Dam inactivation in Neisseria meningitidis: prevalence among diverse hyperinvasive lineages

BACKGROUND: DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci. RESULTS: Of the known meningococcal hyper-invasive lineages investigated, drg was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection (the ST-1, ST-4, ST-5, ST-32 and ST-41/44 clonal complexes). The patterns of sequence diversity observed in drg were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination. CONCLUSION: These findings are consistent with the idea that possession of the drg gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence

The domestication of the probiotic bacterium Lactobacillus acidophilus

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population

A gene-by-gene population genomics platform: de novo assembly, annotation and genealogical analysis of 108 representative Neisseria meningitidis genomes

Background: Highly parallel,‘second generation’ sequencing technologies have rapidly expanded the number of bacterial whole genome sequences available for study, permitting the emergence of the discipline of population genomics. Most of these data are publically available as unassembled short-read sequence files that require extensive processing before they can be used for analysis. The provision of data in a uniform format, which can be easily assessed for quality, linked to provenance and phenotype and used for analysis, is therefore necessary. Results: The performance of de novo short-read assembly followed by automatic annotation using the pubMLST. orgNeisseriadatabase was assessed and evaluated for 108 diverse, representative, and well-characterisedNeisseria meningitidisisolates. High-quality sequences were obtained for >99% of known meningococcal genes among the de novoassembled genomes and four resequenced genomes and less than 1% of reassembled genes had sequence discrepancies or misassembled sequences. A core genome of 1600 loci, present in at least 95% of the population, was determined using the Genome Comparator tool. Genealogical relationships compatible with, but at a higher resolution than, those identified by multilocus sequence typing were obtained with core genome comparisons and ribosomal protein gene analysis which revealed a genomic structure for a number of previously described phenotypes. This unified system for cataloguing Neisseria genetic variation in the genome was implemented and used for multiple analyses and the data are publically available in the PubMLST Neisseria database. Conclusions: The de novo assembly, combined with automated gene-by-gene annotation, generates high quality draft genomes in which the majority of protein-encoding genes are present with high accuracy. The approach catalogues diversity efficiently, permits analyses of a single genome or multiple genome comparisons, and is a practical approach to interpreting WGS data for large bacterial population samples. The method generates novel insights into the biology of the meningococcus and improves our understanding of the whole population structure, not just disease causing lineages.</p

A practical experiment to teach students continuous flow and physico-chemical methods: acetylation of ethylene diamine in liquid bi-phase

Despite growing applications being reported both in academia and industry, continuous flow chemistry remains a relatively untaught field across most chemistry undergraduate courses. This is particularly true in laboratory practical classes, where it is often deemed simpler to carry out synthetic reactions in traditional batch mode using round-bottomed flasks. Herein, we report the development of an undergraduate project that utilises cheap and readily available materials to construct continuous flow reactors. The students compare the performance of different types of reactors and conditions in a biphasic selective acetylation of a symmetrical diamine. Throughout the investigation, the students can vary multiple parameters as they optimise the reaction, thus actively learning and readjusting them based on their improved understanding. The experiments give the students an appreciation of continuous flow techniques in comparison to batch

There are three major Neisseria gonorrhoeae β-lactamase plasmid variants which are associated with specific lineages and carry distinct TEM alleles

Neisseria gonorrhoeae is a significant threat to global health with an estimated incidence of over 80 million cases each year and high levels of antimicrobial resistance. The gonococcal β-lactamase plasmid, pbla, carries the TEM β-lactamase, which requires only one or two amino acid changes to become an extended-spectrum β-lactamase (ESBL); this would render last resort treatments for gonorrhoea ineffective. Although pbla is not mobile, it can be transferred by the conjugative plasmid, pConj, found in N. gonorrhoeae. Seven variants of pbla have been described previously, but little is known about their frequency or distribution in the gonococcal population. We characterised sequences of pbla variants and devised a typing scheme, Ng_pblaST that allows their identification from whole genome short-read sequences. We implemented Ng_pblaST to assess the distribution of pbla variants in 15 532 gonococcal isolates. This demonstrated that only three pbla variants commonly circulate in gonococci, which together account for >99 % of sequences. The pbla variants carry different TEM alleles and are prevalent in distinct gonococcal lineages. Analysis of 2758 pbla-containing isolates revealed the co-occurrence of pbla with certain pConj types, indicating co-operativity between pbla and pConj variants in the spread of plasmid-mediated AMR in N. gonorrhoeae. Understanding the variation and distribution of pbla is essential for monitoring and predicting the spread of plasmid-mediated β-lactam resistance in N. gonorrhoeae

Development of the Pneumococcal Genome Library, a core genome multilocus sequence typing scheme, and a taxonomic life identification number barcoding system to investigate and define pneumococcal population structure

Investigating the genomic epidemiology of major bacterial pathogens is integral to understanding transmission, evolution, colonisation, disease, antimicrobial resistance, and vaccine impact. Furthermore, the recent accumulation of large numbers of whole genome sequences for many bacterial species enhances the development of robust genome-wide typing schemes to define the overall bacterial population structure and lineages within it. Using previously published data, we developed the Pneumococcal Genome Library (PGL), a curated dataset of 30,976 genomes and contextual data for carriage and disease pneumococci recovered between 1916-2018 in 82 countries. We leveraged the size and diversity of the PGL to develop a core genome multilocus sequence typing (cgMLST) scheme comprised of 1,222 loci. Finally, using multilevel single-linkage clustering, we stratified pneumococci into hierarchical clusters based on allelic similarity thresholds, and defined these with a taxonomic life identification number (LIN) barcoding system. The PGL, cgMLST scheme, and LIN barcodes represent a high-quality genomic resource and fine-scale clustering approaches for the analysis of pneumococcal populations, which support the genomic epidemiology and surveillance of this leading global pathogen. Impact statement Many thousands of pneumococcal genomes are available in the public domain, and this creates opportunities for the scientific community to re-use existing data; however, these data are most useful when the contextual data (provenance and phenotype) are also linked to the genomes. Therefore, we created a curated, open-access database in PubMLST that contained nearly 31,000 published pneumococcal genomes and the corresponding contextual data for each genome. This large and diverse pneumococcal database was used to create a novel cgMLST scheme and multilevel clustering method to define genetic lineages with high resolution and a standardised nomenclature. These are open-access resources for all to use and provide a unified framework for the characterisation of global pneumococcal populations

Acinetobacter baumannii IC2 and IC5 isolates with co-existing blaOXA-143-like and blaOXA-72 and exhibiting strong biofilm formation in a Mexican hospital

Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital

Putatively novel serotypes and the potential for reduced vaccine effectiveness: capsular locus diversity revealed among 5405 pneumococcal genomes.

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular (cps) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.This work was supported by a Wellcome Trust Biomedical Research Fund award (04992/Z/14/Z) to M. J. C. M., K. A. J., and A. B. B.; a Wellcome Trust career development fellowship (083511/Z/07/Z) to A. B. B; and a University of Oxford John Fell Fund award (123/734) to A. B. B. Core funding for the Sanger Institute was provided by the Wellcome Trust (098051). Funding for the Icelandic vaccine impact study was provided by GlaxoSmithKline Biologicals SA and the Landspítali University Hospital Research Fund to K. G. K., A. H., H. E., S. D. B., and A. B. B

Species Status of Neisseria gonorrhoeae: Evolutionary and Epidemiological Inferences from MLST

Abstract Background Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. Results A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. Conclusion The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present

ACE and response to pulmonary rehabilitation in COPD: two observational studies.

INTRODUCTION: Skeletal muscle impairment is an important feature of chronic obstructive pulmonary disease (COPD). Renin-angiotensin system activity influences muscle phenotype, so we wished to investigate whether it affects the response to pulmonary rehabilitation. METHODS: Two studies are described; in the first, the response of 168 COPD patients (mean forced expiratory volume in one second 51.9% predicted) to pulmonary rehabilitation was compared between different ACE insertion/deletion polymorphism genotypes. In a second, independent COPD cohort (n=373), baseline characteristics and response to pulmonary rehabilitation were compared between COPD patients who were or were not taking ACE inhibitors or angiotensin receptor antagonists (ARB). RESULTS: In study 1, the incremental shuttle walk distance improved to a similar extent in all three genotypes; DD/ID/II (n=48/91/29) 69(67)m, 61 (76)m and 78 (78)m, respectively, (p>0.05). In study 2, fat free mass index was higher in those on ACE-I/ARB (n=130) than those who were not (n=243), 17.8 (16.0, 19.8) kg m-2 vs 16.5 (14.9, 18.4) kg/m2 (p<0.001). However change in fat free mass, walking distance or quality of life in response to pulmonary rehabilitation did not differ between groups. CONCLUSIONS: While these data support a positive association of ACE-I/ARB treatment and body composition in COPD, neither treatment to reduce ACE activity nor ACE (I/D) genotype influence response to pulmonary rehabilitation.The study was funded by a Trevor Clay grant from The British Lung Foundation (TC 04/4) and supported by the NIHR Respiratory Biomedical Research Unit at Royal Brompton and Harefield Hospital and Imperial College who part fund MIP’s salary. WD-CM is an NIHR Clinician Scientist and supported by a MRC New Investigator Research Grant. DS was funded by the MRC G0701628. ZP is a NIHR Doctorate Research Fellow. SSCK and CJJ were funded by the MR