Showing papers on "Typing published in 2011"

Worldwide Distribution of Major Clones of Listeria monocytogenes

Abstract: Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severe invasive infection in humans with a particularly high case-fatality rate. Listeriosis is a major public health concern in all world regions, with an increasing incidence in Europe, especially among elderly persons (1,2). L. monocytogenes is genetically heterogeneous (3–5). To help epidemiologic investigation and to define clones, i.e., groups of genetically similar isolates descending from a common ancestor, a variety of typing methods have been used, including pulsed-field gel electrophoresis (5,6), single nucleotide polymorphism typing (7), and multiple housekeeping and virulence gene sequencing (8,9). Some clones implicated in multiple outbreaks have been defined as epidemic clones (EC) (3,5,9–11). ECI and ECIV have been described in several countries (3,5), but because of the lack of standardization of genotyping, a definition of clones is not widely accepted, and current knowledge on the global distribution of L. monocytogenes clones is virtually absent. Multilocus sequence typing (MLST) is a reference method for global epidemiology and population biology of bacteria, and its application to L. monocytogenes (12) effectively allows isolate comparisons across laboratories (www.pasteur.fr/mlst). The aim of this study was to investigate the global distribution of L. monocytogenes MLST-defined clones.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterization of type I cell surface-associated antigens.

Abstract: We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

Abstract: SUMMARY Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding.

Staphylococcal cassette chromosome mec (Sccmec) classification and typing methods: an overview

Abstract: Meticillin-resistant Staphylococcus aureus (MRSA) is one of the main causes of hospital-acquired infections, but since late 1990s also the community-acquired. For better understanding of the S.aureus epidemiology there is an urgent need for creation of new typing method for SCCmec element. The molecular typing of MRSA for epidemiological purposes is investigated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing and the SCCmec type assignment. In last few years not only new type of SCCmec (VI to XI) have been identified, but also additional subtypes (i.e. IVg-j) and different variants of already existed one (i.e. 5C2&5 and 2B2&5) were discovered. The aim of this review is to briefly summarize current knowledge about SCCmec classification and to discuss advantages and disadvantages of selected SCCmec typing methods.

[Legionella pneumophila : genetic diversity of patients and environmental isolates].

Abstract: Legionellae are environmental bacteria that can be frequently isolated from technical water supply systems. The most prevalent species is Legionella pneumophila, especially serogroup 1. In the environment, legionellae multiply in amoebae. Since Legionella pneumonias cannot be distinguished from pneumonias caused by other microbial pathogens, special microbiological tests, e.g., urinary antigen assays, are essential to detect Legionella infections. All water supply systems to which the patient is exposed during the incubation time of 2 to 10 days might be the source of the infection. This can be confirmed or excluded by molecular typing of isolates from patients and the environment. The most commonly used techniques are monoclonal antibody typing and sequence-based typing (SBT). Some sequence types (ST) are frequently found among clinical strains but are seldom isolated from the environment, e.g., ST 23, 42, 47, 62, and 146. It is safe to assume that such strains are highly virulent. Conversely, it does not seem to be justified to dedicate the same awareness to all environmental Legionella strains.

Most Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospitals in Eastern France Belong to a Few Clonal Types

Abstract: This study aimed to determine the genetic diversity of clinical multidrug-resistant Pseudomonas aeruginosa. We used pulsed-field gel electrophoresis and multilocus sequence typing to analyze 187 strains isolated in different French hospitals. To illustrate the diversity of resistance mechanisms to antibiotics in a given clone, we identified β-lactamases with an extended spectrum by using phenotypic and genotypic methods. Typing results showed that the majority of our multidrug-resistant isolates belong to a few clonal types (ST235, ST111, and ST175) that are already spreading worldwide. These successful international clones sporadically produced extended-spectrum β-lactamase-encoding genes but mostly became extensively resistant to β-lactams after derepression of intrinsic resistance mechanisms (i.e., AmpC cephalosporinase). Our results indicate that cross-transmission plays a major role in the spread of multidrug-resistant P. aeruginosa in hospital settings.

Plasmid-mediated quinolone resistance and β-lactamases in Escherichia coli from healthy animals from Nigeria

Abstract: Methods: One hundred and sixty-two ampicillin-resistant E. coli were obtained from healthy chickens and pigs at slaughter in Ibadan, Nigeria. Strains were tested for antimicrobial susceptibility by disc diffusion assay. MICs of ciprofloxacin were determined by Etest. Resistance genes were screened by PCR and DNA sequencing. Clonal relatedness of the isolates was determined by enterobacterial repetitive intergenic consensus –PCR. Plasmids were transferred by conjugation and transformation and characterized by PCR-based replicon typing and plasmid multilocus sequence typing. Results: PMQR genes were detected in 18 E. coli strains; 11 of them showed reduced susceptibility to ciprofloxacin. Twelve strains carried qnrS1, three strains carried qnrB19, one strain carried qnrB10 and three strains carried qepA; one strain carried both qepA and qnrB10. All strains carried the blaTEM gene; one strain was positive for the CTX-M-15 extended-spectrum b-lactamase. Conclusions: Our findings suggest that food animals could represent an important reservoir of PMQR in this region of Africa. Previous studies reported high prevalence of qnr genes in clinical isolates from humans in Nigeria, suggesting that the spread of these resistance determinants in this country could be particularly relevant.

Genotypes and Toxin Gene Profiles of Staphylococcus aureus Clinical Isolates from China

Abstract: A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.

Lack of Association between Clinical Outcome of Clostridium difficile Infections, Strain Type, and Virulence-Associated Phenotypes

Abstract: Clostridium difficile strain NAP1/027 (North American pulsed-field gel electrophoresis [PFGE] type 1 and PCR ribotype 027 [R027]) has been associated with recent outbreaks in North America and Europe. It has been associated with more severe disease symptoms, higher mortality rates, and greater risk of relapse. This strain is thought to produce more toxins and sporulate to higher levels. However, recent studies suggest that this may not always be the case. The objective of our study was to assess, in a nonoutbreak situation, whether specific strains, such as NAP1/027, were associated with more severe disease symptoms, higher toxin production, and/or greater sporulation in vitro. We isolated and characterized C. difficile strains from 21 patients with mild to moderate, severe, or complicated symptoms of C. difficile infection (CDI). The isolates were characterized by different molecular typing methods, including PCR ribotyping, tandem repeat sequence typing (TRST), and sequencing of the tcdC gene. Fourteen isolates were of PCR ribotype 027 with deletions in tcdC, but no association with severity or clinical outcome was found. We show by immunodot blot detection of toxins with monoclonal antibodies that all R027 isolates produced more TcdA and TcdB than other strains. On the other hand, they consistently produced fewer spores than non-R027 isolates. Taken together, our data suggest that NAP1/027 isolates are not always associated with more severe disease, even though they may produce larger amounts of toxins. Our study also suggests that current assertions regarding the NAP1/027 may not apply to all isolates and that other factors may come into play.

The M protein of group A Streptococcus is a key virulence factor and a clinically relevant strain identification marker.

Abstract: The M protein coats group A streptococci (GAS) and acts as the primary antigen and determinant of type-specific immunity. M is essential for GAS virulence, providing antiphagocytic functions critical to survival in human tissues and fluids. Specific regions of M protein also serve as shared antigens, and cross-reactivity between these epitopes and human proteins may be the source of autoimmune sequelae such as rheumatic heart disease. The M protein is hypervariable, and has long served as the primary target for epidemiological typing of GAS. Though other markers or genotyping methods may be necessary to increase strain resolution when clones of a given M type differ in clinically critical ways, M typing remains the most directly informative and well-documented method for tracking outbreaks of GAS, predicting clinical outcomes during those outbreaks, and measuring the general threat presented by GAS at any given time and place.

Multilocus Sequence Typing and Phylogenetic Analysis of Propionibacterium acnes

Abstract: Propionibacterium acnes is a commensal of human skin but is also implicated in the pathogenesis of acne vulgaris, in biofilm-associated infections of medical devices and endophthalmitis, and in infections of bone and dental root canals. Recent studies associate P. acnes with prostate cancer. As the species includes evolutionary lineages with distinct association with health and disease, there is a need for a high-resolution typing scheme. Recently, two multilocus sequence typing (MLST) schemes were reported, one based on nine and one based on seven housekeeping genes. In the present study, the two schemes were compared with reference to a phylogenetic tree based on 78 P. acnes genomes and their gene contents. Further support for a basically clonal population structure of P. acnes and a scenario of the global spread of epidemic clones of P. acnes was obtained. Compared to the Belfast scheme, the Aarhus MLST scheme (http://pacnes.mlst.net/), which is based on nine genes, offers significantly enhanced resolution and phylogenetic inferences more concordant with analyses based on a comprehensive sampling of the entire genomes, their gene contents, and their putative pathogenic potential.

Molecular Characterization and Antimicrobial Susceptibility Testing of Escherichia coli Isolates from Patients with Urinary Tract Infections in 20 Chinese Hospitals

Abstract: A total of 222 urinary Escherichia coli isolates from 20 tertiary hospitals in 15 different provinces and 4 municipalities in mainland China were characterized by antimicrobial susceptibility, phylogrouping, and the presence of plasmid-mediated quinolone resistance genes. A subset of 138 suspected extended-spectrum cephalosporinase (ESC) producers were examined for genes encoding cephalosporin resistance. Forty-three isolates harboring blaCTX-M-14 or blaCTX-M-15 were analyzed by pulsed-field gel electrophoresis (PFGE), and plasmids containing these genes were typed using PCR-based replicon typing (PBRT). Thirteen phylogroup B2 blaCTX-M-14- and blaCTX-M-15-positive isolates were analyzed by multilocus sequence typing (MLST). A frequent occurrence of resistance (>46%) was observed toward cephalosporins, gentamicin, and fluoroquinolones. Among the 222 isolates, 4 qnrS1, 4 qepA, and 16 aac(6′)-Ib-cr genes were confirmed. Four major phylogroups (A, B1, B2, and D) and nontypeable isolates (NTs) were found among the isolates, with phylogroup D (54%) being the most common phylogroup. A total of 110 (80%) of the 138 screened isolates harbored blaCTX-M genes, with blaCTX-M-14 (71%) and blaCTX-M-15 (24%) being the most prevalent of these genes. Nine of the 13 CTX-M-15- or CTX-M-14-containing B2 isolates belonged to ST131. PFGE typing showed a high level of diversity, and plasmid analysis indicated a very large pool of different resistance plasmids mediating the spread of blaCTX-M genes in mainland China. An equally very high frequency of resistance and equally high levels of diversity in phylogroups, PFGE types, and plasmids were observed among community- and hospital-acquired E. coli isolates, indicating the presence of a large reservoir in the community and a long-term spread of cephalosporin resistance in China.

Multilocus sequence typing of a global collection of pasteurella multocida isolates from cattle and other host species demonstrates niche association

Abstract: Background Pasteurella multocida causes disease in many host species throughout the world. In bovids, it contributes to bovine respiratory disease (BRD) and causes haemorrhagic septicaemia (HS). Previous studies have suggested that BRD-associated P. multocida isolates are of limited diversity. A multilocus sequence typing (MLST) scheme for P. multocida was used to determine whether the low levels of diversity reported are due to the limited discriminatory power of the typing method used, restricted sample selection or true niche association. Bovine respiratory isolates of P. multocida (n = 133) from the UK, the USA and France, collected between 1984 and 2008 from both healthy and clinically affected animals, were typed using MLST. Isolates of P. multocida from cases of HS, isolates from other host species and data from the MLST database were used as comparison.

Genetic Diversity among Korean Candida albicans Bloodstream Isolates: Assessment by Multilocus Sequence Typing and Restriction Endonuclease Analysis of Genomic DNA by Use of BssHII

Abstract: Multilocus sequence typing (MLST) has been successfully applied to the epidemiology of Candida albicans isolates not only within the hospital setting but also in multiple locations nationwide. We performed MLST to investigate the genetic relatedness among bloodstream infection (BSI) isolates of C. albicans recovered from 10 Korean hospitals over a 12-month period. The 156 isolates yielded 112 unique diploid sequence types (DSTs). While 95 DSTs were each derived from a single isolate, 17 DSTs were shared by 61 isolates (39.1%). Interestingly, 111 (71.1%) isolates clustered within previously known clades, and 29 (18.6%) clustered within a new clade that includes strains of Asian origin previously typed as singletons. This MLST study was complemented by restriction endonuclease analysis of genomic DNA using BssHII (REAG-B) in order to evaluate whether strains with identical DSTs and originating from the same hospital corresponded to nosocomial clusters. Importantly, only those isolates with a strong epidemiological relationship showed ≥95% identical REAG-B types. Our results indicate that REAG-B typing can be complementary to MLST but should be limited to the investigation of isolates of identical DSTs and when interhuman transmission is suspected.

Molecular Typing of Treponema pallidum: A Systematic Review and Meta-Analysis

Abstract: Background Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum) with three objectives: (1) to determine specimen types most suitable for molecular typing; (2) to determine T. pallidum subtype distribution across geographic areas; and (3) to summarize available information on subtypes associated with neurosyphilis and macrolide resistance. Methodology/Principal Findings Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included “Treponema pallidum,” or “syphilis,” combined with the subject headings “molecular,” “subtyping,” “typing,” “genotype,” and “epidemiology.” Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ2 = 247.6, p<0.001) and an average higher efficiency of full molecular typing (80.9% vs. 43.1%, χ2 = 102.3, p<0.001) compared to plasma, whole blood, and cerebrospinal fluid. A pooled analysis of subtype distribution based on country location showed that 14d was the most common subtype, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited. Conclusions/Significance Primary lesion was a better specimen for obtaining T. pallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further validation of ear lobe blood for obtaining T. pallidum DNA would be useful for future molecular studies of syphilis.

Evaluation of Mycobacterium tuberculosis Typing Methods in a 4-Year Study in Schleswig-Holstein, Northern Germany

Abstract: In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci.

Controlled Performance Evaluation of the DiversiLab Repetitive-Sequence-Based Genotyping System for Typing Multidrug-Resistant Health Care-Associated Bacterial Pathogens

Abstract: Fast, reliable, and versatile typing tools are essential to differentiate among related bacterial strains for epidemiological investigation and surveillance of health care-associated infection with multidrug-resistant (MDR) pathogens. The DiversiLab (DL) system is a semiautomated repetitive-sequence-based PCR system designed for rapid genotyping. The DL system performance was assessed by comparing its reproducibility, typeability, discriminatory power, and concordance with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and by assessing its epidemiological concordance on well-characterized MDR bacterial strains (n = 165). These included vanA Enterococcus faecium, extended-spectrum β-lactamase (ESBL)-producing strains of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, and ESBL- or metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa strains. The DL system showed very good performance for E. faecium and K. pneumoniae and good performance for other species, except for a discrimination index of <95% for A. baumannii and E. coli (93.9% and 93.5%, respectively) and incomplete concordance with MLST for P. aeruginosa (78.6%) and E. coli (97.0%). Occasional violations of MLST assignment by DL types were noted for E. coli. Complete epidemiological concordance was observed for all pathogens, as all outbreak-associated strains clustered in identical DL types that were distinct from those of unrelated strains. In conclusion, the DL system showed good to excellent performance, making it a reliable typing tool for investigation of outbreaks caused by study pathogens, even though it was generally less discriminating than PFGE analysis. For E. coli and P. aeruginosa, MLST cannot be reliably inferred from DL type due to phylogenetic group violation or discordance.

International External Quality Assurance for Laboratory Identification and Typing of Streptococcus agalactiae (Group B Streptococci)

Abstract: We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.

The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

Abstract: Background: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. Methodology/Principal Findings: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American- Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. Conclusions/Significance: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.

DNA microarray-based characterisation of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy.

Abstract: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton–Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005–2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

Abstract: The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland.

Abstract: AIMS: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. METHODS AND RESULTS: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which - in combination with MLST - increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37.5, 33.1 and 8.1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone-sensitive human isolates showed overlapping MLST-flaB types with those of chicken origin, resistant strains showed only 39% of matching types. CONCLUSION: Mainly quinolone-sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.

Multilocus sequence typing of Mycoplasma agalactiae

Abstract: Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

SNP-Based Typing: A Useful Tool to Study Bordetella pertussis Populations

Abstract: To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in The Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis.

Use of the accessory genome for characterization and typing of Acinetobacter baumannii.

Abstract: Outbreak strains of Acinetobacter baumannii are highly clonal, and cross-infection investigations can be difficult We sought targets based on AbaR resistance islands and on other genes found in some, but not all, sequenced isolates of A baumannii among a set of clinical isolates (n = 70) that included multiple representatives of a number of pulsed-field gel electrophoresis (PFGE)-defined types These included representatives that varied in their profiles at two variable-number tandem repeat (VNTR) loci, which can provide discrimination within a PFGE cluster Detection, or not, of each element sought provided some degree of discrimination among the set, with the presence or absence of genes coding for a phage terminase (ACICU_02185), a sialic acid synthase (ACICU_00080), a polysaccharide biosynthesis protein (AB57_0094), aphA1, blaTEM, and integron-associated orfX (Kyoto Encyclopedia of Genes and Genomes [KEGG] no K03830) proving the most helpful in discriminating between closely related isolates in our panel The results support VNTR data in describing distinct populations of highly similar isolates Such analysis, in combination with other typing methods, can inform epidemiological investigations and provide additional characterization of isolates Most genotypes carrying blaOXA-23-like were PCR positive for a yeeA-blaOXA-23 fragment found in an AbaR4-type island, suggesting that this is widespread