asian countries belonged to CC239. SCCmec typing showed that most isolates from Korea and Japan were SSmec type II whereas SCCmec type III (or IIIA) was the most common type in strains from other Asian countries. Our data documented a unique geographic distribution and evolutionary pattern of MRSA clones in Asia.

Anti-Bacterial Agents↗

PubMed2005

Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany, from 1984 to 1998.

To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.

Electrophoresis, Gel, Pulsed-Field↗