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Molecular techniques for MRSA typing: current issues and perspectives.

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially for therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Bacterial Typing Techniques↗

The 1998 Senegal epidemic of meningitis was due to the clonal expansion of A:4:P1.9, clone III-1, sequence type 5 Neisseria meningitidis strains.

Between January and April 1998, a meningitis outbreak due to serogroup A meningococcus took place in Senegal. The outbreak began in Gandiaye, 165 km to the east of Dakar, and progressed towards the towns of Gossas, Niakkhar, Guinguineo, Fatik, Foundiougne, Dioffior, Sokone, Kaolack, and Nioro. At the same time, the outbreak reached regions of Kaffrine, Koungheul, and Tambacounda in the east of Senegal. A total of 1,350 cases and 200 deaths were reported. The WHO Collaborating Center in Marseilles received 24 strains for analysis. All were serogroup A Neisseria meningitidis, type 4 and subtype P1.9. Multilocus enzyme electrophoresis, performed by Institut Pasteur Paris, showed that the strains belonged to clone III-1. DNA restriction fragments generated by endonuclease BglII and analyzed by pulsed-field gel electrophoresis showed 24 indistinguishable fingerprint patterns similar to those of meningococcus strains isolated from African outbreaks since 1988. Three strains were studied by multilocus sequence typing (MLST) with seven loci. The comparison between sequences and existing alleles on the MLST website () allowed us to assign these strains to sequence type 5 (ST5), as their sequences were identical to the consensus at seven loci. All 24 strains were susceptible to penicillin, amoxicillin, chloramphenicol, and rifampin. Subgroup III is finishing its spread towards west of the meningitis belt of Africa. To our knowledge, this is the first time subgroup III, and more precisely ST5, strains are reported as being responsible for a meningitis outbreak in Senegal.

Bacterial Typing Techniques↗

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

Bacterial Typing Techniques↗

Salmonella Pullorum strain SPullorum-YN-07 from dead embryos of Yanjin black-bone chickens: Complete genome with IncFII(S) and Col(pVC) plasmids and pathogenicity.

Salmonella Pullorum is a host-adapted pathogen that causes Pullorum disease in chickens and can be vertically transmitted via eggs, leading to embryonic mortality. The susceptibility and vertical transmission of S. Pullorum may vary among chicken breeds, yet genomic characterization of strains from dead embryos of indigenous breeds remains limited. This study isolated and characterized a Gram-negative short rod, designated Salmonella Pullorum strain SPullorum-YN-07, from dead embryos of Yanjin black-bone chickens, a native breed in Yunnan, China. The strain formed colorless colonies on MacConkey agar and red, non-H2S colonies on XLD agar, with biochemical reactions consistent with the genus Salmonella. Whole-genome sequencing using Illumina and PacBio platforms generated a complete genome consisting of one circular chromosome and four circular plasmids; plasmid replicon types IncFII(S) and Col(pVC) were identified in two of the plasmids. On the chromosome, a total of 340 virulence-associated genes were detected, including those involved in secretion systems, adhesion, motility, and immune modulation. Resistance gene analysis identified the acquired aminoglycoside resistance gene aac(6')-Iaa, alongside multiple intrinsic resistance determinants related to efflux pumps and target alteration. Multilocus sequence typing (MLST) assigned the strain to sequence type ST92, and core-genome phylogenetic analysis confirmed its clustering within the Salmonella Pullorum lineage. In a chick infection model, the strain induced depression, white diarrhea, and growth retardation, with clinical scores peaking at 10 days post-infection and a mortality rate of 10%. Bacterial colonization was highest in the cecum, and histopathological lesions were observed in the liver, spleen, and cecum. This study provides the first complete genomic characterization and pathogenicity assessment of an S. Pullorum strain isolated from dead embryos of Yanjin black-bone chickens, offering a foundation for understanding host-pathogen interactions in indigenous breeds and assessing cross-transmission risks to commercial poultry populations.

Complete genome↗

Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database.

Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to beta-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.

Databases, Factual↗

Streptococcal toxic shock syndrome by an iMLS resistant M type 77 Streptococcus pyogenes in the Netherlands.

An increasing number of group A streptococci (GAS) with constitutive or inducible resistance to macrolide-lincosamide-streptogramin B antibiotics (cMLS or iMLS phenotype) is observed in Europe, but MLS resistant GAS associated with streptococcal toxic shock syndrome (STSS) has not been reported. We describe a patient admitted with STSS caused by an iMLS resistant T28 M77 Streptococcus pyogenes carrying the ermA [subclass TR] gene. A 2-y retrospective analysis among 701 nationwide collected GAS strains revealed an incidence of 3.1% of this M type 77 GAS. Analysis of 17 available M77 strains (12 T28 and 5 T13) indicated that 2 (12%) were MLS resistant due to the ermA [TR] gene. Both MLS resistant strains were cultured from blood and belonged to T28 serotype. Multilocus sequence typing (MLST) showed that all M77 isolates belonged to sequence type 63. We conclude that 17 M77 GAS collected in the Netherlands in a 2-y period were associated with invasive disease and belonged to the same clonal complex. Since only 12% carried the ermA [TR] resistance gene, it is very likely that the gene has been acquired by horizontal transmission rather than from spread of a resistant circulating clone.

Adult↗

Diversity of penicillin-nonsusceptible Streptococcus pneumoniae circulating in Iceland after the introduction of penicillin-resistant clone Spain(6B)-2.

After the introduction and extensive dissemination of the multidrug-resistant Streptococcus pneumoniae clone Spain(6B)-2 between 1989 and the early to mid-1990s, the prevalence of pneumococcal isolates expressing intermediate resistance to penicillin, mainly of capsular types 6, 19, and 23, also began to increase in Iceland. The purpose of this study was to investigate whether these isolates originated in Iceland or represented strains imported to the country. Isolates were characterized by pulsed-field gel electrophoresis; multilocus sequence typing; determination of pbp1a, pbp2b, and pbp2x gene restriction patterns; and partial sequencing of these pbp genes. The results indicate that, although singular events suggesting horizontal transfer of pbp genes (and capsular genes) were detected, the majority of clones circulating in the country had genetic backgrounds also detected abroad. The major mechanism of dissemination of penicillin resistance in Iceland appears to be the repeated introduction of multiple lineages, followed by clonal spread.

Bacterial Capsules↗

Sequence diversity of Neisseria meningitidis 16S rRNA genes and use of 16S rRNA gene sequencing as a molecular subtyping tool.

We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.

Bacterial Typing Techniques↗

Probe hybridization array typing: a binary typing method for Escherichia coli.

The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., "library on a slide"), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

Adolescent↗

Molecular typing methods for Neisseria meningitidis.

Neisseria meningitidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningitidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods - PFGE, MLEE and MLST - is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.

Bacterial Typing Techniques↗

Serotype III Streptococcus agalactiae from bovine milk and human neonatal infections.

Streptococcus agalactiae (group B streptococcus [GBS]) causes invasive human infections and bovine mastitis. This study examined the genetic relationship between bovine and human serotype III GBS by using molecular techniques that classify human serotype III GBS into four distinct phylogenetic lineages. Bovine serotype III GBS were largely contained in two lineages, which are distinct from the two major lineages (restriction digest types III-2 and III-3) that infect human neonates. One of the bovine lineages closely resembles the human III-1 lineage, whose members occasionally cause human neonatal infections. The bovine strains in the other lineage characteristically have an initiation factor IF2 gene (infB) H allele and multilocus sequence types that are not found in human GBS strains. Evidence suggests that this "H allele" lineage is related to the human III-3 lineage. These results support the assertion that human and bovine GBS are largely unrelated and provide further insight into the genetic relation between human and bovine GBS.

Alleles↗

Staphylococcal cassette chromosome mec (SCCmec) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements.

A description of staphylococcal cassette chromosome mec (SCCmec) elements carried by 615 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 11 Asian countries is reported, and a novel nomenclatural system based on their structures is proposed. The 615 strains were classified as type 3A (370 strains), type 2A (207 strains), type 2B (32 strains), type 1B (1 strain), and nontypeable (5 strains). The previously reported type III SCCmec (DDBJ/EMBL/GenBank accession no. AB037671) carried by the MRSA strain 85/2082 was ascertained to be composed of two SCC elements, type 3A SCCmec and SCCmercury. PCR analysis indicated that 310 of 370 type 3A SCCmec strains carried both SCC elements. These strains were prevalent in eight countries: Thailand, Sri Lanka, Indonesia, Vietnam, Philippines, Saudi Arabia, India, and Singapore. The remaining 60 type 3A SCCmec strains differed with respect to the left extremity polymorphism or to the presence of ccrC. Among these, two were identified as carrying only type 3A SCCmec elements, but their left extremities differed. Type 2A SCCmec strains predominated in Korea and Japan, although the frequency of the presence of ant(4')-1 gene downstream of mecA varied (53% for Korean strains; 93% for Japanese strains). Various SCCmec elements were identified in the tested strains, and limited numbers were identified by their multilocus sequence typing genotypes. These data suggest that numerous MRSA clones are disseminated in Asian hospitals, and these consist of minor clones that are presumed to have arisen locally and major clones that are presumed to have been introduced from other countries.

Asia↗

Comparison of emm typing and ribotyping with three restriction enzymes to characterize clinical isolates of Streptococcus pyogenes.

A total of 336 Streptococcus pyogenes isolates recently recovered from patients with pharyngitis from 13 countries were characterized by emm typing and riboprinting using an automated Riboprinter (Dupont/Qualicon) based on the patterns produced by three restriction enzymes, EcoRI, PstI, and HindIII. Three enzymes were necessary to increase the discrimination of ribogroups formed by each enzyme. A total of 40 ribogroups and 38 emm sequences (not counting allelic variations) were identified. Multilocus sequence typing was performed on a sampling of the isolates, and those results were consistent with those of both emm typing and ribotyping. Correlations were observed among all three methods.

Antigens, Bacterial↗

Secrets of success of a human pathogen: molecular evolution of pandemic clones of meticillin-resistant Staphylococcus aureus.

The first European isolate of meticillin-resistant Staphylococcus aureus (MRSA) was detected in 1960. Since then MRSA has become a leading cause of nosocomial infections worldwide. Using molecular typing techniques--primarily pulsed-field gel electrophoresis (PFGE)--we identified five major MRSA clones that accounted for almost 70% of the over 3000 MRSA isolates recovered in hospitals mainly in southern and eastern Europe, South America, and the USA. Most of our surveillance studies were done in these areas. Multilocus sequencing typing (MLST) of representative isolates of this collection showed that these five pandemic MRSA clones have evolved from only two distinct ancestral genetic backgrounds, one of which can be traced back to the very first European MRSA isolates and also to meticillin susceptible S aureus strains circulating in Danish hospitals during the mid to late 1950s--i.e., shortly before the introduction of meticillin into therapy. The second lineage with a completely different MLST profile included MRSA frequently recovered in the USA, Japan, and among paediatric isolates from several parts of the world. A few isolates with a third distinct MLST type corresponding to that of EMRSA-16 were also detected in the early Danish isolates. The four structural types of mec element, the heterologous DNA segment containing the meticillin resistance determinant mecA, were present in unique combinations with the MRSA clonal types. Our findings establish evolutionary associations in the most widely spread pandemic clones of MRSA. The epidemiological factors that contributed to the massive dissemination of a few MRSA clones are not well understood. We suggest, however, that the secrets of effectiveness of MRSA could be hidden in the unique genetic background of a surprisingly few lineages of S aureus particularly well able to cope with the contemporary clinical environment.

DNA, Bacterial↗

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus-induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA-polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands.

Animals↗

National surveillance of methicillin-resistant Staphylococcus aureus in Belgian hospitals indicates rapid diversification of epidemic clones.

Methicillin-resistant Staphylococcus aureus (MRSA) strains (n = 455) collected in 2001 from 100 Belgian hospitals were characterized by molecular typing and by resistance gene distribution to macrolides-lincosamides-streptogramins and to aminoglycoside antibiotics. Rapid diversification of MRSA clones, compared with results of previous surveys, was evidenced by the broad geographic distribution of seven major clones belonging to the pandemic MRSA clonal complexes 5, 8, 22, 30, and 45 by multilocus sequence typing.

Aminoglycosides↗

Evaluation of molecular typing methods for Escherichia coli O157:H7 isolates from cattle, food, and humans.

Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

Animals↗

Clonal origin of the type 37 Streptococcus pneumoniae.

It has been recently reported that different type 37 clinical isolates of Streptococcus pneumoniae have an identical tts gene directing the formation of type 37 capsular polysaccharide. Here we show that type 37 S. pneumoniae strains isolated in two different continents (Europe and America) some 60 years apart frequently gave rise to nontypable variants upon in vitro cultivation. The tts gene from three independent nontypable mutants was PCR amplified and sequenced showing different classes of inactivating mutations. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that the type 37 pneumococcal isolates studied so far constitute a highly related strain cluster (a clonal complex), and strongly suggested that every type 37 pneumococcus has spread globally from a single, old clone.

Bacterial Typing Techniques↗