Genomic determination of minimum multi-locus sequence typing schemas to represent the genomic phylogeny of Mycoplasma hominis.

Background: Mycoplasma hominis is an opportunistic human pathogen, associated with clinically diverse disease. Currently, there is no standardised method for typing M. homins, which would aid in understanding pathogen epidemiology and transmission. Due to availability and costs of whole genome sequencing and the challenges in obtaining adequate M. hominis DNA, the use of whole genome sequence analysis to provide clinical guidance is unpractical for this bacterial species as well as other fastidious organisms. Results: This study identified pan-genome set of 700 genes found to be present in four published reference genomes. A subset of 417 genes was identified to be core genome for 18 isolates and 1 reference. Leave-one-out analysis of the core genes highlighted set of 48 genes that are required to recapture the original phylogenetic relationships observed using whole genome SNP analysis. Three 7-locus MLST schemas with high diversity index (97%) and low dN/dS ratios (0.1, 0.13, and 0.11) were derived that could be used to confer good discrimination between strains and could be of practical use in future studies direct on clinical specimens. Conclusions: The genes proposed in this study could be utilised to design a costeffective and rapid PCR-based MLST assay that could be applied directly to clinical isolates, without prior isolation. This study includes additional genomic analysis revealing high levels of genetic heterogeneity among this species. This provides a novel and evidence based approach for the development of MLST schema that accurately represent genomic phylogeny for use in epidemiology and transmission studies

Spatial structuring of a Legionella pneumophila population within the water system of a large occupational building.

The diversity of Legionella pneumophila populations within single water systems is not well understood, particularly in those unassociated with cases of Legionnaires' disease. Here, we performed genomic analysis of 235 L. pneumophila isolates obtained from 28 water samples in 13 locations within a large occupational building. Despite regular treatment, the water system of this building is thought to have been colonized by L. pneumophila for at least 30 years without evidence of association with Legionnaires' disease cases. All isolates belonged to one of three sequence types (STs), ST27 (n=81), ST68 (n=122) and ST87 (n=32), all three of which have been recovered from Legionnaires' disease patients previously. Pairwise single nucleotide polymorphism differences amongst isolates of the same ST were low, ranging from 0 to 19 in ST27, from 0 to 30 in ST68 and from 0 to 7 in ST87, and no homologous recombination was observed in any lineage. However, there was evidence of horizontal transfer of a plasmid, which was found in all ST87 isolates and only one ST68 isolate. A single ST was found in 10/13 sampled locations, and isolates of each ST were also more similar to those from the same location compared with those from different locations, demonstrating spatial structuring of the population within the water system. These findings provide the first insights into the diversity and genomic evolution of a L. pneumophila population within a complex water system not associated with disease

Antibiotic resistance among clinical Ureaplasma Isolates Recovered from Neonates in England and Wales between 2007 and 2013

Ureaplasma spp. are associated with numerous clinical sequelae with treatment options being limited due to patient and pathogen factors. This report examines the prevalence and mechanisms of antibiotic resistance among clinical strains isolated from 95 neonates, 32 women attending a sexual health clinic, and 3 patients under investigation for immunological disorders, between 2007 and 2013 in England and Wales. MICs were determined by using broth microdilution assays, and a subset of isolates were compared using the broth microdilution method and the Mycoplasma IST2 assay. The underlying molecular mechanisms for resistance were determined for all resistant isolates. Three isolates carried the tet(M) tetracycline resistance gene (2.3%; confidence interval [CI], 0.49 to 6.86%); two isolates were ciprofloxacin resistant (1.5%; CI, 0.07 to 5.79%) but sensitive to levofloxacin and moxifloxacin, while no resistance was seen to any macrolides tested. The MIC values for chloramphenicol were universally low (2 μg/ml), while inherently high-level MIC values for gentamicin were seen (44 to 66 μg/ml). The Mycoplasma IST2 assay identified a number of false positives for ciprofloxacin resistance, as the method does not conform to international testing guidelines. While antibiotic resistance among Ureaplasma isolates remains low, continued surveillance is essential to monitor trends and threats from importation of resistant clones

Development of a multilocus sequence typing scheme for the molecular typing of Mycoplasma pneumoniae

This work was funded by Public Health England. These studies were supported by funding initiatives by the National Institute for Social Care and Health Research (NISCHR; research support from the Welsh Government) via the registered research group Microbial and Infection Translational Research Group (MITReG) and Children and Young Persons Research Network (CYPRN).Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).PostprintPeer reviewe

Mycoplasma pneumoniae epidemiology in England and Wales: a national perspective

Investigations of patients with suspected Mycoplasma pneumoniae infection have been undertaken in England since the early 1970s. M. pneumoniae is a respiratory pathogen that is a common cause of pneumonia and may cause serious sequelae such as encephalitis and has been documented in children with persistent cough. The pathogen is found in all age groups, with higher prevalence in children aged 5–14 years. In England, recurrent epidemic periods have occurred at ~4-yearly intervals. In addition, low-level sporadic infection occurs with seasonal peaks from December to February. Voluntarily reports from regional laboratories and hospitals in England from 1975 to 2015 were collated by Public Health England for epidemiological analysis. Further data pertaining cases of note and specimens submitted to Public Health England from 2005 to 2015 for confirmation, molecular typing is included

Legionella antimicrobial sensitivity testing: comparison of microbroth dilution with BCYE and LASARUS solid media

Objectives There is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance. Methods Antibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline. Results The deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene. Conclusions BMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates

Identifying large-scale recombination and capsular switching events in Streptococcus agalactiae strains causing disease in adults in the UK between 2014 and 2015

Cases of invasive group B streptococcal infection in the adult UK population have steadily increased over recent years, with the most common serotypes being V, III and Ia, but less is known of the genetic background of these strains. We have carried out in-depth analysis of the whole-genome sequences of 193 clinically important group B Streptococcus (GBS) isolates (184 were from invasive infection, 8 were from non-invasive infection and 1 had no information on isolation site) isolated from adults and submitted to the National Reference Laboratory at the UK Health Security Agency between January 2014 and December 2015. We have determined that capsular serotypes III (26.9%), Ia (26.4%) and V (15.0%) were most commonly identified, with slight differences in gender and age distribution. Most isolates (n=182) grouped to five clonal complexes (CCs), CC1, CC8/CC10, CC17, CC19 and CC23, with common associations between specific serotypes and virulence genes. Additionally, we have identified large recombination events mediating potential capsular switching events between sequence type (ST)1 serotype V and serotypes Ib (n=2 isolates), II (n=2 isolates) and VI (n=2 isolates); between ST19 serotype III and serotype V (n=5 isolates); and between CC17 serotype III and serotype IV (n=1 isolate). The high genetic diversity of disease-causing isolates and multiple recombination events reported in this study highlight the need for routine surveillance of the circulating disease-causing GBS strains. This information is crucial to better understand the global spread of GBS serotypes and genotypes, and will form the baseline information for any future GBS vaccine research in the UK and worldwide

Seeding and Establishment of Legionella pneumophila in Hospitals: Implications for Genomic Investigations of Nosocomial Legionnaires' Disease.

BACKGROUND: Legionnaires' disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1. METHODS: WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires' disease. RESULTS: Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1. CONCLUSIONS: WGS can be used to support or refute suspected links between hospitals and Legionnaires' disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations