WITHDRAWN: Evaluation of Fourier Transform Infrared spectroscopy (IR Biotyper) as a complement to Whole genome sequencing (WGS) to characterise Enterobacter cloacae , Citrobacter freundii and Klebsiella pneumoniae isolates recovered from hospital sinks.

The authors have withdrawn their manuscript due to becoming aware of methodology issues related to the curation of the training set used to determine cut-off values for Biotyper cluster assignation and lack of replicate measurements on different days for the isolates analysed. It is therefore unclear whether the conclusions of the manuscript are founded and no further work is possible to correct these issues as the instrument is no longer available to the authors. If you have any questions, please contact the corresponding author.

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.

Tocilizumab for treatment of SARS-CoV-2 infection at home: A case report.

SARS-CoV-2 virtual wards have successfully developed to monitor and escalate patients to hospital throughout the pandemic. Here we describe the case of an 84 year old man who received his complete care for severe SARS-CoV-2 pneumonitis at home, including the administration of oxygen, dexamethasone and tocilizumab.

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.

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.

Occurrence and characterization of Escherichia coli ST410 co-harbouring blaNDM-5, blaCMY-42 and blaTEM-190 in a dog from the UK.

BACKGROUND/OBJECTIVES: Carbapenemase-producing Enterobacteriaceae (CPE) are a public health threat, and have been found in humans, animals and the environment. Carbapenems are not authorized for use in EU or UK companion animals, and the prevalence of carbapenem-resistant Gram-negative bacilli (CRGNB) in this population is unknown. METHODS: We investigated CRGNB isolated from animal specimens received by one diagnostic laboratory from 34 UK veterinary practices (September 2015-December 2016). Any Gram-negative isolates from clinical specimens showing reduced susceptibility to fluoroquinolones and/or aminoglycosides and/or cephalosporins were investigated phenotypically and genotypically for carbapenemases. A complete genome assembly (Illumina/Nanopore) was generated for the single isolate identified to investigate the genetic context for carbapenem resistance. RESULTS: One ST410 Escherichia coli isolate [(CARB35); 1/191, 0.5%], cultured from a wound in a springer spaniel, harboured a known carbapenem resistance gene (blaNDM-5). The gene was located in the chromosome on an integrated 100 kb IncF plasmid, also harbouring other drug resistance genes (mrx, sul1, ant1 and dfrA). The isolate also contained blaCMY-42 and blaTEM-190 on two separate plasmids (IncI1 and IncFII, respectively) that showed homology with other publicly available plasmid sequences from Italy and Myanmar. CONCLUSIONS: Even though the use of carbapenems in companion animals is restricted, the concurrent presence of blaCMY-42 and other antimicrobial resistance genes could lead to co-selection of carbapenemase genes in this population. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst humans and companion animals are needed.

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.

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.

Extensive Within-Host Diversity in Fecally Carried Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolates: Implications for Transmission Analyses.

Studies of the transmission epidemiology of antimicrobial-resistant Escherichia coli, such as strains harboring extended-spectrum beta-lactamase (ESBL) genes, frequently use selective culture of rectal surveillance swabs to identify isolates for molecular epidemiological investigation. Typically, only single colonies are evaluated, which risks underestimating species diversity and transmission events. We sequenced the genomes of 16 E. coli colonies from each of eight fecal samples (n = 127 genomes; one failure), taken from different individuals in Cambodia, a region of high ESBL-producing E. coli prevalence. Sequence data were used to characterize both the core chromosomal diversity of E. coli isolates and their resistance/virulence gene content as a proxy measure of accessory genome diversity. The 127 E. coli genomes represented 31 distinct sequence types (STs). Seven (88%) of eight subjects carried ESBL-positive isolates, all containing blaCTX-M variants. Diversity was substantial, with a median of four STs/individual (range, 1 to 10) and wide genetic divergence at the nucleotide level within some STs. In 2/8 (25%) individuals, the same blaCTX-M variant occurred in different clones, and/or different blaCTX-M variants occurred in the same clone. Patterns of other resistance genes and common virulence factors, representing differences in the accessory genome, were also diverse within and between clones. The substantial diversity among intestinally carried ESBL-positive E. coli bacteria suggests that fecal surveillance, particularly if based on single-colony subcultures, will likely underestimate transmission events, especially in high-prevalence settings.

Dynamics of MDR Enterobacter cloacae outbreaks in a neonatal unit in Nepal: insights using wider sampling frames and next-generation sequencing.

OBJECTIVES: There are limited data on Enterobacter cloacae outbreaks and fewer describing these in association with NDM-1. With whole-genome sequencing, we tested the hypothesis that a cluster of 16 E. cloacae bacteraemia cases in a Nepali neonatal unit represented a single clonal outbreak, using a wider set of epidemiologically unrelated clinical E. cloacae isolates for comparison. METHODS: Forty-three isolates were analysed, including 23 E. cloacae and 3 Citrobacter sp. isolates obtained from blood cultures from 16 neonates over a 3 month period. These were compared with two contemporaneous community-associated drug-resistant isolates from adults, a unit soap dispenser isolate and a set of historical invasive isolates (n=14) from the same geographical locality. RESULTS: There were two clear neonatal outbreaks and one isolated case in the unit. One outbreak was associated with an NDM-1 plasmid also identified in a historical community-associated strain. The smaller, second outbreak was likely associated with a contaminated soap dispenser. The two community-acquired adult cases and three sets of historical hospital-associated neonatal isolates represented four additional genetic clusters. CONCLUSIONS: E. cloacae infections in this context represent several different transmission networks, operating at the community/hospital and host strain/plasmid levels. Wide sampling frames and high-resolution typing methods are needed to describe the complex molecular epidemiology of E. cloacae outbreaks, which is not appropriately reflected by routine susceptibility phenotypes. Soap dispensers may represent a reservoir for E. cloacae and bacterial strains and plasmids may persist in hospitals and in the community for long periods, sporadically being involved in outbreaks of disease.

Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting.

NDM-producing Klebsiella pneumoniae strains represent major clinical and infection control challenges, particularly in resource-limited settings with high rates of antimicrobial resistance. Determining whether transmission occurs at a gene, plasmid, or bacterial strain level and within hospital and/or the community has implications for monitoring and controlling spread. Whole-genome sequencing (WGS) is the highest-resolution typing method available for transmission epidemiology. We sequenced carbapenem-resistant K. pneumoniae isolates from 26 individuals involved in several infection case clusters in a Nepali neonatal unit and 68 other clinical Gram-negative isolates from a similar time frame, using Illumina and PacBio technologies. Within-outbreak chromosomal and closed-plasmid structures were generated and used as data set-specific references. Three temporally separated case clusters were caused by a single NDM K. pneumoniae strain with a conserved set of four plasmids, one being a 304,526-bp plasmid carrying bla(NDM-1). The plasmids contained a large number of antimicrobial/heavy metal resistance and plasmid maintenance genes, which may have explained their persistence. No obvious environmental/human reservoir was found. There was no evidence of transmission of outbreak plasmids to other Gram-negative clinical isolates, although bla(NDM) variants were present in other isolates in different genetic contexts. WGS can effectively define complex antimicrobial resistance epidemiology. Wider sampling frames are required to contextualize outbreaks. Infection control may be effective in terminating outbreaks caused by particular strains, even in areas with widespread resistance, although this study could not demonstrate evidence supporting specific interventions. Larger, detailed studies are needed to characterize resistance genes, vectors, and host strains involved in disease, to enable effective intervention.

Septic arthritis of the hip in a Cambodian child caused by multidrug-resistant Salmonella enterica serovar Typhi with intermediate susceptibility to ciprofloxacin treated with ceftriaxone and azithromycin.

Septic arthritis is a rare complication of typhoid fever. A 12-year-old boy without pre-existing disease attended a paediatric hospital in Cambodia with fever and left hip pain. A hip synovial fluid aspirate grew multidrug-resistant Salmonella enterica ser. Typhi with intermediate susceptibility to ciprofloxacin. Arthrotomy, 2 weeks of intravenous ceftriaxone and 4 weeks of oral azithromycin led to resolution of symptoms. The optimum management of septic arthritis in drug-resistant typhoid is undefined.

Comparison of multilocus variable-number tandem-repeat analysis and whole-genome sequencing for investigation of Clostridium difficile transmission.

No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ≥2 SNVs but ≤10 STRDs, and 69 (5%) had ≤2 SNVs but ≥10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data.

OBJECTIVES: Whole-genome sequencing potentially represents a single, rapid and cost-effective approach to defining resistance mechanisms and predicting phenotype, and strain type, for both clinical and epidemiological purposes. This retrospective study aimed to determine the efficacy of whole genome-based antimicrobial resistance prediction in clinical isolates of Escherichia coli and Klebsiella pneumoniae. METHODS: Seventy-four E. coli and 69 K. pneumoniae bacteraemia isolates from Oxfordshire, UK, were sequenced (Illumina HiSeq 2000). Resistance phenotypes were predicted from genomic sequences using BLASTn-based comparisons of de novo-assembled contigs with a study database of >100 known resistance-associated loci, including plasmid-associated and chromosomal genes. Predictions were made for seven commonly used antimicrobials: amoxicillin, co-amoxiclav, ceftriaxone, ceftazidime, ciprofloxacin, gentamicin and meropenem. Comparisons were made with phenotypic results obtained in duplicate by broth dilution (BD Phoenix). Discrepancies, either between duplicate BD Phoenix results or between genotype and phenotype, were resolved with gradient diffusion analyses. RESULTS: A wide variety of antimicrobial resistance genes were identified, including blaCTX-M, blaLEN, blaOKP, blaOXA, blaSHV, blaTEM, aac(3')-Ia, aac-(3')-IId, aac-(3')-IIe, aac(6')-Ib-cr, aadA1a, aadA4, aadA5, aadA16, aph(6')-Id, aph(3')-Ia, qnrB and qnrS, as well as resistance-associated mutations in chromosomal gyrA and parC genes. The sensitivity of genome-based resistance prediction across all antibiotics for both species was 0.96 (95% CI: 0.94-0.98) and the specificity was 0.97 (95% CI: 0.95-0.98). Very major and major error rates were 1.2% and 2.1%, respectively. CONCLUSIONS: Our method was as sensitive and specific as routinely deployed phenotypic methods. Validation against larger datasets and formal assessments of cost and turnaround time in a routine laboratory setting are warranted.

The value of intermittent point-prevalence surveys of healthcare-associated infections for evaluating infection control interventions at Angkor Hospital for Children, Siem Reap, Cambodia.

BACKGROUND: There are limited data on the epidemiology of paediatric healthcare-associated infection (HCAI) and infection control in low-income countries. We describe the value of intermittent point-prevalence surveys for monitoring HCAI and evaluating infection control interventions in a Cambodian paediatric hospital. METHODS: Hospital-wide, point-prevalence surveys were performed monthly in 2011. Infection control interventions introduced during this period included a hand hygiene programme and a ventilator-associated pneumonia (VAP) care bundle. RESULTS: Overall HCAI prevalence was 13.8/100 patients at-risk, with a significant decline over time. The highest HCAI rates (50%) were observed in critical care; the majority of HCAIs were respiratory (61%). Klebsiella pneumoniae was most commonly isolated and antimicrobial resistance was widespread. Hand hygiene compliance doubled to 51.6%, and total VAP cases/1000 patient-ventilator days fell from 30 to 10. CONCLUSION: Rates of HCAI were substantial in our institution, and antimicrobial resistance a major concern. Point-prevalence surveys are effective for HCAI surveillance, and in monitoring trends in response to infection control interventions.