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We have employed whole genome sequencing to define and evaluate a core genome multilocus sequence typing (cgMLST) scheme for Acinetobacter baumannii. To define a core genome we downloaded a total of 1,573 putative A. baumannii genomes from NCBI as well as representative isolates belonging to the eight previously described international A. baumannii clonal lineages. The core genome was then employed against a total of fifty-three carbapenem-resistant A. baumannii isolates that were previously typed by PFGE and linked to hospital outbreaks in eight German cities. We defined a core genome of 2,390 genes of which an average 98.4% were called successfully from 1,339 A. baumannii genomes, while Acinetobacter nosocomialis, Acinetobacter pittii, and Acinetobacter calcoaceticus resulted in 71.2%, 33.3%, and 23.2% good targets, respectively. When tested against the previously identified outbreak strains, we found good correlation between PFGE and cgMLST clustering, with 0-8 allelic differences within a pulsotype, and 40-2,166 differences between pulsotypes. The highest number of allelic differences was between the isolates representing the international clones. This typing scheme was highly discriminatory and identified separate A. baumannii outbreaks. Moreover, because a standardised cgMLST nomenclature is used, the system will allow inter-laboratory exchange of data.

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation.

Assessing the genetic variation of bacteria has become ever more complex as more sequencing data has become available. Here, Maiden and colleagues propose a gene-by-gene approach of analysing whole-genome data; this approach is based on their experience with multilocus sequence typing (MLST) and reflects the functional and evolutionary relationships among bacteria. Multilocus sequence typing (MLST) was proposed in 1998 as a portable sequence-based method for identifying clonal relationships among bacteria. Today, in the whole-genome era of microbiology, the need for systematic, standardized descriptions of bacterial genotypic variation remains a priority. Here, to meet this need, we draw on the successes of MLST and 16S rRNA gene sequencing to propose a hierarchical gene-by-gene approach that reflects functional and evolutionary relationships and catalogues bacteria 'from domain to strain'. Our gene-based typing approach using online platforms such as the Bacterial Isolate Genome Sequence Database (BIGSdb) allows the scalable organization and analysis of whole-genome sequence data.

Background Previous phylogenetic analyses of species within the genus Golovinomyces (Ascomycota , Erysiphales), based on ITS and 28S rDNA sequence data, revealed a co-evolutionary relationship between powdery mildew species and hosts of certain tribes of the plant family Asteraceae. Golovinomyces growing on host plants belonging to the Heliantheae formed a single lineage, comprised of a morphologically differentiated complex of species, which included G. ambrosiae , G. circumfusus , and G. spadiceus . However, the lineage also encompassed sequences retrieved from Golovinomyces specimens on other Asteraceae tribes as well as other plant families, suggesting the involvement of a plurivorous species. A multilocus phylogenetic examination of this complex, using ITS, 28S, IGS (intergenic spacer), TUB2 (beta-tubulin), and CHS1 (chitin synthase I) sequence data was carried out to clarify the discrepancies between ITS and 28S rDNA sequence data and morphological differences. Furthermore, the circumscription of species and their host ranges were emended. Results The phylogenetic and morphological analyses conducted in this study revealed three distinct species named, viz., (1) G. ambrosiae emend. (including G. spadiceus ), a plurivorous species that occurs on a multitude of hosts including, Ambrosia spp., multiple species of the Heliantheae and plant species of other tribes of Asteraceae including the Asian species of Eupatorium ; (2) G. latisporus comb. nov. (≡ Oidium latisporum ), the closely related, but morphologically distinct species confined to hosts of the Heliantheae genera Helianthus , Zinnia , and most likely Rudbeckia ; and (3) G. circumfusus confined to Eupatorium cannabinum in Europe. Conclusions The present results provide strong evidence that the combination of multi-locus phylogeny and morphological analysis is an effective way to identify species in the genus Golovinomyces .

A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63–90, range of point estimates 73–87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. Novartis Vaccines and Diagnostics.

The control of Clostridium difficile infections is an international clinical challenge. The incidence of C difficile in England declined by roughly 80% after 2006, following the implementation of national control policies; we tested two hypotheses to investigate their role in this decline. First, if C difficile infection declines in England were driven by reductions in use of particular antibiotics, then incidence of C difficile infections caused by resistant isolates should decline faster than that caused by susceptible isolates across multiple genotypes. Second, if C difficile infection declines were driven by improvements in hospital infection control, then transmitted (secondary) cases should decline regardless of susceptibility. Regional (Oxfordshire and Leeds, UK) and national data for the incidence of C difficile infections and antimicrobial prescribing data (1998–2014) were combined with whole genome sequences from 4045 national and international C difficile isolates. Genotype (multilocus sequence type) and fluoroquinolone susceptibility were determined from whole genome sequences. The incidence of C difficile infections caused by fluoroquinolone-resistant and fluoroquinolone-susceptible isolates was estimated with negative-binomial regression, overall and per genotype. Selection and transmission were investigated with phylogenetic analyses. National fluoroquinolone and cephalosporin prescribing correlated highly with incidence of C difficile infections (cross-correlations >0·88), by contrast with total antibiotic prescribing (cross-correlations <0·59). Regionally, C difficile decline was driven by elimination of fluoroquinolone-resistant isolates (approximately 67% of Oxfordshire infections in September, 2006, falling to approximately 3% in February, 2013; annual incidence rate ratio 0·52, 95% CI 0·48–0·56 vs fluoroquinolone-susceptible isolates: 1·02, 0·97–1·08). C difficile infections caused by fluoroquinolone-resistant isolates declined in four distinct genotypes (p<0·01). The regions of phylogenies containing fluoroquinolone-resistant isolates were short-branched and geographically structured, consistent with selection and rapid transmission. The importance of fluoroquinolone restriction over infection control was shown by significant declines in inferred secondary (transmitted) cases caused by fluoroquinolone-resistant isolates with or without hospital contact (p<0·0001) versus no change in either group of cases caused by fluoroquinolone-susceptible isolates (p>0·2). Restricting fluoroquinolone prescribing appears to explain the decline in incidence of C difficile infections, above other measures, in Oxfordshire and Leeds, England. Antimicrobial stewardship should be a central component of C difficile infection control programmes. UK Clinical Research Collaboration (Medical Research Council, Wellcome Trust, National Institute for Health Research); NIHR Oxford Biomedical Research Centre; NIHR Health Protection Research Unit on Healthcare Associated Infection and Antimicrobial Resistance (Oxford University in partnership with Public Health England [PHE]), and on Modelling Methodology (Imperial College, London in partnership with PHE); and the Health Innovation Challenge Fund.

The family Streptomycetaceae , notably species in the genus Streptomyces , have long been the subject of investigation due to their well-known ability to produce secondary metabolites. The emergence of drug resistant pathogens and the relative ease of producing genome sequences has renewed the importance of Streptomyces as producers of new natural products and resulted in revived efforts in isolating and describing strains from novel environments. A previous large study of the phylogeny in the Streptomycetaceae based on 16S rRNA gene sequences provided a useful framework for the relationships among species, but did not always have sufficient resolution to provide definitive identification. Multi-locus sequence analysis of 5 house-keeping genes has been shown to provide improved taxonomic resolution of Streptomyces species in a number of previous reports so a comprehensive study was undertaken to evaluate evolutionary relationships among species within the family Streptomycetaceae where type strains are available in the ARS Culture Collection or genome sequences are available in GenBank. The results of the analysis supported the distinctiveness of Kitasatospora and Streptacidiphilus as validly named genera since they cluster outside of the phylogenetic radiation of the genus Streptomyces . There is also support for the transfer of a number of Streptomyces species to the genus Kitasatospora as well for reducing at least 31 species clusters to a single taxon. The multi-locus sequence database resulting from the study is a useful tool for identification of new isolates and the phylogenetic analysis presented also provides a road map for planning future genome sequencing efforts in the Streptomycetaceae .

Campylobacteriosis is among the world’s most common foodborne illnesses, caused predominantly by the bacterium Campylobacter jejuni . Effective interventions require determination of the infection source which is challenging as transmission occurs via multiple sources such as contaminated meat, poultry, and drinking water. Strain variation has allowed source tracking based upon allelic variation in multi-locus sequence typing (MLST) genes allowing isolates from infected individuals to be attributed to specific animal or environmental reservoirs. However, the accuracy of probabilistic attribution models has been limited by the ability to differentiate isolates based upon just 7 MLST genes. Here, we broaden the input data spectrum to include core genome MLST (cgMLST) and whole genome sequences (WGS), and implement multiple machine learning algorithms, allowing more accurate source attribution. We increase attribution accuracy from 64% using the standard iSource population genetic approach to 71% for MLST, 85% for cgMLST and 78% for kmerized WGS data using the classifier we named aiSource. To gain insight beyond the source model prediction, we use Bayesian inference to analyse the relative affinity of C . jejuni strains to infect humans and identified potential differences, in source-human transmission ability among clonally related isolates in the most common disease causing lineage (ST-21 clonal complex). Providing generalizable computationally efficient methods, based upon machine learning and population genetics, we provide a scalable approach to global disease surveillance that can continuously incorporate novel samples for source attribution and identify fine-scale variation in transmission potential.

Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.

Objectives To establish the epidemiological correlation between the study isolates, Indian and World isolates of Acinetobacter baumannii by performing Multilocus Sequence typing. Materials and methods A total of 181 isolates (Sputum ( n  = 116), lower respiratory tract other than sputum ( n  = 36), upper respiratory tract ( n  = 20), Environment (medical) ( n  = 4), and Blood ( n  = 5) of Acinetobacter baumannii were retrieved from our repository. DNA was isolated and Multilocus Sequence Typing was performed according to the Pasteur scheme. The amplified fragments were sequenced by outsourcing, and the locus and the sequence types were determined as given in the PUBMLST site. The clonal complexes were assigned using eBURST. Results and conclusion Of the 181 isolates, 20 were colonizers and 4 were from hospital environments. All the study isolates were multidrug-resistant (MDR) and 4 of them were extensively drug-resistant (XDR). 23 sequence types were unique and were assigned new sequence types. Among them, 2125 ( n  = 12), an SLV (Single Locus Variant) of 2, was the commonest followed by 2126 ( n  = 2) which was a DLV (Double Locus Variant) of 2 and an SLV of 2125. Others were singletons. Among the known STs, 149 ( n  = 72) was the commonest followed by ST 2 ( n  = 62) & 415 ( n  = 5), ST 10 ( n  = 4), ST 15, ST622 and ST1482 (3 each). ST149 had 1SLV ST1482 ( n  = 3). ST 2 has 5 SLVs (415, 1555, 2125, 2128, & 2131, and 2 DLVs (2130 & 2126). eBURST analysis of the study isolates showed three groups: Group I (86 isolates) with ST 2 as the primary founder, group II (6 isolates) and group III (79 isolates) with ST 149 as the primary founder. All the other 11 isolates were singletons. There was no difference in antimicrobial sensitivity or sequence types of clinical and colonizing isolates. The sequence types of study isolates were compared to the world isolates in the PUBMLST database. To conclude, MLST is an important tool for establishing isolates’ phylogenetic relationships. Acinetobacter baumannii being an important nosocomial pathogen should be routinely screened for the frequent changes in the sequence types to demonstrate the emerging resistance patterns and other changes.