Molecular characterization of plasmids encoding blaCTX-M from faecal Escherichia coli in travellers returning to the UK from South Asia.

BACKGROUND: The global prevalence of extended-spectrum beta-lactamase-producing Escherichia coli is rising and is dominated by blaCTX-M spread by plasmids. Travellers to South Asia from Western Europe have high rates of acquisition of faecal CTX-M-producing E. coli (CTX-M-EC). AIMS: To determine the conjugative ability of CTX-M-EC acquired by healthy volunteers after travel to South Asia, the proportion of travel-acquired CTX-M-EC where blaCTX-M is encoded on a plasmid vs on the bacterial chromosome, and the relatedness of travel-acquired CTX-M-EC plasmids to previously sequenced plasmids. METHODS: Faecal samples were collected pre- and post-travel from 23 volunteers who visited South Asia, and CTX-M-EC were cultured. After short- and long-read sequencing, 10 plasmid sequences were identified and compared with previously sequenced plasmids in GenBank. Conjugation to E. coli K-12 was undertaken using filter mating. FINDINGS: Thirty-five percent of CTX-M-EC isolates tested transferred the blaCTX-M plasmid by conjugation. Travel-acquired CTX-M-EC carried blaCTX-M on a plasmid in 62% of isolates, whereas 38% of isolates had blaCTX-M on the chromosome. CTX-M-EC plasmids acquired after travel to South Asia had close homology to previously described epidemic plasmids which are widely disseminated in humans, animals and the natural environment. CONCLUSION: Globally successful epidemic plasmids are involved in the spread of CTX-M-EC. Targeted strategies may be used to displace such plasmids from the host strain as part of efforts in infection prevention and control in healthcare settings. Bacteria with blaCTX-M plasmids were readily acquired by healthy volunteers, and were carried on return to the UK, providing opportunities for onward dissemination.

Results from the first English stool bank using faecal microbiota transplant as a medicinal product for the treatment of Clostridioides difficile infection.

BACKGROUND: Faecal Microbiota Transplant (FMT) has improved outcomes for the treatment of Clostridioides difficile infection (CDI) compared to antibiotic therapy. FMT is classified as a medicinal product in the United Kingdom, similar to the USA and Canada, limiting supply via stool banks without appropriate licencing. In the largest UK cohort to date, we describe the clinical outcomes for 124 patients receiving FMT for recurrent or refractory CDI and present a framework to produce FMT as a licenced medicinal product. METHODS: Anonymous unrelated healthy donors, screened via health assessment and microbiological testing donated stool. In aerobic conditions FMT aliquots were prepared for immediate use or frozen storage, following a production framework developed to comply with Good Manufacturing Practice. Outcome measures were clinical response to FMT defined as resolution of diarrhoea within seven days and clinical cure defined as response without diarrhoea recurrence at 90 days. FINDINGS: Clinical response was 83·9% (95% CI 76·0%-90·0%) after one treatment. Clinical cure was 78·2% (95% CI 67·4%-89·0%) across the cohort. Refractory cases appeared to have a lower initial clinical response rate compared to recurrent cases, however at day 90 there were no differences observed between these groups. INTERPRETATION: The methodology developed here enabled successful licencing of FMT by The Medicines and Healthcare products Regulatory Agency as a medicinal product. This has widened the availability of FMT in the National Health Service via a stool bank and can be applied in other centres across the world to improve access to safe and quality assured treatments.

Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening.

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum ß-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important ß-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel ß-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.

Investigation of community carriage rates of Clostridium difficile and Hungatella hathewayi in healthy volunteers from four regions of England.

Faecal samples from 1365 healthy asymptomatic volunteers from four regions in England were screened for the presence of Clostridium difficile between December 2013 and July 2014. The carriage rate of C. difficile in healthy patients was 0.5%, which is lower than reported previously. This study demonstrates that the true community reservoir of C. difficile in the healthy UK population is very low and is, therefore, unlikely to be a reservoir for infections diagnosed in the hospital setting.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

Programmatic utility of tuberculosis cluster investigation using a social network approach in Birmingham, United Kingdom.

SETTING: Birmingham, United Kingdom, 2010-2014. OBJECTIVE: To investigate predictors for clustering of tuberculosis (TB) cases and cluster size and to evaluate the impact of cluster investigation using social network data. DESIGN: Retrospective observational cohort study. Prioritised cases linked using 24-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) were interviewed using a social network approach to find epidemiological links. RESULTS: Of 2055 TB cases notified, 56% could be typed. Clustering was associated with younger age, UK birth, Black Caribbean ethnicity, social risk factors, pulmonary TB and negative human immunodeficiency virus status. Only UK birth and presence of more than one social risk factor were associated with larger cluster size, while drug resistance was associated with smaller cluster size. Social network data from 139/431 clustered cases found new epidemiological links in 11/19 clusters with ⩾5 members (undirected median network density 0.09, interquartile range 0.05-0.4). Ninety-eight additional contacts were assessed, with one case of active TB and 24 with latent tuberculous infection diagnosed. CONCLUSION: A social network approach increased knowledge of likely transmission events, but few additional TB cases were diagnosed. Obtaining social network data for all typed and untyped TB cases may improve contact tracing and reduce unexpected transmission detected from molecular data.

Multidrug-resistant Gram-negative bacteria: a product of globalization.

Global trade and mobility of people has increased rapidly over the last 20 years. This has had profound consequences for the evolution and the movement of antibiotic resistance genes. There is increasing exposure of populations all around the world to resistant bacteria arising in the emerging economies. Arguably the most important development of the last two decades in the field of antibiotic resistance is the emergence and spread of extended-spectrum ß-lactamases (ESBLs) of the CTX-M group. A consequence of the very high rates of ESBL production among Enterobacteriaceae in Asian countries is that there is a substantial use of carbapenem antibiotics, resulting in the emergence of plasmid-mediated resistance to carbapenems. This article reviews the emergence and spread of multidrug-resistant Gram-negative bacteria, focuses on three particular carbapenemases--imipenem carbapenemases, Klebsiella pneumoniae carbapenemase, and New Delhi metallo-ß-lactamase--and highlights the importance of control of antibiotic use.

Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment.

OBJECTIVES: Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river. METHODS: River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate bla(CTX-M-15) mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology. RESULTS: We report the first examples of bla(CTX-M-15) in UK river sediment; the prevalence of bla(CTX-M-15) was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The bla(CTX-M-15) -gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river. CONCLUSIONS: The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.

Faecal transplantation for the treatment of Clostridium difficile infection: a review.

Clostridium difficile infection (CDI) remains a major healthcare burden despite recent global falls in its prevalence. The risk of recurrence is high when using antibiotics such as vancomycin, particularly in already recurrent disease. In light of this, new therapy options are being perused, including novel antibiotics such as fidaxomicin, probiotics, intravenous immunoglobulin and faecal transplantation. Faecal transplantation, referred to here as human probiotic infusion (HPI), is attracting an increasing amount of interest from physicians and patients. Its use has been documented in ca. 500 cases for the treatment of CDI, with overall efficacy rates reported to be ca. 91%. The first randomised controlled trial (RCT) demonstrated that HPI was superior to a 14-day course of vancomycin (89% vs. 31%; P<0.001) and reported no deaths or serious adverse events. Safety and patient acceptability are often cited as limitations to the widespread use of this technique. However, data suggest that the short-term safety profile is encouraging, and concerns over patient acceptability are not warranted in the majority of cases. It seems appropriate to treat an infection which is caused by a major disturbance in the gut microbiota with a treatment that reverses this disturbance, rather than antibiotics that may exacerbate the problem. However, to fully understand the role of HPI in the management of CDI, further RCTs are needed with comparator antibiotics such as fidaxomicin and to establish the most efficacious HPI protocol for administration and preparation.

Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common ß-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.

Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.

A two-stage algorithm for Clostridium difficile including PCR: can we replace the toxin EIA?

A two step, three-test algorithm for Clostridium difficile infection (CDI) was reviewed. Stool samples were tested by enzyme immunoassays for C. difficile common antigen glutamate dehydrogenase (G) and toxin A/B (T). Samples with discordant results were tested by polymerase chain reaction detecting the toxin B gene (P). The algorithm quickly identified patients with detectable toxin A/B, whereas a large group of patients excreting toxigenic C. difficile but with toxin A/B production below detection level (G(+)T(-)P(+)) was identified separately. The average white blood cell count in patients with a G(+)T(+) result was higher than in those with a G(+)T(-)P(+) result.

Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission.

PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.

Integron prevalence and diversity in manured soil.

The levels of integron abundance and diversity in soil amended with pig slurry were studied. Real-time PCR illustrated a significant increase in class 1 integron prevalence after slurry application, with increased prevalence still evident at 10 months after application. Culture-dependent data revealed 10 genera, including putative human pathogens, carrying class 1 and 2 integrons.

Community and nosocomial transmission of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus: implications for healthcare.

In the UK, infections due to Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus (PVL-MRSA) have been reported sporadically. In September 2006, a fatal PVL-MRSA infection occurred in a Filipino healthcare worker (HCW) after she underwent caesarean section. Throat and nasal swabs were obtained from contacts of cases in community and hospital. MRSA with an antibiogram similar to the PVL-MRSA strain were characterised including toxin gene profiling, polymerase chain reaction- and sequence-based typing. Carriers underwent decolonisation treatment, and HCWs were restricted from patient care until they and their household members were considered negative for PVL-MRSA. The PVL-MRSA belonged to ST30, was protein A gene (spa) type t019, SCCmec IVc, agr 3, and resistant only to beta-lactam antibiotics. Representatives of the same lineage were identified among a further 16 individuals in community and hospital. Infections likely to be caused by PVL-MRSA had occurred in 12 cases, and were likely to be hospital-acquired in two patients (one fatal) and occupationally acquired in one HCW. Nine cases worked as nursing staff in the hospital. Eight of these had emigrated from the Philippines in the previous five years and were linked socially. Thus, PVL-MRSA-ST30 was detected in a HCW community in the UK. This is the first report of nosocomial transmission of this pandemic clone in the UK associated with a fatality. Increased vigilance in healthcare and community is needed in response to this emerging threat.

Reducing Clostridium difficile through early identification of clusters and the use of a standardised set of interventions.

In recent years the rates of Clostridium difficile infection (CDI) have increased worldwide with several large outbreaks occurring within the UK. New guidance from the UK Department of Health describes measures to investigate periods of increased incidence (PII) of CDI which include informing staff, ribotyping isolates, enhanced cleaning, audits and monitoring of antibiotic prescribing. This study aimed to determine whether a standardised set of measures could be used to control the incidence of CDI within an acute hospital setting over an 18 month period. During the study period a total of 102 PII involving 439 patients were investigated. The number of PII per month ranged from 14 in February 2008 to one in June 2009. From January 2008 to September 2008, ribotyping of patient isolates was only carried out on PII involving more than 10 patients, but from October 2008 it was carried out on all PII. During the period October 2008 to June 2009, 28 PII were investigated on 21 different wards, with seven wards having two PII. Ribotyping of the isolates confirmed nine (32%) of these PII to be outbreaks, with three being due to ribotype 027, two ribotype 078 and the others distinct ribotypes. Use of a set of standardised interventions has resulted in a decrease in the incidence of PII and a reduction in the number of patients involved. By taking early action with a set of standardised measures the incidence of hospital-acquired CDI can be reduced.

Low-level glycopeptide resistance in methicillin-resistant Staphylococcus aureus and how to test it.

Vancomycin resistance in Staphylococcus aureus has emerged over the last ten years due to varying mechanisms and giving variable levels of resistance to vancomycin. The most resistant strains (fortunately rare) bear the vanA gene cluster and these are generally recognisable as MICs of vancomycin are usually found to be in the range 32-64mg/L. It should be noted that some automated systems have failed to detect these isolates. The much more commonly encountered GISA and hGISA vancomycin resistant strains of MRSA and methicillin sensitive Staph. aureus (MSSA) exhibit lower levels of resistance and difficulty is encountered in reliably defining and identifying these strains in clinical laboratories. No single completely reliable, convenient test either phonotypical genetic currently exists which can be readily applied in the clinical laboratory for the detection of hGISA/GISA. The population analysis profile (PAP) method is currently regarded as the reference method but is slow and tedious to perform on a large number of isolates. This enables the differentiation of hGISA and GISA from fully vancomycin sensitive strains. In the clinical laboratory the use of Meuller-Hinton agar with 5mg/L teicoplanin and a 10microL innoculum of MacFarland 0.5 incubated for 48h represents the most reliable and economical screening test. Further confirmation would be required using either macrodilution Etest methodology using an MIC >or= 8mg/L of vancomycin and/or teicoplanin as the cut off for hGISA or the newer GRD (glycopeptide resistance detection) strip.

Beta-lactams and beta-lactamase-inhibitors in current- or potential-clinical practice: a comprehensive update.

The use of successive generations of beta-lactams has selected successive generations of beta-lactamases including CTX-M ESBLs, AmpC beta-lactamases, and KPC carbapenamases in Enterobacteriaceae. Moreover, this cephalosporin resistance, along with rising resistance to fluoroquinolones, is now driving the use of carbapenems and unfortunately the carbapenem resistance has emerged markedly, especially in Acinetobacter spp. due to OXA- and metallo-carbapenemases. The industry responded to the challenge of rising resistance and recently developed some novel beta-lactams such as ceftobiprole, ceftaroline etc. and many beta-lactam compounds, including beta-lactamase-inhibitors, such as BMS-247243, S-3578, RWJ-54428, CS-023, SMP-601, NXL 104, BAL 30376, LK 157, and so on are under trials. This review provides the comprehensive accounts of the developments in penicillins, cephalosporins, carbapenems, and beta-lactamase-inhibitors, and the insight about medicinal chemistry, mechanism(s) of action and resistance, potential strategies to overcome resistance due to beta-lactamases, and also the recent advancements in the development of newer beta-lactam compounds; some of which are still under trials and yet to be classified. This review will fill the gap since previously published reviews and will serve as a comprehensive update on the current topic.