A workflow for the detection of antibiotic residues, measurement of water chemistry and preservation of hospital sink drain samples for metagenomic sequencing.

BACKGROUND: Hospital sinks are environmental reservoirs that harbour healthcare-associated (HCA) pathogens. Selective pressures in sink environments, such as antibiotic residues, nutrient waste and hardness ions, may promote antibiotic resistance gene (ARG) exchange between bacteria. However, cheap and accurate sampling methods to characterize these factors are lacking. AIMS: To validate a workflow to detect antibiotic residues and evaluate water chemistry using dipsticks. Secondarily, to validate boric acid to preserve the taxonomic and ARG ('resistome') composition of sink trap samples for metagenomic sequencing. METHODS: Antibiotic residue dipsticks were validated against serial dilutions of ampicillin, doxycycline, sulfamethoxazole and ciprofloxacin, and water chemistry dipsticks against serial dilutions of chemical calibration standards. Sink trap aspirates were used for a 'real-world' pilot evaluation of dipsticks. To assess boric acid as a preservative of microbial diversity, the impact of incubation with and without boric acid at ∼22 °C on metagenomic sequencing outputs was evaluated at Day 2 and Day 5 compared with baseline (Day 0). FINDINGS: The limits of detection for each antibiotic were: 3 µg/L (ampicillin), 10 µg/L (doxycycline), 20 µg/L (sulfamethoxazole) and 8 µg/L (ciprofloxacin). The best performing water chemistry dipstick correctly characterized 34/40 (85%) standards in a concentration-dependent manner. One trap sample tested positive for the presence of tetracyclines and sulphonamides. Taxonomic and resistome composition were largely maintained after storage with boric acid at ∼22 °C for up to five days. CONCLUSIONS: Dipsticks can be used to detect antibiotic residues and characterize water chemistry in sink trap samples. Boric acid was an effective preservative of trap sample composition, representing a low-cost alternative to cold-chain transport.

High-resolution characterization of short-term temporal variability in the taxonomic and resistome composition of wastewater influent.

Wastewater-based epidemiology (WBE) for population-level surveillance of antimicrobial resistance (AMR) is gaining significant traction, but the impact of wastewater sampling methods on results is unclear. In this study, we characterized taxonomic and resistome differences between single-timepoint-grab and 24 h composites of wastewater influent from a large UK-based wastewater treatment work [WWTW (population equivalent: 223 435)]. We autosampled hourly influent grab samples (n=72) over three consecutive weekdays, and prepared additional 24 h composites (n=3) from respective grabs. For taxonomic profiling, metagenomic DNA was extracted from all samples and 16S rRNA gene sequencing was performed. One composite and six grabs from day 1 underwent metagenomic sequencing for metagenomic dissimilarity estimation and resistome profiling. Taxonomic abundances of phyla varied significantly across hourly grab samples but followed a repeating diurnal pattern for all 3 days. Hierarchical clustering grouped grab samples into four time periods dissimilar in both 16S rRNA gene-based profiles and metagenomic distances. 24H-composites resembled mean daily phyla abundances and showed low variability of taxonomic profiles. Of the 122 AMR gene families (AGFs) identified across all day 1 samples, single grab samples identified a median of six (IQR: 5-8) AGFs not seen in the composite. However, 36/36 of these hits were at lateral coverage <0.5 (median: 0.19; interquartile range: 0.16-0.22) and potential false positives. Conversely, the 24H-composite identified three AGFs not seen in any grab with higher lateral coverage (0.82; 0.55-0.84). Additionally, several clinically significant human AGFs (bla VIM, bla IMP, bla KPC) were intermittently or completely missed by grab sampling but captured by the 24 h composite. Wastewater influent undergoes significant taxonomic and resistome changes on short timescales potentially affecting interpretation of results based on sampling strategy. Grab samples are more convenient and potentially capture low-prevalence/transient targets but are less comprehensive and temporally variable. Therefore, we recommend 24H-composite sampling where feasible. Further validation and optimization of WBE methods is vital for its development into a robust AMR surveillance approach.

Systematic review of wastewater surveillance of antimicrobial resistance in human populations.

OBJECTIVES: We systematically reviewed studies using wastewater for AMR surveillance in human populations, to determine: (i) evidence of concordance between wastewater-human AMR prevalence estimates, and (ii) methodological approaches which optimised identifying such an association, and which could be recommended as standard. We used Lin's concordance correlation coefficient (CCC) to quantify concordance between AMR prevalence estimates in wastewater and human compartments (where CCC = 1 reflects perfect concordance), and logistic regression to identify study features (e.g. sampling methods) associated with high agreement studies (defined as >70% of within-study wastewater-human AMR prevalence comparisons within ±10%). RESULTS: Of 8,867 records and 441 full-text methods reviewed, 33 studies were included. AMR prevalence data was extractable from 24 studies conducting phenotypic-only (n = 7), genotypic-only (n = 1) or combined (n = 16) AMR detection. Overall concordance of wastewater-human AMR prevalence estimates was reasonably high for both phenotypic (CCC = 0.85 [95% CI 0.8-0.89]) and genotypic approaches (CCC = 0.88 (95% CI 0.84-0.9)) despite diverse study designs, bacterial species investigated and phenotypic/genotypic targets. No significant relationships between methodological approaches and high agreement studies were identified using logistic regression; however, this was limited by inconsistent reporting of study features, significant heterogeneity in approaches and limited sample size. Based on a secondary, descriptive synthesis, studies conducting composite sampling of wastewater influent, longitudinal sampling >12 months, and time-/location-matched sampling of wastewater and human compartments generally had higher agreement. CONCLUSION: Wastewater-based surveillance of AMR appears promising, with high overall concordance between wastewater and human AMR prevalence estimates in studies irrespective of heterogenous approaches. However, our review suggests future work would benefit from: time-/location-matched sampling of wastewater and human populations, composite sampling of influent, and sampling >12 months for longitudinal studies. Further research and clear and consistent reporting of study methods is required to identify optimal practice.

Barriers and facilitators to infection prevention and control in a neonatal unit in Zimbabwe - a theory-driven qualitative study to inform design of a behaviour change intervention.

BACKGROUND: Hospital-acquired infection (HAI) is an increasing cause of neonatal morbidity/mortality in low-income settings. Hospital staff behaviours (e.g., hand hygiene) are key contributors to HAI. Understanding the drivers of these can inform interventions to improve infection prevention and control (IPC). AIM: To explore barriers/facilitators to IPC in a neonatal unit in Harare, Zimbabwe. METHODS: Interviews were conducted with 15 staff members of neonatal and maternity units alongside ethnographic observations. The interview guide and data analysis were informed by the COM-B (Capability, Opportunity, Motivation-Behaviour) model and explored individual, socio-cultural, and organizational barriers/facilitators to IPC. Potential interventions were identified using the Behaviour-Change Wheel. FINDINGS: Enablers within Capability included awareness of IPC, and within Motivation beliefs that IPC was crucial to one's role, and concerns about consequences of poor IPC. Staff were optimistic that IPC could improve, contingent upon resource availability (Opportunity). Barriers included: limited knowledge of guidelines, no formal feedback on performance (Capability), lack of resources (Opportunity), often leading to improvization and poor habit formation. Further barriers included the unit's hierarchy, e.g., low engagement of cleaners and mothers in IPC, and staff witnessing implementation of poor practices by other team members (Opportunity). Potential interventions could include role-modelling, engaging mothers and staff across cadres, audit and feedback and flexible protocols (adaptable to water/handrub availability). CONCLUSIONS: Most barriers to IPC fell within Opportunity, whilst most enablers fell under Capability and Motivation. Theory-based investigation provides the basis for systematically identifying and developing interventions to address barriers and enablers to IPC in low-income settings.

Laccases 2 & 3 as biomarkers of Botrytis cinerea infection in sweet white wines.

Botrytized sweet wines are made with berries infected by the fungus Botrytis cinerea. The aim of this study was to identify biomarkers of B. cinerea infection in sweet wines with a focus on laccases which are exocellular oxidase enzymes produced by this fungus during fruit contamination. Total proteins from six commercial sweet wines, including three naturally botrytized wines and three non-botrytized wines were analysed by LC-QTOF-MS. Five laccases, namely laccase-1-BcLCC1, laccase-2-BcLCC2, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12, were identified in both types of wine. Then, a targeted proteomic approach by LC-MRM was used to semi-quantify laccase-2-BcLCC2 and laccase-3-BcLCC7, in the six samples. LC-MRM targeted analysis of the two enzymes allowed the discrimination of botrytized versus non-botrytized sweet white wines.

Genomic dynamics of species and mobile genetic elements in a prolonged blaIMP-4-associated carbapenemase outbreak in an Australian hospital.

BACKGROUND: Hospital outbreaks of carbapenemase-producing organisms, such as blaIMP-4-containing organisms, are an increasing threat to patient safety. OBJECTIVES: To investigate the genomic dynamics of a 10 year (2006-15) outbreak of blaIMP-4-containing organisms in a burns unit in a hospital in Sydney, Australia. METHODS: All carbapenem-non-susceptible or MDR clinical isolates (2006-15) and a random selection of equivalent or ESBL-producing environmental isolates (2012-15) were sequenced [short-read (Illumina), long-read (Oxford Nanopore Technology)]. Sequence data were used to assess genetic relatedness of isolates (Mash; mapping and recombination-adjusted phylogenies), perform in silico typing (MLST, resistance genes and plasmid replicons) and reconstruct a subset of blaIMP plasmids for comparative plasmid genomics. RESULTS: A total of 46/58 clinical and 67/96 environmental isolates contained blaIMP-4. All blaIMP-4-positive organisms contained five or more other resistance genes. Enterobacter cloacae was the predominant organism, with 12 other species mainly found in either the environment or patients, some persisting despite several cleaning methods. On phylogenetic analysis there were three genetic clusters of E. cloacae containing both clinical and environmental isolates, and an additional four clusters restricted to either reservoir. blaIMP-4 was mostly found as part of a cassette array (blaIMP-4-qacG2-aacA4-catB3) in a class 1 integron within a previously described IncM2 plasmid (pEl1573), with almost complete conservation of this cassette across the species over the 10 years. Several other plasmids were also implicated, including an IncF plasmid backbone not previously widely described in association with blaIMP-4. CONCLUSIONS: Genetic backgrounds disseminating blaIMP-4 can persist, diversify and evolve amongst both human and environmental reservoirs during a prolonged outbreak despite intensive prevention efforts.

Risk factors for Klebsiella pneumoniae carbapenemase (KPC) gene acquisition and clinical outcomes across multiple bacterial species.

INTRODUCTION: Risk factors for carbapenemase-producing Enterobacterales (CPE) acquisition/infection and associated clinical outcomes have been evaluated in the context of clonal, species-specific outbreaks. Equivalent analyses for complex, multi-species outbreaks, which are increasingly common, are lacking. METHODS: Between December 2010 and January 2017, a case-control study of Klebsiella pneumoniae carbapenemase (KPC)-producing organism (KPCO) acquisition was undertaken using electronic health records from inpatients in a US academic medical centre and long-term acute care hospital (LTACH) with ongoing multi-species KPCO transmission despite a robust CPE screening programme. Cases had a first KPCO-positive culture >48 h after admission, and included colonizations and infections (defined by clinical records). Controls had at least two negative perirectal screens and no positive cultures. Risk factors for KPCO acquisition, first infection following acquisition, and 14-day mortality following each episode of infection were identified using multi-variable logistic regression. RESULTS: In 303 cases (89 with at least one infection) and 5929 controls, risk factors for KPCO acquisition included: longer inpatient stay, transfusion, complex thoracic pathology, mechanical ventilation, dialysis, and exposure to carbapenems and ß-lactam/ß-lactamase inhibitors. Exposure to other KPCO-colonized patients was only a risk factor for acquisition in a single unit, suggesting that direct patient-to-patient transmission did not play a major role. There were 15 species of KPCO; 61 (20%) cases were colonized/infected with more than one species. Fourteen-day mortality following non-urinary KPCO infection was 20% (20/97 episodes) and was associated with failure to achieve source control. CONCLUSIONS: Healthcare exposures, antimicrobials and invasive procedures increased the risk of KPCO colonization/infection, suggesting potential targets for infection control interventions in multi-species outbreaks. Evidence for patient-to-patient transmission was limited.

Auditing use of antibiotics in Zimbabwean neonates.

BACKGROUND: Neonatal sepsis is a major cause of morbidity and mortality in low-income settings. As signs of sepsis are non-specific and deterioration precipitous, antibiotics are often used profusely in these settings where diagnostics may not be readily available. Harare Central Hospital, Zimbabwe, delivers 12000 babies per annum admitting ∼4800 to the neonatal unit. Overcrowding, understaffing and rapid staff turnover are consistent problems. Suspected sepsis is highly prevalent, and antibiotics widely used. We audited the impact of training and benchmarking intervention on rationalizing antibiotic prescription using local, World Health Organization-derived, guidelines as the standard. METHODS: An initial audit of admission diagnosis and antibiotic use was performed between 8th May - 6th June 2018 as per the audit cycle. An intern training programme, focusing on antimicrobial stewardship and differentiating between babies 'at risk of' versus 'with' clinically-suspected sepsis was instituted post-primary audit. Re-audit was conducted after 5 months. RESULTS: Sepsis was the most common admitting diagnosis by interns at both time points but reduced at repeat audit (81% versus 59%, P<0.0001). Re-audit after 5 months demonstrated a decrease in antibiotic prescribing at admission and discharge. Babies prescribed antibiotics at admission decreased from 449 (98%) to 96 (51%), P<0.0001. Inpatient days of therapy (DOT) reduced from 1243 to 1110/1000 patient-days. Oral amoxicillin prescription at discharge reduced from 349/354 (99%) to 1% 1/161 (P<0.0001). CONCLUSION: A substantial decrease in antibiotic use was achieved by performance feedback, training and leadership, although ongoing performance review will be key to ensuring safety and sustainability.

Optimizing design of research to evaluate antibiotic stewardship interventions: consensus recommendations of a multinational working group.

BACKGROUND: Antimicrobial stewardship interventions and programmes aim to ensure effective treatment while minimizing antimicrobial-associated harms including resistance. Practice in this vital area is undermined by the poor quality of research addressing both what specific antimicrobial use interventions are effective and how antimicrobial use improvement strategies can be implemented into practice. In 2016 we established a working party to identify the key design features that limit translation of existing research into practice and then to make recommendations for how future studies in this field should be optimally designed. The first part of this work has been published as a systematic review. Here we present the working group's final recommendations. METHODS: An international working group for design of antimicrobial stewardship intervention evaluations was convened in response to the fourth call for leading expert network proposals by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR). The group comprised clinical and academic specialists in antimicrobial stewardship and clinical trial design from six European countries. Group members completed a structured questionnaire to establish the scope of work and key issues to develop ahead of a first face-to-face meeting that (a) identified the need for a comprehensive systematic review of study designs in the literature and (b) prioritized key areas where research design considerations restrict translation of findings into practice. The working group's initial outputs were reviewed by independent advisors and additional expertise was sought in specific clinical areas. At a second face-to-face meeting the working group developed a theoretical framework and specific recommendations to support optimal study design. These were finalized by the working group co-ordinators and agreed by all working group members. RESULTS: We propose a theoretical framework in which consideration of the intervention rationale the intervention setting, intervention features and the intervention aims inform selection and prioritization of outcome measures, whether the research sets out to determine superiority or non-inferiority of the intervention measured by its primary outcome(s), the most appropriate study design (e.g. experimental or quasi- experimental) and the detailed design features. We make 18 specific recommendation in three domains: outcomes, objectives and study design. CONCLUSIONS: Researchers, funders and practitioners will be able to draw on our recommendations to most efficiently evaluate antimicrobial stewardship interventions.

Adaptation and implementation of the ARK (Antibiotic Review Kit) intervention to safely and substantially reduce antibiotic use in hospitals: a feasibility study.

BACKGROUND: Antimicrobial stewardship initiatives in secondary care depend on clinicians undertaking antibiotic prescription reviews but decisions to limit antibiotic treatment at review are complex. AIM: To assess the feasibility and acceptability of implementing ARK (Antibiotic Review Kit), a behaviour change intervention made up of four components (brief online tool, prescribing decision aid, regular data collection and feedback process, and patient leaflet) to support stopping antibiotic treatment when it is safe to do so among hospitalized patients; before definitive evaluation through a stepped-wedge cluster-randomized controlled trial. METHODS: Acceptability of the different intervention elements was assessed for a period of 12 weeks by uptake of the online tool, adoption of the decision aid into prescribing practice, and rates of decisions to stop antibiotics at review (assessed through repeated point-prevalence surveys). Patient perceptions of the information leaflet were assessed through a brief questionnaire. FINDINGS: All elements of the intervention were successfully introduced into practice. A total of 132 staff encompassing a broad range of prescribers and non-prescribers completed the online tool (19.4 per 100 acute beds), including 97% (32/33) of the pre-specified essential clinical staff. Among 588 prescription charts evaluated in seven point-prevalence surveys over the 12-week implementation period, 82% overall (76-90% at each survey) used the decision aid. The median antibiotic stop rate post implementation was 36% (range: 29-40% at each survey) compared with 9% pre implementation (P < 0.001). CONCLUSION: ARK provides a feasible and acceptable mechanism to support stopping antibiotics safely at post-prescription reviews in an acute hospital setting.

Intervention planning for Antibiotic Review Kit (ARK): a digital and behavioural intervention to safely review and reduce antibiotic prescriptions in acute and general medicine.

BACKGROUND: Hospital antimicrobial stewardship strategies, such as 'Start Smart, Then Focus' in the UK, balance the need for prompt, effective antibiotic treatment with the need to limit antibiotic overuse using 'review and revise'. However, only a minority of review decisions are to stop antibiotics. Research suggests that this is due to both behavioural and organizational factors. OBJECTIVES: To develop and optimize the Antibiotic Review Kit (ARK) intervention. ARK is a complex digital, organizational and behavioural intervention that supports implementation of 'review and revise' to help healthcare professionals safely stop unnecessary antibiotics. METHODS: A theory-, evidence- and person-based approach was used to develop and optimize ARK and its implementation. This was done through iterative stakeholder consultation and in-depth qualitative research with doctors, nurses and pharmacists in UK hospitals. Barriers to and facilitators of the intervention and its implementation, and ways to address them, were identified and then used to inform the intervention's development. RESULTS: A key barrier to stopping antibiotics was reportedly a lack of information about the original prescriber's rationale for and their degree of certainty about the need for antibiotics. An integral component of ARK was the development and optimization of a Decision Aid and its implementation to increase transparency around initial prescribing decisions. CONCLUSIONS: The key output of this research is a digital and behavioural intervention targeting important barriers to stopping antibiotics at review (see http://bsac-vle.com/ark-the-antibiotic-review-kit/ and http://antibioticreviewkit.org.uk/). ARK will be evaluated in a feasibility study and, if successful, a stepped-wedge cluster-randomized controlled trial at acute hospitals across the NHS.

Protocol of a two arm randomised, multi-centre, 12-month controlled trial: evaluating the impact of a Cognitive Behavioural Therapy (CBT)-based intervention Supporting UPtake and Adherence to antiretrovirals (SUPA) in adults with HIV.

BACKGROUND: Delay to start antiretroviral therapy (ART) and nonadherence compromise the health and wellbeing of people living with HIV (PLWH), raise the cost of care and increase risk of transmission to sexual partners. To date, interventions to improve adherence to ART have had limited success, perhaps because they have failed to systematically elicit and address both perceptual and practical barriers to adherence. The primary aim of this study is to determine the efficacy of the Supporting UPtake and Adherence (SUPA) intervention. METHODS: This study comprises 2 phases. Phase 1 is an observational cohort study, in which PLWH who are ART naïve and recommended to take ART by their clinician complete a questionnaire assessing their beliefs about ART over 12 months. Phase 2 is a randomised controlled trial (RCT) nested within the observational cohort study to investigate the effectiveness of the SUPA intervention on adherence to ART. PLWH at risk of nonadherence (based on their beliefs about ART) will be recruited and randomised 1:1 to the intervention (SUPA intervention + usual care) and control (usual care) arms. The SUPA intervention involves 4 tailored treatment support sessions delivered by a Research Nurse utilising a collaborative Cognitive Behavioural Therapy (CBT) and Motivational Interviewing (MI) approach. Sessions are tailored to individual needs and preferences based on the individual patient's perceptions and practical barriers to ART. An animation series and intervention manual have been developed to communicate a rationale for the personal necessity for ART and illustrate concerns and potential solutions. The primary outcome is adherence to ART measured using Medication Event Monitoring System (MEMS). Three hundred seventy-two patients will be sufficient to detect a 15% difference in adherence with 80% power and an alpha of 0.05. Costs will be compared between intervention and control groups. Costs will be combined with the primary outcome in cost-effectiveness analyses. Quality adjusted life-years (QALYs) will also be estimated over the follow-up period and used in the analyses. DISCUSSION: The findings will enable patients, healthcare providers and policy makers to make informed decisions about the value of the SUPA intervention. TRIAL REGISTRATION: The trial was retrospectively registered 21/02/2014, ISRCTN35514212 .

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. OBJECTIVES: To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. METHODS: Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. RESULTS: CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CONCLUSIONS: CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.

Epidemiology of paediatric gastrointestinal colonisation by extended spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates in north-west Cambodia.

BACKGROUND: Extended-spectrum cephalosporin resistance (ESC-R) in Escherichia coli and Klebsiella pneumoniae is a healthcare threat; high gastrointestinal carriage rates are reported from South-east Asia. Colonisation prevalence data in Cambodia are lacking. The aim of this study was to determine gastrointestinal colonisation prevalence of ESC-resistant E. coli (ESC-R-EC) and K. pneumoniae (ESC-R-KP) in Cambodian children/adolescents and associated socio-demographic risk factors; and to characterise relevant resistance genes, their genetic contexts, and the genetic relatedness of ESC-R strains using whole genome sequencing (WGS). RESULTS: Faeces and questionnaire data were obtained from individuals < 16 years in north-western Cambodia, 2012. WGS of cultured ESC-R-EC/KP was performed (Illumina). Maximum likelihood phylogenies were used to characterise relatedness of isolates; ESC-R-associated resistance genes and their genetic contexts were identified from de novo assemblies using BLASTn and automated/manual annotation. 82/148 (55%) of children/adolescents were ESC-R-EC/KP colonised; 12/148 (8%) were co-colonised with both species. Independent risk factors for colonisation were hospitalisation (OR: 3.12, 95% CI [1.52-6.38]) and intestinal parasites (OR: 3.11 [1.29-7.51]); school attendance conferred decreased risk (OR: 0.44 [0.21-0.92]. ESC-R strains were diverse; the commonest ESC-R mechanisms were blaCTX-M 1 and 9 sub-family variants. Structures flanking these genes were highly variable, and for blaCTX-M-15, - 55 and - 27 frequently involved IS26. Chromosomal blaCTX-M integration was common in E. coli. CONCLUSIONS: Gastrointestinal ESC-R-EC/KP colonisation is widespread in Cambodian children/adolescents; hospital admission and intestinal parasites are independent risk factors. The genetic contexts of blaCTX-M are highly mosaic, consistent with rapid horizontal exchange. Chromosomal integration of blaCTX-M may result in stable propagation in these community-associated pathogens.

The quality of studies evaluating antimicrobial stewardship interventions: a systematic review.

BACKGROUND: Antimicrobial stewardship aims to optimize antibiotic use and minimize selection of antimicrobial resistance. The methodological quality of published studies in this field is unknown. AIMS: Our objective was to perform a comprehensive systematic review of antimicrobial stewardship research design and identify features which limit validity and translation of research findings into clinical practice. SOURCES: The following online database was searched: PubMed. STUDY ELIGIBILITY CRITERIA: Studies published between January 1950 and January 2017, evaluating any antimicrobial stewardship intervention in the community or hospital setting, without restriction on study design or outcome. CONTENT: We extracted data on pre-specified design quality features and factors that may influence design choices including (1) clinical setting, (2) age group studied, (3) when the study was conducted, (4) geographical region, and (5) financial support received. The initial search yielded 17 382 articles; 1008 were selected for full-text screening, of which 825 were included. Most studies (675/825, 82%) were non-experimental; 104 (15%) used interrupted time series analysis, 41 (6%) used external controls, and 19 (3%) used both. Studies in the community setting fulfilled a median of five out of 10 quality features (IQR 3-7) and 3 (IQR 2-4) in the hospital setting. Community setting studies (25%, 205/825) were significantly more likely to use randomization (OR 5.9; 95% CI 3.8-9.2), external controls (OR 5.6; 95% CI 3.6-8.5), and multiple centres (OR 10.5; 95% CI 7.1-15.7). From all studies, only 48% (398/825) reported clinical and 23% (190/825) reported microbiological outcomes. Quality did not improve over time. IMPLICATIONS: Overall quality of antimicrobial stewardship studies is low and has not improved over time. Most studies do not report clinical and microbiological outcome data. Studies conducted in the community setting were associated with better quality. These limitations should inform the design of future stewardship evaluations so that a robust evidence base can be built to guide clinical practice.

A Large, Refractory Nosocomial Outbreak of Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli Demonstrates Carbapenemase Gene Outbreaks Involving Sink Sites Require Novel Approaches to Infection Control.

Carbapenem-resistant Enterobacteriaceae (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing E. coli isolates (n = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n = 268 isolates) identified the spread of a KPC-producing E. coli outbreak clone (strain A, sequence type 216 [ST216]; n = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing E. coli outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of blaKPC in E. coli, including pathogenic lineages, are of concern.

A national quality incentive scheme to reduce antibiotic overuse in hospitals: evaluation of perceptions and impact.

Background: In 2016/2017, a financially linked antibiotic prescribing quality improvement initiative Commissioning for Quality and Innovation (AMR-CQUIN) was introduced across acute hospitals in England. This aimed for >1% reductions in DDDs/1000 admissions of total antibiotics, piperacillin/tazobactam and carbapenems compared with 2013/2014 and improved review of empirical antibiotic prescriptions. Objectives: To assess perceptions of staff leading antimicrobial stewardship activity regarding the AMR-CQUIN, the investments made by hospitals to achieve it and how these related to achieving reductions in antibiotic use. Methods: We invited antimicrobial stewardship leads at acute hospitals across England to complete a web-based survey. Antibiotic prescribing data were downloaded from the PHE Antimicrobial Resistance Local Indicators resource. Results: Responses were received from 116/155 (75%) acute hospitals. Owing to yearly increases in antibiotic use, most trusts needed to make >5% reductions in antibiotic consumption to achieve the AMR-CQUIN goal of 1% reduction. Additional funding was made available at 23/113 (20%) trusts and, in 18 (78%), this was <10% of the AMR-CQUIN value. Nationally, the annual trend for increased antibiotic use reversed in 2016/2017. In 2014/2015, year-on-year changes were +3.7% (IQR -0.8%, +8.4%), +9.4% (+0.2%, +19.5%) and +5.8% (-6.2%, +18.2%) for total antibiotics, piperacillin/tazobactam and carbapenems, respectively, and +0.1% (-5.4%, +4.0%), -4.8% (-16.9%, +3.2%) and -8.0% (-20.2%, +4.0%) in 2016/2017. Hospitals where staff believed they could reduce antibiotic use were more likely to do so (P < 0.001). Conclusions: Introducing the AMR-CQUIN was associated with a reduction in antibiotic use. For individual hospitals, achieving the AMR-CQUIN was associated with favourable perceptions of staff and not availability of funding.

Illumina short-read and MinION long-read WGS to characterize the molecular epidemiology of an NDM-1 Serratia marcescens outbreak in Romania.

Background and Objectives: Serratia marcescens is an emerging nosocomial pathogen, and the carbapenemase blaNDM has been reported in several surveys in Romania. We aimed to investigate the molecular epidemiology of S. marcescens in two Romanian hospitals over 2010-15, including a neonatal NDM-1 S. marcescens outbreak. Methods: Isolates were sequenced using Illumina technology together with carbapenem-non-susceptible NDM-1-positive and NDM-1-negative Klebsiella pneumoniae and Enterobacter cloacae to provide genomic context. A subset was sequenced with MinION to fully resolve NDM-1 plasmid structures. Resistance genes, plasmid replicons and ISs were identified in silico for all isolates; an annotated phylogeny was reconstructed for S. marcescens. Fully resolved study NDM-1 plasmid sequences were compared with the most closely related publicly available NDM-1 plasmid reference. Results: 44/45 isolates were successfully sequenced (S. marcescens, n = 33; K. pneumoniae, n = 7; E. cloacae, n = 4); 10 with MinION. The S. marcescens phylogeny demonstrated several discrete clusters of NDM-1-positive and -negative isolates. All NDM-1-positive isolates across species harboured a pKOX_NDM1-like plasmid; more detailed comparisons of the plasmid structures demonstrated a number of differences, but highlighted the largely conserved plasmid backbones across species and hospital sites. Conclusions: The molecular epidemiology is most consistent with the importation of a pKOX_NDM1-like plasmid into Romania and its dissemination amongst K. pneumoniae/E. cloacae and subsequently S. marcescens across hospitals. The data suggested multiple acquisitions of this plasmid by S. marcescens in the two hospitals studied; transmission events within centres, including a large outbreak on the Targu Mures neonatal unit; and sharing of the pKOX_NDM1-like plasmid between species within outbreaks.

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10 kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n = 21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (≥9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

Whole-Genome Sequencing Reveals the Contribution of Long-Term Carriers in Staphylococcus aureus Outbreak Investigation.

Whole-genome sequencing (WGS) makes it possible to determine the relatedness of bacterial isolates at a high resolution, thereby helping to characterize outbreaks. However, for Staphylococcus aureus, the accumulation of within-host diversity during carriage might limit the interpretation of sequencing data. In this study, we hypothesized the converse, namely, that within-host diversity can in fact be exploited to reveal the involvement of long-term carriers (LTCs) in outbreaks. We analyzed WGS data from 20 historical outbreaks and applied phylogenetic methods to assess genetic relatedness and to estimate the time to most recent common ancestor (TMRCA). The findings were compared with the routine investigation results and epidemiological evidence. Outbreaks with epidemiological evidence for an LTC source had a mean estimated TMRCA (adjusted for outbreak duration) of 243 days (95% highest posterior density interval [HPD], 143 to 343 days) compared with 55 days (95% HPD, 28 to 81 days) for outbreaks lacking epidemiological evidence for an LTC (P = 0.004). A threshold of 156 days predicted LTC involvement with a sensitivity of 0.875 and a specificity of 1. We also found 6/20 outbreaks included isolates with differing antimicrobial susceptibility profiles; however, these had only modestly increased pairwise diversity (mean 17.5 single nucleotide variants [SNVs] [95% confidence interval {CI}, 17.3 to 17.8]) compared with isolates with identical antibiograms (12.7 SNVs [95% CI, 12.5 to 12.8]) (P < 0.0001). Additionally, for 2 outbreaks, WGS identified 1 or more isolates that were genetically distinct despite having the outbreak pulsed-field gel electrophoresis (PFGE) pulsotype. The duration-adjusted TMRCA allowed the involvement of LTCs in outbreaks to be identified and could be used to decide whether screening for long-term carriage (e.g., in health care workers) is warranted. Requiring identical antibiograms to trigger investigation could miss important contributors to outbreaks.