Clonal expansion and rapid characterization of Klebsiella pneumoniae ST1788, an otherwise uncommon strain spreading in Wales, UK.

A multidrug-resistant strain of Klebsiella pneumoniae (Kp) sequence type (ST) 1788, an otherwise uncommon ST worldwide, was isolated from 65 patients at 11 hospitals and 11 general practices across South and West Wales, UK, between February 2019 and November 2021. A collection of 97 Kp ST1788 isolates (including 94 from Wales) was analysed to investigate the diversity and spread across Wales and to identify molecular marker(s) to aid development of a strain-specific real-time PCR. Whole genome sequencing (WGS) was performed with Illumina technology and the data were used to perform phylogenetic analyses. Pan-genome analysis of further Kp genome collections was used to identify an ST1788-specific gene target; a real-time PCR was then validated against a panel of 314 strains and 218 broth-enriched screening samples. Low genomic diversity was demonstrated amongst the 94 isolates from Wales. Evidence of spread within and across healthcare facilities was found. A yersiniabactin locus and the KL2 capsular locus were identified in 85/94 (90.4 %) and 94/94 (100 %) genomes respectively; bla SHV-232, bla TEM-1, bla CTX-M-15 and bla OXA-1 were simultaneously carried by 86/94 (91.5 %) isolates; 4/94 (4.3 %) isolates also carried bla OXA-48 carbapenemase. Aminoglycoside and fluoroquinolone resistance markers were found in 94/94 (100 %) and 86/94 (91.5 %) isolates respectively. The ST1788-specific real-time PCR was 100 % sensitive and specific. Our analyses demonstrated recent clonal expansion and spread of Kp ST1788 in the community and across healthcare facilities in South and West Wales with isolates carrying well-defined antimicrobial resistance and virulence markers. An ST1788-specific marker was also identified, enabling rapid and reliable preliminary characterization of isolates by real-time PCR. This study confirms the utility of WGS in investigating novel strains and in aiding proactive implementation of molecular tools to assist infection control specialists.

First large-scale study of antimicrobial susceptibility data, and genetic resistance determinants, in Fusobacterium necrophorum highlighting the importance of continuing focused susceptibility trend surveillance.

OBJECTIVES: The objective of the study was to explore antimicrobial resistance gene determinant, and phenotypic antibiotic susceptibility, data for Fusobacterium necrophorum from a collection of UK strains. Antimicrobial resistance genes detected in publicly available assembled whole genome sequences were investigated for comparison. METHODS: Three hundred and eighty five F. necrophorum strains (1982-2019) were revived from cryovials (Prolab). Subsequent to sequencing (Illumina) and quality checking, 374 whole genomes were available for analysis. Genomes were interrogated, using BioNumerics (bioMérieux; v 8.1), for the presence of known antimicrobial resistance genes (ARGs). Agar dilution susceptibility results for 313 F. necrophorum isolates (2016-2021) were also examined. RESULTS: The phenotypic data for the 313 contemporary strains demonstrated potential resistance to penicillin in three isolates, using EUCAST v 11.0 breakpoints, and 73 (23%) strains using v 13.0 analysis. All strains were susceptible to multiple agents using v 11.0 guidance other than clindamycin (n = 2). Employing v 13.0 breakpoints, metronidazole (n = 3) and meropenem (n = 13) resistance were also detected. The tet(O), tet(M), tet(40), aph(3')-III, ant(6)-la and blaOXA-85 ARGs were present in publicly available genomes. tet(M), tet(32), erm(A) and erm(B) were found within the UK strains, with correspondingly raised clindamycin and tetracycline minimum inhibitory concentrations. CONCLUSIONS: Susceptibility to antibiotics recommended for the treatment of F. necrophorum infections should not be assumed. With evidence of potential ARG transmission from oral bacteria, and the detection of a transposon-mediated beta-lactamase resistance determinant in F. necrophorum, surveillance of both phenotypic and genotypic antimicrobial susceptibility trends must continue, and increase.

Influenza classification from short reads with VAPOR facilitates robust mapping pipelines and zoonotic strain detection for routine surveillance applications.

MOTIVATION: Influenza viruses represent a global public health burden due to annual epidemics and pandemic potential. Due to a rapidly evolving RNA genome, inter-species transmission, intra-host variation, and noise in short-read data, reads can be lost during mapping, and de novo assembly can be time consuming and result in misassembly. We assessed read loss during mapping and designed a graph-based classifier, VAPOR, for selecting mapping references, assembly validation and detection of strains of non-human origin. RESULTS: Standard human reference viruses were insufficient for mapping diverse influenza samples in simulation. VAPOR retrieved references for 257 real whole-genome sequencing samples with a mean of >99.8% identity to assemblies, and increased the proportion of mapped reads by up to 13.3% compared to standard references. VAPOR has the potential to improve the robustness of bioinformatics pipelines for surveillance and could be adapted to other RNA viruses. AVAILABILITY AND IMPLEMENTATION: VAPOR is available at https://github.com/connor-lab/vapor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Collect, boil and amplify--a simple approach for the detection of three common viruses associated with epidemic keratoconjunctivitis, conjunctivitis and dendritic ulcers.

During 2011' an outbreak of epidemic keratoconjunctivitis led to increased clinical requests for molecular screening of viruses from conjunctival swabs. To maximise throughput with minimal cost, a simple boil extraction on dry swabs followed by amplification and real-time detection using 'in-house' assays for herpes simplex viruses (HSV) and adenoviruses with RNaseP as an internal control was validated and introduced. Data from 541 patients who were tested for one or more viral targets was analysed. Adenovirus was most frequently detected accounting for 30% of all cases including the community outbreak. Genotyping of the hexon gene identified the cause as an adenovirus type 8. HSV was detected in 7% of the samples tested, predominantly HSV-1 with a single case of HSV-2. Invalid results due to poor RNaseP signals were reported in 10.5% of samples but for the HSV-1 assay 23% of the samples were invalid due to interference of the fluorescein dye used by ophthalmologists resulting in repeat sampling to obtain a valid result. Despite this, when compared to conventional techniques such as direct immunofluorescence, collect, boil and amplify increased significantly the detection of DNA viruses in conjunctival samples ensuring improved diagnosis, patient management and infection control measures at a modest cost.

Comparison of galactomannan enzyme immunoassay performance levels when testing serum and plasma samples.

Diagnostic galactomannan (GM) enzyme immunoassay (EIA) testing is formally validated only for serum, though in practice, plasma is occasionally tested. It is assumed, but not confirmed, that results will be comparable to those for serum. GM EIA when testing plasma was evaluated, providing sensitivity (85.7%) and specificity (85.4%) comparable to those for serum. Plasma index values were higher than those for serum; if plasma GM EIA were used to define probable cases, four additional cases would have been diagnosed.