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PubMed2005

International spread of major clones of methicillin resistant Staphylococcus aureus: nosocomial endemicity of multi locus sequence type 239 in Saudi Arabia and Romania.

Phenotypically identified methicillin resistant Staphylococcus aureus (MRSA) strains from several hospitals in Romania and Saudi Arabia (n = 103 and 68, respectively) were confirmed to be MRSA by mecA PCR and PBP-2' based latex agglutination. Subsequently, strains were differentiated at the sub-species level using pulsed field gel electrophoresis (PFGE) of SmaI DNA macro-restriction fragments. Comparison of the PFGE fingerprints identified major clusters of strains, persistently present in the various hospitals. Endemicity of certain strains was identified, amongst others one due to a particularly methicillin resistant type in the burn wound sector of the Romanian hospital. No PFGE-based overlap was found between the Saudi and Romanian strains. However, multi locus sequence typing (MLST), performed for 20% of all strains, revealed that genuine genetic similarity was obscured by the PFGE analysis. In both the Romanian and Saudi hospitals the renowned sequence type (ST) 239 was very over-represented. This was especially apparent in Saudi Arabia, where all strains except two shared the ST 239 genotype. This clonal type has previously been identified in a variety of other countries. Despite the MLST concordance, PFGE data indicate that ST 239 diversifies while maintaining its core genome intact. ST 80, another previously but less frequently identified clone, was introduced in 2000 in the Romanian institutes and persisted over the past 3 years as a frequent cause of infections in a surgical department. The successful MRSA types can acquire prominent positions in hospitals of previously low-endemicity MRSA status.

Bacterial Typing Techniques↗

PubMed2006

Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance.

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.

Ampicillin Resistance↗

PubMed2003

Multi-locus sequence typing: a tool for global epidemiology.

The characterization of pathogenic isolates plays a pivotal role in the epidemiology of infectious diseases, generating the information necessary for identifying, tracking, and intervening against disease outbreaks. In 1998 multi-locus sequence typing (MLST) was proposed as a nucleotide sequence-based approach that could be applied to many bacterial pathogens. It combined developments in high-throughput sequencing and bioinformatics with established population genetics techniques to provide a portable, reproducible, and scalable typing system that reflected the population and evolutionary biology of bacterial pathogens. MLST schemes have been developed for a variety of procaryotic and eucaryotic pathogens and the data generated have contributed to both epidemiological surveillance and fundamental studies of pathogen biology.

Bacteria↗

PubMed2004

Multilocus sequence typing--what is resolved?

Nucleotide sequence-based methods for bacterial typing (multilocus sequence typing; MLST) allow rapid and global comparisons between results from different laboratories. Combining this advantage with the reduced cost of high throughput sequencing, increasing automation and the amenability of sequence data for evolutionary analysis, it seems inevitable that sequence-based typing will eventually predominate over gel-based methods such as pulsed-field gel electrophoresis (PFGE) for most bacterial species. The increasing availability of multiple genome sequences for single pathogenic species, and the recent development of many new MLST schemes, means that a re-examination of the utility of multilocus sequencing, and in particular the choice of gene loci, is now appropriate.

Bacteria↗

PubMed2006

Clonal relationships among Clostridium perfringens of porcine origin as determined by multilocus sequence typing.

Clostridium perfringens is ubiquitous in the environment and the intestinal tracts of most mammals, but this organism also causes gas gangrene and enteritis in human and animal hosts. While expression of specific toxins correlates with specific disease in certain hosts, the other factors involved in commensalism and host pathogenesis have not been clearly identified. A multilocus sequence typing (MLST) scheme was developed for C. perfringens with the aim of grouping isolates with respect to disease presentation and/or host preference. Sequence data were obtained from one virulence and seven housekeeping genes for 132 C. perfringens isolates that comprised all five toxin types and were isolated from 10 host species. Eighty sequence types (STs) were identified, with the majority (75%) containing only one isolate. eBURST analysis identified three clonal complexes, which contained 59.1% of the isolates. Clonal complex (CC) 1 contained 31, predominantly type A isolates from diverse host species. Clonal complex 2 contained 75% of the bovine type E isolates examined in this study. Clonal complex 3 consisted predominantly of porcine type A and type C isolates. Interestingly, these porcine isolates (n=32) all carried consensus cpb2 and cna genes, encoding beta2 toxin and CpCna, a collagen binding protein, respectively. This compares to carriage of both these genes by only 3.6% of porcine isolates not present in clonal complex 3 (n=28). The data obtained indicates that MLST may be used to identify host species relationships with respect to these C. perfringens isolates.

Animals↗

PubMed2006

Characteristics of methicillin resistant Staphylococcus aureus isolated from chicken meat and hospitalized dogs in Korea and their epidemiological relatedness.

Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important pathogens in human and veterinary hospitals. The isolation of MRSA from animals and foodstuffs has been reported with an increased incidence. However, methicillin (oxacillin) is not used in animal husbandry or in animal hospitals in Korea. In this study, three pre-MRSA and one silent mecA-carrying methicillin susceptible S. aureus (smMSSA) were isolated from retail chicken meat, and three MRSA were isolated from hospitalized dogs in Korea. The three pre-MRSA isolates were determined to have a staphylococcal cassette chromosome mec (SCCmec) type III, and the smMSSA isolate was not classified. The animal hospital isolates were found to contain SCCmec type II. Seven and 15 S. aureus isolated from hospitalized humans and bovine milk, respectively, were also examined in this study in order to determine the epidemiological origins of MRSA. Multilocus sequencing typing (MLST) revealed that the chicken meat and bovine milk isolates were closely related to the animal hospital isolates. The SCCmec characteristics and MLST analyses indicated the possibility of the human to animal transmission of MRSA. These results highlight the importance of identifying MRSA carriers as well as intercepting MRSA transmission because MRSA is becoming increasingly widespread without any plausible relationship with the use of methicillin (oxacillin).

Animals↗

PubMed2006

Identification of a new Neisseria meningitidis serogroup C clone from Anhui province, China.

BACKGROUND: Outbreaks of a new serogroup C meningococcal disease emerged during 2003-04 (five outbreaks with 43 cases) and in 2004-05 (five outbreaks with 29 cases), all in Anhui province, China. We describe the molecular epidemiology and features of the causative bacterial strains. METHODS: We used pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to analyse the strains. FINDINGS: Of 34 strains of Neisseria meningitidis cultured during 2003-04 from Anhui province, 31 were group C meningococci, 28 of which were associated with three of five outbreaks; one from a patient and 27 from close contacts of eight patients. Of 30 strains isolated from Anhui province during 2004-05, 17 were identified as serogroup C meningococci, ten of which were associated with four of five outbreaks. In a nationwide survey, 542 strains were isolated during 2004-05; 58 were serogroup C meningococci interspersed among 11 other provinces where no serogroup C outbreak occurred. Of the 106 serogroup C strains analysed, 89 had identical PFGE patterns, designated AH1. Of 28 strains selected for MLST analyses, 25 were sequence type 4821 (ST-4821), which did not belong to any of the previously reported sequence types that can form a new hypervirulent lineage. INTERPRETATION: ST-4821 seems to be unique and caused the serogroup C meningitis outbreaks during the two seasons from 2003 to 2005 in Anhui province. The emergence of this sequence type has epidemiological importance that should be monitored for future spread in China and the rest of the world.

Child↗

PubMed2003

Multi locus fingerprinting of Listeria monocytogenes by sequence-specific labeling of DNA probes combined with array hybridization.

We have developed an alternative multi locus sequence typing (MLST) approach that targets the variable genetic changes directly in a DNA array format. Our approach is based on DNA array hybridization in combination with sequence-specific labeling of oligonucleotide probes. Listeria monocytogenes was chosen for the development and evaluation of the assay. The genes hlyA, iap, flaA, inlA and actA were targeted. Twenty-nine suitable probe regions were identified within these genes. The DNA array results from 32 different strains were compared to serotype and amplified fragment length polymorphism data. This comparison showed that our DNA array method gave good discrimination between the strains analyzed. In conclusion, the DNA array-based MLST method is a promising tool for fingerprint bacteria.

Bacterial Typing Techniques↗

PubMed1999

Multilocus sequence typing.

Multilocus sequence typing (MLST) provides a new approach to molecular epidemiology that can identify and track the global spread of virulent or antibiotic-resistant isolates of bacterial pathogens using the Internet. MLST databases, together with interrogation software, are available for Neisseria meningitidis and Streptococcus pneumoniae and databases for Streptococcus pyogenes and Staphylococcus aureus will be released shortly.

Bacteria↗

PubMed2002

Multilocus sequence typing of Neisseria meningitidis directly from cerebrospinal fluid.

MLST typing of Neisseria meningitidis directly from clinical material was introduced in the National Reference Laboratory for Meningococcal Infections in Prague. Four cerebrospinal fluid samples were obtained from patients with suspected meningococcal invasive disease. In all samples, all classical laboratory methods gave negative results and the only positive method was PCR, which revealed Neisseria meningitidis group C (two specimens) and group B (two specimens), respectively. MLST performed directly from cerebrospinal fluid revealed that the strains causing the two group C infections were of sequence type (ST) 11, while the two group B infections were characterized as ST-32 and ST-33 respectively. Multi-locus sequence typing of meningococci directly from clinical material offers the opportunity to improve further the surveillance of meningococcal disease and has now been introduced into the routine portfolio of tests employed at the national reference laboratory of the Czech Republic.

Adolescent↗

PubMed2001

Multilocus sequence typing of Streptococcus pneumoniae clones with unusual drug resistance patterns: genetic backgrounds and relatedness to other epidemic clones.

Six drug-resistant Streptococcus pneumoniae clones were previously identified from day care centers in Portugal, primarily on the basis of common pulsed-field gel electrophoresis (PFGE) patterns. These clones were susceptible to penicillin or had only very low-level resistance to it (most MICs, < or =0.25 microg/mL) and accounted for a large proportion (35%) of all drug-resistant pneumococci colonizing the nasopharynx of healthy children attending day care. Five of the 6 clones were identified among pneumococcal clinical isolates collected in other countries. In this study, we applied multilocus sequence typing (MLST) to describe the genetic background of these clones. MLST confirmed previous findings obtained by PFGE and allowed for the extension of the international clonal relationships by showing that each of the 6 clones was internationally disseminated and was able to cause pneumococcal disease.

Anti-Bacterial Agents↗

PubMed2004

Evolution of erythromycin-resistant Streptococcus pneumoniae from Asian countries that contains erm(B) and mef(A) genes.

To investigate the genetic characteristics of erythromycin-resistant Streptococcus pneumoniae in Asian countries, 110 pneumococcal isolates were analyzed by multilocus sequence typing (MLST). MLST analysis showed that 2 clonal complexes--CC236 (Taiwan19F-14 clone) and CC81 (Spain23F-1 clone)--are the major lineages of erythromycin-resistant S. pneumoniae in the Asian region. Pneumococcal isolates containing both the erm(B) and the mef(A) genes are thought to have originated from the Taiwan19F-14 clone containing the mef(A) gene, after introduction of the erm(B) gene. Further evolution of this variant clone has generated resistant strains with different sequence types. Dissemination of these variant clones of the Taiwan19F-14 could be the main reason for the high frequency of pneumococcal isolates containing both erm(B) and mef(A) in some Asian countries. Data suggest that the high prevalence of erythromycin-resistant S. pneumoniae in the Asian region is partly due to the clonal spread of a few multidrug-resistant clones.

Asia↗

PubMed2004

Emergence of penicillin-nonsusceptible Streptococcus pneumoniae clones expressing serotypes not present in the antipneumococcal conjugate vaccine.

BACKGROUND: Penicillin-nonsusceptible Streptococcus pneumoniae isolates are confined mainly to a few serogroups. Capsular transformation may serve as a mechanism for spreading antibiotic resistance to new serotypes. METHODS: Antibiogram and molecular typing, by pulsed-field gel electrophoresis (PFGE), were performed on 46 nasopharyngeal and middle ear fluid (MEF) isolates expressing serotype 11A, 45 MEF isolates expressing serotype 15B/C (recovered during 1998-2003 from Israeli children <5 years old), and 57 MEF isolates expressing serotype 19F (recovered during 1998-2001 from Costa Rican children <7.5 years old). RESULTS: PFGE patterns showed that 49 (86%) of 57 serotype 19F isolates and 19 (41%) of 46 serotype 15B/C isolates were closely related. The vast majority of these isolates (80% of serotype 19F and 100% of serotype 15B/C isolates) were nonsusceptible to penicillin. Multilocus sequence typing (MLST) data show that the serotype 15B/C isolates belonged to the ST346 cluster, whereas the serotype 19F isolates were a single-locus variant of ST346. For serotype 11A isolates, PFGE patterns and MLST analysis showed that 8 (80%) of the 10 penicillin-nonsusceptible isolates belonged to a single clone--namely, ST156--which was identical to the international Spain9V-3 clone. CONCLUSIONS: Penicillin-nonsusceptible pneumococcal clones of serotypes not related to those included in the 11-valent conjugate vaccines may derive from capsular transformation of vaccine-related serotypes. Of particular concern was the detection of serotype 11A variants of the successful international Spain9V-3 clone. This phenomenon, although seemingly rare at present, can have implications for the long-term effectiveness of the conjugate vaccines.

Child↗

PubMed2005

An outbreak of serotype 1 Streptococcus pneumoniae meningitis in northern Ghana with features that are characteristic of Neisseria meningitidis meningitis epidemics.

BACKGROUND: The Kassena-Nankana District (KND) of northern Ghana lies in the African meningitis belt, where epidemics of bacterial meningitis have been reoccurring every 8-12 years. These epidemics are generally caused by Neisseria meningitidis, an organism that is considered to be uniquely capable of causing meningitis epidemics. METHODS: We recruited all patients with suspected meningitis in the KND between 1998 and 2003. Cerebrospinal fluid samples were collected and analyzed by standard microbiological techniques. Bacterial isolates were subjected to serotyping, multilocus sequence typing (MLST), and antibiotic-resistance testing. RESULTS: A continual increase in the incidence of pneumococcal meningitis was observed from 2000 to 2003. This outbreak exhibited strong seasonality, a broad host age range, and clonal dominance, all of which are characteristic of meningococcal meningitis epidemics in the African meningitis belt. The case-fatality rate for pneumococcal meningitis was 44.4%; the majority of pneumococcal isolates were antibiotic sensitive and expressed the serotype 1 capsule. MLST revealed that these isolates belonged to a clonal complex dominated by sequence type (ST) 217 and its 2 single-locus variants, ST303 and ST612. CONCLUSIONS: The S. pneumoniae ST217 clonal complex represents a hypervirulent lineage with a high propensity to cause meningitis, and our results suggest that this lineage might have the potential to cause an epidemic. Serotype 1 is not included in the currently licensed pediatric heptavalent pneumococcal vaccine. Mass vaccination with a less complex conjugate vaccine that targets hypervirulent serotypes should, therefore, be considered.

Drug Resistance, Bacterial↗

PubMed2004

Vancomycin-resistant Enterococcus faecium isolates causing hospital outbreaks in northern Italy belong to the multilocus sequence typing C1 lineage.

Multilocus sequence typing (MLST) was used to obtain insights into the genetic relationships between 14 vancomycin-resistant Enterococcus faecium (VREF) isolates from humans (hospitalized patients, 5 strains) and nonhuman sources (meat and poultry, 9 strains) in northern Italy over the period 1993-2001. The typing scheme (Homan et al., 2002, J. Clin. Microb., 40:1963-1971) based on seven housekeeping genes--adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine-D-alanine ligase), gyd (glyceraldehyde-3-phosphate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), purK (phosphoribosylaminoimidazole carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter)--was used. In the 14 VREF analyzed, the number of unique alleles ranged from 1 (gyd) to 8 (atpA). Isolates from hospitalized patients were defined by the unique allele purK 1. Nine sequence types (STs) were identified. All of the epidemic strains isolated over the period 2000-2001 showed identical or closely related pulsed-field gel electrophoresis (PFGE) patterns and clustered in the same ST78. These strains shared six of the seven alleles with the strain CA20 representative of the 1993-1999 outbreaks, which PFGE indicated as being unrelated to those of the recent outbreaks. MLST confirmed the unrelatedness of human and nonhuman strains already detected by PFGE. All isolates clustered in three main genetic lineages: group A comprised two of the three isolates from meat; group C the human strains of all outbreaks and one poultry strain; and group B four of the five poultry strains and one meat strain. All human strains carried the esp gene and clustered in the C1 sublineage that has been described as having emerged recently worldwide.

Alleles↗

PubMed2006

Genomic backgrounds of drug-resistant Streptococcus pneumoniae in Ankara, Turkey: Identification of emerging new clones.

Streptococcus pneumoniae exhibiting decreased susceptibility to penicillin are isolated with an increasing prevalence in Turkey during the last decade. This study was undertaken to investigate the molecular epidemiology of non-penicillin-susceptible pneumococci isolated in Ankara, Turkey. Among a population of 246 pneumococci, 90 pneumococci with penicillin MIC > or = 0.1 microg/ml were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE), and sequence typed by multilocus sequence typing (MLST). The overall resistance to penicillin, cefotaxime, erythromycin, clindamycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, and vancomycin were 36.6%, 4%, 27.6%, 10.9%, 5.3%, 22.4%, 4.5%, 2%, and 0, respectively. The most frequent serotypes were 14, 23B, 9V, 19F, 19A, and 23F. PFGE types represented 17 genetic clusters. PFGE and MLST data revealed that there were isolates identical or closely related to the Spain(9V)-3 ST 156 clone, Portugal(19F)- 21 ST 177 clone, and Spain(23F)-1 ST81 clone. Eleven serotype 14 isolates with emerging resistance to penicillin belonged to the ST 230 complex, a predominantly susceptible clone. Serotype 19A, 19F, and 7F variants of the ST 230 clone were also identified in the study population. Eight serotype 23B isolates with a new ST 1349 (18-13-8-6-3-6-8) created another clone with no relation to the currently defined international clones. Although the pandemic clones Spain(9V)-3, Portugal1(9F)-21, and Spain(23F)-1 are present in our region, the emergence of a new 23B clone with a unique ST and the emergence of resistance in the ST230 clone, has presumably contributed to the increase in the prevalence of drug-resistant pneumococci in Turkey.

Anti-Bacterial Agents↗

PubMed2003

Intra-hospital dissemination of quinupristin/dalfopristin- and vancomycin-resistant Enterococcus faecium in a paediatric ward of a German hospital.

OBJECTIVES: To demonstrate nosocomial transmission of Enterococcus faecium resistant to quinupristin/dalfopristin and vancomycin/teicoplanin among paediatric patients in a German hospital ward. MATERIALS AND METHODS: Multiply-resistant E. faecium were isolated from three female patients aged 9 months, 2 and 15 years during a 10 day time span. Antibiotic susceptibilities were determined by microbroth dilution. Clonal relatedness among the isolates was investigated via SmaI-macrorestriction analysis by PFGE, multilocus sequence typing (MLST), and plasmid profiling. Presence of virulence and resistance determinants was tested by polymerase chain reaction (PCR). Selected resistance genes were localized by Southern hybridizations. RESULTS: A single E. faecium isolate per patient was investigated. All exhibited resistances to quinupristin/dalfopristin, vancomycin/teicoplanin, streptomycin (high-level), penicillin/ampicillin, erythromycin, oxytetracycline, chloramphenicol, rifampicin and fusidic acid. The isolates were susceptible to linezolid only and intermediately resistant to fluoroquinolones including moxifloxacin. PFGE revealed identical patterns for all three isolates. PCRs for virulence determinants hyaluronidase and enterococcal surface protein, esp, were negative, whereas PCR for the enterocin A gene was positive. MLST identified clonal type [8-5-1-1-1-1-1] belonging to a clonal subgroup C1 of hospital- and outbreak-related E. faecium. Southern hybridizations located several resistance genes (erm(B), vat(D), vanA) on a large plasmid, which was transferable in mating experiments with an E. faecium recipient. CONCLUSIONS: These data show routes of dissemination of resistance to multiple antibiotics including streptogramins and glycopeptides in E. faecium via vertical and/or horizontal gene transfer. The isolates spread in the absence of a direct selective pressure, as none of the patients had received earlier streptogramin or glycopeptide therapy.

Anti-Bacterial Agents↗

PubMed2004

Emergence of invasive erythromycin-resistant Streptococcus pneumoniae strains in Portugal: contribution and phylogenetic relatedness of serotype 14.

OBJECTIVES: To study the phenotype and phylogenetic relatedness of invasive Streptococcus pneumoniae strains isolated in Portugal. METHODS: A total of 614 invasive S. pneumoniae strains, isolated in Portugal from 1999 to the first 6 months of 2002, were characterized using serotyping and antimicrobial susceptibility testing. National outpatient sale data for macrolides were compared with erythromycin resistance. We investigated the main clonal lineages from erythromycin-resistant strains of serotype 14 by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: The emergence of erythromycin-resistant strains correlated well with the usage of azithromycin in Portugal during the period of the study (r=0.900, P=0.001). Serotype 14 made the largest contribution to this emergence. We found two clonal complexes (CC) among erythromycin-resistant strains: CC1 was formed by three sequence types (ST), ST156, ST143 and ST1042, and CC2 by ST15 and ST9. All STs described, except ST1042, are putative founders of clonal groups or subgroups from serotype 14 as defined in the pneumococcal MLST database. CONCLUSIONS: This study suggests that macrolides usage is an important factor enhancing the spread of invasive erythromycin-resistant S. pneumoniae clones in Portugal. The study also makes a contribution to the understanding of spread of erythromycin-resistant clones in an international context.

Anti-Bacterial Agents↗