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Results for “Multilocus sequence typing”

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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↗

PubMed2003

Invasive disease due to nontypeable Haemophilus influenzae among children in Arkansas.

In this study, we reviewed cases of invasive disease due to nontypeable Haemophilus influenzae among children hospitalized at Arkansas Children's Hospital from 1993 to 2001. A total of 28 cases were examined, including 21 associated with bacteremia and 4 associated with meningitis. Of the patients examined, 86% were </=4 years of age, and 68% had underlying medical conditions. Characterization of the bacterial isolates by multilocus sequence type genotyping revealed significant overall genetic diversity, similar to the diversity in the general population structure for nontypeable H. influenzae. However, four separate pairs of isolates were closely related genetically, a relationship confirmed by pulsed-field gel electrophoresis and Southern hybridization studies using probes for the major H. influenzae adhesin genes. These results suggest that selected strains of nontypeable H. influenzae may have more invasive potential, especially in young children and patients with underlying medical conditions. At this point, the specific factors that contribute to enhanced virulence remain unclear.

Adolescent↗

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

Distribution of serogroups and genotypes among disease-associated and carried isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway.

The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes.

Carrier State↗

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

Molecular epidemiology of multiresistant Streptococcus pneumoniae with both erm(B)- and mef(A)-mediated macrolide resistance.

Of a total of 1043 macrolide-resistant Streptococcus pneumoniae isolates collected from 24 countries as part of PROTEKT 1999-2000, 71 isolates tested positive for both the mef(A) and erm(B) genes. Of 69 isolates subjected to further molecular investigations, all were resistant to tetracycline, 63 (91.3%) were resistant to penicillin, and 57 (82.6%) were resistant to trimethoprim-sulfamethoxazole. One isolate was also fluoroquinolone resistant, and another was resistant to quinupristin-dalfopristin. The ketolide telithromycin retained activity against all of the isolates. Of the 69 of these 71 isolates viable for further testing, 46 were from South Korea, 13 were from the United States, 8 came from Japan, and 1 each came from Mexico and Hungary. One major clonal complex (59 [85.5%] of 69 isolates) was identified by serotyping (with 85.5% of the isolates being 19A or 19F), pulsed-field gel electrophoresis, and multilocus sequence typing. The remaining isolates were less clonal in nature. Representative isolates were shown to carry the mobile genetic elements Tn1545 and mega, were negative for Tn1207.1, had tetracycline resistance mediated by tet(M), and contained the mef(E) variant of mef(A). All isolates were positive for mel, a homologue of the msr(A) efflux gene. These clones are obviously very efficient at global dissemination, and hence it will be very important to monitor their progress through continued surveillance. Telithromycin demonstrated high levels of activity (MIC for 90% of the strains tested, 0.5 micro g/ml; MIC range, 0.06 to 1 micro g/ml) against all isolates.

Anti-Bacterial Agents↗

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

Hyperinvasive neonatal group B streptococcus has arisen from a bovine ancestor.

The genetic relatedness and evolutionary relationships between group B streptococcus (GBS) isolates from humans and those from bovines were investigated by phylogenetic analysis of multilocus sequence typing data. The collection of isolates consisted of 111 GBS isolates from cows with mastitis and a diverse global collection of GBS isolates from patients with invasive disease (n = 83) and carriers (n = 69). Cluster analysis showed that the majority of the bovine isolates (93%) grouped into one phylogenetic cluster. The human isolates showed greater diversity and clustered separately from the bovine population. However, the homogeneous human sequence type 17 (ST-17) complex, known to be significantly associated with invasive neonatal disease, was the only human lineage found to be clustered within the bovine population and was distinct from all the other human lineages. Split decomposition analysis revealed that the human isolate ST-17 complex, the major hyperinvasive neonatal clone, has recently arisen from a bovine lineage.

Animals↗

PubMed2004

Clonality of Streptococcus pneumoniae serotype 1 isolates from pediatric patients in the United States.

We compared Streptococcus pneumoniae serotype 1 isolates causing disease among children in six geographic regions of the United States to determine genetic relatedness. Genomic fingerprints were determined by repetitive element polymorphism PCR (Rep-PCR). Multilocus sequence type characterization was performed on selected isolates. Four different genomic banding patterns were identified by Rep-PCR. One profile (clone 1) was predominant and matched sequence type 227.

Adolescent↗

PubMed2004

Widespread dissemination in The Netherlands of the epidemic berlin methicillin-resistant Staphylococcus aureus clone with low-level resistance to oxacillin.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and represents a growing public health burden due to the emergence and spread of epidemic strains, particularly within the hospital environment. An epidemic MRSA clone, with characteristic low-level resistance to oxacillin, emerged in the year 2000 and became endemic in the Netherlands. Multilocus sequence typing characterized the strain as sequence type 45, which was previously designated the Berlin epidemic MRSA clone. In 2 years, this strain has become the predominant MRSA clone in the Netherlands.

Drug Resistance, Bacterial↗

PubMed2004

Uses of Staphylococcus aureus GeneChips in genotyping and genetic composition analysis.

Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical.

Algorithms↗

PubMed2005

Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III.

Two new species, Candida orthopsilosis and C. metapsilosis, are proposed to replace the existing designations of C. parapsilosis groups II and III, respectively. The species C. parapsilosis is retained for group I isolates. Attempts to construct a multilocus sequence typing scheme to differentiate individual strains of C. parapsilosis instead revealed fixed DNA sequence differences between pairs of subgroups in four genes: COX3, L1A1, SADH, and SYA1. PCR amplicons for sequencing were obtained for these four plus a further seven genes from 21 group I isolates. For nine group II isolates, PCR products were obtained from only 5 of the 11 genes, and for two group III isolates PCR products were obtained from a different set of 5 genes. Three of the PCR products from group II and III isolates differed in size from the group I products. Cluster analysis of sequence polymorphisms from COX3, SADH, and SYA1, which were common to the three groups, consistently separated the isolates into three distinct sets. All of these differences, together with DNA sequence similarities <90% in the ITS1 sequence, suggest the subgroups should be afforded species status. The near absence of DNA sequence variability among isolates of C. parapsilosis and relatively high levels of sequence variability among isolates of C. orthopsilosis suggest that the former species may have evolved very recently from the latter.

Base Sequence↗

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

Clonal association between Streptococcus pneumoniae serotype 23A, circulating within the United States, and an internationally dispersed clone of serotype 23F.

Streptococcus pneumoniae is an important pathogen in the United States and is associated with significant morbidity and mortality. Since the introduction of the seven-valent conjugate vaccine, a significant decline in pneumococcal disease has been reported. However, surveillance for pneumococcal disease remains essential, as the extent of cross protection against vaccine-related serotypes is still unclear. Further, any increase in non-vaccine-related serotypes also needs monitoring. We report on a new clonal association between a vaccine-related serotype, serotype 23A, obtained as part of the Active Bacterial Core surveillance, with an established internationally dispersed Pneumococcal Molecular Epidemiology Network (PMEN) clone, clone Colombia(23F)-26. Sixty-two isolates of serotype 23A collected from sterile sites during a 2-year period (2002 and 2003) were characterized. Twenty-one (34%) isolates were penicillin nonsusceptible, although none were fully resistant. Pulsed-field gel electrophoresis and multilocus sequence typing analysis showed that 24 (39%) of the serotype 23A isolates shared either genetic identity or high genetic relatedness with PMEN clone Colombia(23F)-26. Extensive variability was noted within the sequenced region of pbp2b in two penicillin-nonsusceptible isolates as well as in PMEN clone Colombia(23F)-26, suggesting that these isolates probably acquired penicillin resistance independently. The emergence of such new serotype and genotype associations highlights the dynamic nature of the pneumococcal population, necessitating continuous monitoring in the post-vaccine era.

Adolescent↗

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↗

PubMed2005

Campylobacter coli in swine production: antimicrobial resistance mechanisms and molecular epidemiology.

The aim of this study was to determine antimicrobial resistance, to evaluate and compare the use of two genotyping methods for molecular epidemiology purposes, and to determine the genotypic diversity of Campylobacter coli of porcine origin. A total of 100 C. coli isolates from swine were tested for susceptibility to six antimicrobials using the agar dilution method and genotyped using two high-resolution fingerprinting approaches: multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Evaluation of the methods was based on their resistance patterns, discriminatory indexes (DI), high test throughputs, costs, and turnaround times. Resistance to erythromycin and tetracycline was the most common. Both genotypic methods were found to have high discriminatory power, although MLST had a higher DI (0.936) than PFGE (DI = 0.889). It also had a higher throughput than PFGE. Isolates were clustered into 27 groups by MLST compared to 11 by PFGE. MLST was able to further discriminate the isolates grouped under the same cluster by PFGE. Out of the 65 MLST sequence types (STs) identified among the total isolates, 50 were reported for the first time. Most STs were found to be specific to the farm (n = 38) and to slaughter (n = 22). Resistance against tetracycline and erythromycin was encoded by the tet(O) gene and a A2075G point mutation in the 23S rRNA gene, respectively. A high ciprofloxacin MIC (>64 microg/liter) was conferred by a point mutation in the gyrA gene. The weak clonal structure of the C. coli population among swine was further highlighted by the index of association value of 0.293. The findings of this study indicate that multidrug-resistant diverse C. coli strains exhibiting resistance to ciprofloxacin and erythromycin are concerning, since these are the drugs of choice for treating invasive campylobacteriosis cases in humans.

Abattoirs↗

PubMed2005

Recurrent melioidosis in patients in northeast Thailand is frequently due to reinfection rather than relapse.

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (P = 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (P < 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.

Bacterial Typing Techniques↗