Investigation of two cases of Mycobacterium chelonae infection in haemato-oncology patients using whole-genome sequencing and a potential link to the hospital water supply.

BACKGROUND: Haemato-oncology patients are at increased risk of infection from atypical mycobacteria such as Mycobacterium chelonae which are commonly found in both domestic and hospital water systems. AIMS: To describe the investigation and control measures following two patient cases of M. chelonae and positive water samples in the study hospital. METHODS: Water testing was undertaken from outlets, storage tanks and mains supply. Whole-genome sequencing (WGS) was used to compare patient and positive water samples. The subsequent infection control measures implemented are described. FINDINGS: The WGS results showed two main populations of M. chelonae within the group of sampled isolates. The results showed that the patient strains were unrelated to each other, but that the isolate from one patient was closely related to environmental samples from water outlets, supporting nosocomial acquisition. CONCLUSIONS: WGS was used to investigate two patient cases of M. chelonae and positive water samples from a hospital water supply. Relevant control measures and the potential for chemical dosing of water systems to enhance proliferation of atypical mycobacteria are discussed.

Clinical perspectives in integrating whole-genome sequencing into the investigation of healthcare and public health outbreaks - hype or help?

Outbreaks pose a significant risk to patient safety as well as being costly and time consuming to investigate. The implementation of targeted infection prevention and control measures relies on infection prevention and control teams having access to rapid results that detect resistance accurately, and typing results that give clinically useful information on the relatedness of isolates. At present, determining whether transmission has occurred can be a major challenge. Conventional typing results do not always have sufficient granularity or robustness to define strains unequivocally, and sufficient epidemiological data are not always available to establish links between patients and the environment. Whole-genome sequencing (WGS) has emerged as the ultimate genotyping tool, but has not yet fully crossed the divide between research method and routine clinical diagnostic microbiological technique. A clinical WGS service was officially established in 2014 as part of the Scottish Healthcare Associated Infection Prevention Institute to confirm or refute outbreaks in hospital settings from across Scotland. This article describes the authors' experiences with the aim of providing new insights into practical application of the use of WGS to investigate healthcare and public health outbreaks. Solutions to overcome barriers to implementation of this technology in a clinical environment are proposed.

Chlorhexidine and octenidine use, carriage of qac genes, and reduced antiseptic susceptibility in methicillin-resistant Staphylococcus aureus isolates from a healthcare network.

OBJECTIVES: With the widespread use of antiseptics in healthcare facilities for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) transmission, there are concerns for antiseptic tolerance and resistance. We sought to understand the use of chlorhexidine and octenidine, carriage of qac genes, and reduced antiseptic susceptibilities. METHODS: A serial cross-sectional study was conducted in an acute care hospital and three extended-care facilities of a healthcare network in June-July, 2014-2016. Two of the extended-care facilities were exposed to intranasal octenidine and universal daily chlorhexidine/octenidine bathing. The minimum inhibitory concentration (MIC) levels and qac genes were determined by broth microdilution tests and whole genome sequencing respectively. Multivariable logistic regression was used to assess for the independent associations between antiseptic exposures, qac genes, and reduced antiseptic susceptibilities. RESULTS: A total of 878 MRSA isolates were obtained. There were associations between qacA/B carriage and chlorhexidine (adjusted odds ratio (aOR) 7.80; 95% confidence interval (CI) 3.25-18.71) and octenidine (aOR 11.79; 95% CI 5.14-27.04) exposures. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility (MIC ≥4 mg/L) (aOR 3.15; 95% CI 1.14-8.74). Carriage of qacA/B (aOR 10.65; 95% CI 4.14-27.40) or qacC (aOR 2.55; 95% CI 1.22-5.32) had an association with reduced chlorhexidine susceptibility; while MRSA sequence type modified the association. However, we found no direct association between (i) antiseptics use and qacC carriage, (ii) octenidine exposure and reduced susceptibility, and (iii) reduced octenidine susceptibility and qacA/B or qacC carriage. CONCLUSIONS: Antiseptic exposures were associated with carriage of qac genes. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility, requiring continued surveillance for the emergence of resistance.

The widespread use of topical antimicrobials enriches for resistance in Staphylococcus aureus isolated from patients with atopic dermatitis.

BACKGROUND: Carriage rates of Staphylococcus aureus on affected skin in atopic dermatitis (AD) are approximately 70%. Increasing disease severity during flares and overall disease severity correlate with increased burden of S. aureus. Treatment in AD therefore often targets S. aureus with topical and systemic antimicrobials. OBJECTIVES: To determine whether antimicrobial sensitivities and genetic determinants of resistance differed in S. aureus isolates from the skin of children with AD and healthy child nasal carriers. METHODS: In this case-control study, we compared S. aureus isolates from children with AD (n = 50) attending a hospital dermatology department against nasal carriage isolates from children without skin disease (n = 49) attending a hospital emergency department for noninfective conditions. Using whole genome sequencing we generated a phylogenetic framework for the isolates based on variation in the core genome, then compared antimicrobial resistance phenotypes and genotypes between disease groups. RESULTS: Staphylococcus aureus from cases and controls had on average similar numbers of phenotypic resistances per isolate. Case isolates differed in their resistance patterns, with fusidic acid resistance (FusR ) being significantly more frequent in AD (P = 0·009). The genetic basis of FusR also differentiated the populations, with chromosomal mutations in fusA predominating in AD (P = 0·049). Analysis revealed that FusR evolved multiple times and via multiple mechanism in the population. Carriage of plasmid-derived qac genes, which have been associated with reduced susceptibility to antiseptics, was eight times more frequent in AD (P = 0·016). CONCLUSIONS: The results suggest that strong selective pressure drives the emergence and maintenance of specific resistances in AD.

Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing.

BACKGROUND: Pseudomonas aeruginosa healthcare outbreaks can be time consuming and difficult to investigate. Guidance does not specify which typing technique is most practical for decision-making. AIM: To explore the usefulness of whole-genome sequencing (WGS) in the investigation of a P. aeruginosa outbreak, describing how it compares with pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) analysis. METHODS: Six patient isolates and six environmental samples from an intensive care unit (ICU) positive for P. aeruginosa over two years underwent VNTR, PFGE and WGS. FINDINGS: VNTR and PFGE were required to fully determine the potential source of infection and rule out others. WGS results unambiguously distinguished linked isolates, giving greater assurance of the transmission route between wash-hand basin water and two patients, supporting the control measures employed. CONCLUSION: WGS provided detailed information without the need for further typing. When allied to epidemiological information, WGS can be used to understand outbreak situations rapidly and with certainty. Implementation of WGS in real-time would be a major advance in day-to-day practice. It could become a standard of care as it becomes more widespread due to its reproducibility and lower costs.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee.

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

Genomic insights into the emergence and spread of international clones of healthcare-, community- and livestock-associated meticillin-resistant Staphylococcus aureus: Blurring of the traditional definitions.

The evolution of meticillin-resistant Staphylococcus aureus (MRSA) from meticillin-susceptible S. aureus has been a result of the accumulation of genetic elements under selection pressure from antibiotics. The traditional classification of MRSA into healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) is no longer relevant as there is significant overlap of identical clones between these groups, with an increasing recognition of human infection caused by livestock-associated MRSA (LA-MRSA). Genomic studies have enabled us to model the epidemiology of MRSA along these lines. In this review, we discuss the clinical relevance of genomic studies, particularly whole-genome sequencing, in the investigation of outbreaks. We also discuss the blurring of each of the three epidemiological groups (HA-MRSA, CA-MRSA and LA-MRSA), demonstrating the limited relevance of this classification.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129.

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.