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.

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.

Caprine arthritis-encephalitis virus: an efficient method for the large scale production of serological antigens.

Caprine arthritis-encephalitis virus (CAEV) was grown on a large scale in lamb corneal cell cultures. Antigens for use in two different CAEV agar gel immuno-diffusion tests were prepared from the same culture fluid. These antigens were separated on the basis of size by membrane filtration.

Caprine retrovirus infection in New South Wales: virus isolations, clinical and histopathological findings and prevalence of antibody.

Caprine arthritis-encephalitis virus (CAEV) was isolated by explant cultures of carpal synovial membranes and lung from 7 goats in New South Wales. These goats were clinically affected with the arthritic, neurologic, and pneumonic forms of CAEV infection either singly or in combination. CAEV antibody was detected by the gel immunodiffusion precipitin (GDP) test in 5 of the 7 goats. Serum samples from 2,708 goats, from 115 herds, were examined for CAEV antibody using the GDP test. Approximately one-third of the animals and 82% of the herds tested had CAEV antibody. The infection was common in all breeds of dairy goats with an indication of a significantly lower prevalence in the Saanen breed (24.4%) compared to Nubians, British Alpines and Toggenbergs (43.8%, 38.7% and 39.1% respectively). CAEV antibody was also demonstrated in 11 of 230 Angora goats. The infection was equally common in all age groups, with slightly higher prevalence in males (83 of 230, 36%) compared to females (648 of 2,232, 29%). Among seropositive animals 85% were clinically normal. Of 280 clinically affected goats tested only 42% had detectable antibody. One of 5 sheep that had been in contact with infected goats in one herd had CAEV serum antibody.

Healthcare-associated outbreak of meticillin-resistant Staphylococcus aureus bacteraemia: role of a cryptic variant of an epidemic clone.

BACKGROUND: New strains of meticillin-resistant Staphylococcus aureus (MRSA) may be associated with changes in rates of disease or clinical presentation. Conventional typing techniques may not detect new clonal variants that underlie changes in epidemiology or clinical phenotype. AIM: To investigate the role of clonal variants of MRSA in an outbreak of MRSA bacteraemia at a hospital in England. METHODS: Bacteraemia isolates of the major UK lineages (EMRSA-15 and -16) from before and after the outbreak were analysed by whole-genome sequencing in the context of epidemiological and clinical data. For comparison, EMRSA-15 and -16 isolates from another hospital in England were sequenced. A clonal variant of EMRSA-16 was identified at the outbreak hospital and a molecular signature test designed to distinguish variant isolates among further EMRSA-16 strains. FINDINGS: By whole-genome sequencing, EMRSA-16 isolates during the outbreak showed strikingly low genetic diversity (P < 1 × 10(-6), Monte Carlo test), compared with EMRSA-15 and EMRSA-16 isolates from before the outbreak or the comparator hospital, demonstrating the emergence of a clonal variant. The variant was indistinguishable from the ancestral strain by conventional typing. This clonal variant accounted for 64/72 (89%) of EMRSA-16 bacteraemia isolates at the outbreak hospital from 2006. CONCLUSIONS: Evolutionary changes in epidemic MRSA strains not detected by conventional typing may be associated with changes in disease epidemiology. Rapid and affordable technologies for whole-genome sequencing are becoming available with the potential to identify and track the emergence of variants of highly clonal organisms.

Microtiter hemagglutination-inhibition test for the detection of encephalomyocarditis virus antibodies.

Several variables were found to affect the agglutination of sheep erythrocytes by encephalomyocarditis virus. A satisfactory and reliable microtiter hemagglutination-inhibition test is described.