The Characterisation of the Genus Micrococcus and the Genus Stomatococcus Using Phenotypic and Genotypic Techniques

This study shows that the biochemical inertness of the genus Micrococcus makes the API microtitre strip an unsuitable technique by which to identify species within the genus and that colony pigmentation is an invalid character with which to speciate the genus Micrococcus. The genus Micrococcus forms a heterogeneous group of organisms when ribotype group and pulsed-field gel electrophoresis (PFGE) is considered. The use of two restriction enzymes forms different ribotype groupings within the genus. In this study M. kristinae, M. varians and the majority of M. sedentarius formed homogeneous core ribotype profiles. M. luteus and M. lylae formed heterogeneous ribotype and PFGE groups. The ability of M. lylae and M. sedentarius to belong to M. luteus ribotype groups demonstrates a genetic relationship between these species and M. luteus. Restriction enzyme analysis (REA) and PFGE were the most useful techniques for typing the micrococci. By PFGE M. roseus has a genome size of 3,387 kilobases (kb) which is approximately 30% larger than other members of the genus, which have genomes of between 1,823 and 2,598 kb, and may warrant generic status. By REA and ribotyping it was shown that people carry a clonal population of M. luteus over a period of time

Genomic analysis of global Staphylococcus argenteus strains reveals distinct lineages with differing virulence and antibiotic resistance gene content

This work was funded by the Scottish Executive via the Chief Scientists Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI).Infections due to Staphylococcus argenteus have been increasingly reported worldwide and the microbe cannot be distinguished from Staphylococcus aureus by standard methods. Its complement of virulence determinants and antibiotic resistance genes remain unclear, and how far these are distinct from those produced by S. aureus remains undetermined. In order to address these uncertainties, we have collected 132 publicly available sequences from fourteen different countries, including the United Kingdom, between 2005 and 2018 to study the global genetic structure of the population. We have compared the genomes for antibiotic resistance genes, virulence determinants and mobile genetic elements such as phages, pathogenicity islands and presence of plasmid groups between different clades. 20% (n = 26) isolates were methicillin resistant harboring a mecA gene and 88% were penicillin resistant, harboring the blaZ gene. ST2250 was identified as the most frequent strain, but ST1223, which was the second largest group, contained a marginally larger number of virulence genes compared to the other STs. Novel S. argenteus pathogenicity islands were identified in our isolates harboring tsst-1, seb, sec3, ear, selk, selq toxin genes, as well as chromosomal clusters of enterotoxin and superantigen-like genes. Strain-specific type I modification systems were widespread which would limit interstrain transfer of genetic material. In addition, ST2250 possessed a CRISPR/Cas system, lacking in most other STs. S. argenteus possesses important genetic differences from S. aureus, as well as between different STs, with the potential to produce distinct clinical manifestations.Publisher PDFPeer reviewe

Origin, maintenance and spread of antibiotic resistance genes within plasmids and chromosomes of bloodstream isolates of Escherichia coli

The work was funded by the Scottish Executive via the Chief Scientists Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI).Blood stream invasion by Escherichia coli is the commonest cause of bacteremia in the UK and elsewhere with an attributable mortality of about 15–20 %; antibiotic resistance to multiple agents is common in this microbe and is associated with worse outcomes. Genes conferring antimicrobial resistance, and their frequent location on horizontally transferred genetic elements is well-recognised, but the origin of these determinants, and their ability to be maintained and spread within clinically-relevant bacterial populations is unclear. Here, we set out to examine the distribution of antimicrobial resistance genes in chromosomes and plasmids of 16 bloodstream isolates of E. coli from patients within Scotland, and how these genes are maintained and spread. Using a combination of short and long-read whole genome sequencing methods, we were able to assemble complete sequences of 44 plasmids, with 16 Inc group F and 20 col plasmids; antibiotic resistance genes located almost exclusively within the F group. blaCTX-M15 genes had re-arranged in some strains into the chromosome alone (five strains), while others contained plasmid copies alone (two strains). Integrons containing multiple antibiotic genes were widespread in plasmids, notably many with a dfrA7 gene encoding resistance to trimethoprim, thus linking trimethoprim resistance to the other antibiotic resistance genes within the plasmids. This will allow even narrow spectrum antibiotics such as trimethoprim to act as a selective agent for plasmids containing antibiotic resistance genes mediating much broader resistance, including blaCTX-M15. To our knowledge, this is the first analysis to provide complete sequence data of chromosomes and plasmids in a collection of pathogenic human bloodstream isolates of E. coli. Our findings reveal the interplay between plasmids and integrative and conjugative elements in the maintenance and spread of antibiotic resistance genes within pathogenic E. coli.Publisher PDFPeer reviewe

How do we evaluate the cost of nosocomial infection? The ECONI protocol: an incidence study with nested case-control evaluating cost and quality of life

Introduction Healthcare-associated or nosocomial infection (HAI) is distressing to patients and costly for the National Health Service (NHS). With increasing pressure to demonstrate cost-effectiveness of interventions to control HAI and notwithstanding the risk from antimicrobial-resistant infections, there is a need to understand the incidence rates of HAI and costs incurred by the health system and for patients themselves. Methods and analysis The Evaluation of Cost of Nosocomial Infection study (ECONI) is an observational incidence survey with record linkage and a nested case-control study that will include postdischarge longitudinal follow-up and qualitative interviews. ECONI will be conducted in one large teaching hospital and one district general hospital in NHS Scotland. The case mix of these hospitals reflects the majority of overnight admissions within Scotland. An incidence survey will record all HAI cases using standard case definitions. Subsequent linkage to routine data sets will provide information on an admission cohort which will be grouped into HAI and non-HAI cases. The case-control study will recruit eligible patients who develop HAI and twice that number without HAI as controls. Patients will be asked to complete five questionnaires: the first during their stay, and four others during the year following discharge from their recruitment admission (1, 3, 6 and 12 months). Multiple data collection methods will include clinical case note review; patient-reported outcome; linkage to electronic health records and qualitative interviews. Outcomes collected encompass infection types; morbidity and mortality; length of stay; quality of life; healthcare utilisation; repeat admissions and postdischarge prescribing. Ethics and dissemination The study has received a favourable ethical opinion from the Scotland A Research Ethics Committee (reference 16/SS/0199). All publications arising from this study will be published in open-access peer-reviewed journal. Lay-person summaries will be published on the ECONI website. Trial registration number NCT03253640; Pre-results

Cost burden of Clostridioides difficile infection to the health service:A retrospective cohort study in Scotland

BACKGROUND:Clostridioides difficile infection (CDI) is associated with high healthcare demands and related costs. AIM:To evaluate the healthcare and economic burden of CDI in hospitalised patients with community- (HOCA-CDI) or hospital-associated CDI (HOHA-CDI) in the National Health Service in Scotland. METHODS:A retrospective cohort study was conducted, examining data between August 2010 and July 2013 from four patient-level Scottish datasets, linked to death data. Data examined included prior antimicrobial prescriptions in the community, hospitalisations, length of stay and mortality. Each CDI case was matched to three hospital-based controls on the basis of age, gender, hospital and date of admission. Descriptive economic evaluations were based on bed-day costs for different types of wards. FINDINGS:Overall, 3304 CDI cases were included in the study. CDI was associated with additional median lengths of stay of 7.2 days for HOCA-CDI and 12.0 days for HOHA-CDI compared with their respective, matched controls. The 30-day mortality rate was 6.8% for HOCA-CDI and 12.4% for HOHA-CDI. Overall, recurrence within 90 days of the first CDI episode occurred in 373/2740 (13.6%) survivors. The median additional expenditure for each initial CDI case compared with matched controls was £1713. In the 6 months after the index hospitalisation, the cost associated with a CDI case was £5126 higher than for controls. CONCLUSION:Using routinely collected national data, we demonstrate the substantial burden of CDI on healthcare services, including lengthy hospital stays and readmissions, which increase the costs of managing patients with CDI compared with matched controls

Genetic analysis of invasive Escherichia coli in Scotland reveals determinants of healthcare-associated versus community-acquired infections

The work was funded by the Scottish Executive via the Chief Scientist Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI).Bacteraemia caused by Escherichia coli is a growing problem with a significant mortality. The factors that influence the acquisition and outcome of these infections are not clear. Here, we have linked detailed genetic data from the whole-genome sequencing of 162 bacteraemic isolates collected in Scotland, UK, in 2013-2015, with clinical data in order to delineate bacterial and host factors that influence the acquisition in hospital or the community, outcome and antibiotic resistance. We identified four major sequence types (STs) in these isolates: ST131, ST69, ST73 and ST95. Nearly 50% of the bacteraemic isolates had a urinary origin. ST69 was genetically distinct from the other STs, with significantly less sharing of accessory genes and with a distinct plasmid population. Virulence genes were widespread and diversely distributed between the dominant STs. ST131 was significantly associated with hospital-associated infections (HAIs), and ST69 with those from the community. However, there was no association of ST with outcome, although patients with HAI had a higher immediate mortality compared to those with community-associated infections (CAIs). Genome-wide association studies revealed genes involved in antibiotic persistence as significantly associated with HAIs and those encoding elements of a type VI secretion system with CAIs. Antibiotic resistance was common, and there were networks of correlated resistance genes and phenotypic antibiotic resistance. This study has revealed the complex interactions between the genotype of E. coli and its ability to cause bacteraemia, and some of the determinants influencing hospital or community acquisition. In part, these are shaped by antibiotic usage, but strain-specific factors are also important.Publisher PDFPeer reviewe

Consideration of within-patient diversity highlights transmission pathways and antimicrobial resistance gene variability in vancomycin-resistant Enterococcus faecium

BackgroundWGS is increasingly being applied to healthcare-associated vancomycin-resistant Enterococcus faecium (VREfm) outbreaks. Within-patient diversity could complicate transmission resolution if single colonies are sequenced from identified cases.ObjectivesDetermine the impact of within-patient diversity on transmission resolution of VREfm.Materials and methodsFourteen colonies were collected from VREfm positive rectal screens, single colonies were collected from clinical samples and Illumina WGS was performed. Two isolates were selected for Oxford Nanopore sequencing and hybrid genome assembly to generate lineage-specific reference genomes. Mapping to closely related references was used to identify genetic variations and closely related genomes. A transmission network was inferred for the entire genome set using Phyloscanner.Results and discussionIn total, 229 isolates from 11 patients were sequenced. Carriage of two or three sequence types was detected in 27% of patients. Presence of antimicrobial resistance genes and plasmids was variable within genomes from the same patient and sequence type. We identified two dominant sequence types (ST80 and ST1424), with two putative transmission clusters of two patients within ST80, and a single cluster of six patients within ST1424. We found transmission resolution was impaired using fewer than 14 colonies.ConclusionsPatients can carry multiple sequence types of VREfm, and even within related lineages the presence of mobile genetic elements and antimicrobial resistance genes can vary. VREfm within-patient diversity could be considered in future to aid accurate resolution of transmission networks

A highly conserved complete accessory Escherichia coli type III secretion system 2 is widespread in bloodstream isolates of the ST69 lineage

The work was funded by the Scottish Executive via the Chief Scientists Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.Bacterial type III secretion systems (T3SSs) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagic Escherichia coli contain a well-defined T3SS but in addition a second T3SS termed E. coli T3SS 2 (ETT2) has been described in a number of strains of E. coli. The majority of pathogenic E. coli contain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenic E. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that 22 of 26 sequence type (ST) 69 isolates from this collection contained an intact ETT2 together with an associated eip locus which encodes putative secreted ETT2 effectors as well as eilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene for eilA activation, we defined conditions under which this gene was differentially activated. Analysis of published E. coli genomes with worldwide representation showed that ST69 contained an intact ETT2 in these strains as well. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has importance in infection, although its exact functional role remains obscure.Publisher PDFPeer reviewe