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.

UK Bacteroides species surveillance survey: Change in antimicrobial resistance over 16 years (2000-2016).

OBJECTIVES: To assess the differences in antimicrobial susceptibility of UK Bacteroides species across two distinct cohorts from 2000 to 2016. METHODS: Strain identification was performed using matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) or by partial 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) were determined using agar dilution, following CLSI guidelines (CLSI, 2012; 2017). RESULTS: 224 isolates were included from 2000 to 168 from 2016. Bacteroides fragilis was the most common species, comprising 68% of the 2000 cohort, and 77% in 2016. For all antimicrobials tested, there was an overall increase in the rates of non-susceptible isolates between the cohorts. CONCLUSIONS: The antibiogram of Bacteroides species in the UK is no longer predictable. Multi-drug resistant isolates although rare, are on the rise, and require testing to guide therapy. The monitoring and surveillance of resistance trends is imperative, as is the development of standardised, robust and accessible antimicrobial susceptibility testing methodology for clinical laboratories.

Development of a EUCAST disk diffusion method for the susceptibility testing of rapidly growing anaerobic bacteria using Fastidious Anaerobe Agar (FAA): a development study using Bacteroides species.

OBJECTIVES: Antimicrobial resistance among anaerobic bacteria is increasing, leading to a growing demand for inexpensive and reliable susceptibility testing methods. The aim of this study was to determine the suitability of Fastidious Anaerobe Agar (FAA) as a medium for disk diffusion for rapidly growing anaerobic bacteria. METHODS: Reproducibility of zone diameters and quality of growth were tested using six quality control (QC) strains. We compared four anaerobic incubation systems, two incubation temperatures (35°C and 37°C), and FAA from four manufacturers. The effect of incubation for 16-20 hours instead of 24 hours was tested on ten randomly selected isolates of the Bacteroides fragilis group. The final method was tested on 170 clinical B. fragilis-group isolates and compared to agar dilution MICs. RESULTS: After 24 hours' incubation, all QC strains demonstrated confluent growth. The different anaerobic incubation systems were equal regarding quality of growth and zone diameters. Incubation at 35°C resulted in slightly larger zones (1-2 mm) than at 37°C. Except for Acumedia FAA, the different manufacturers showed good agreement in zone diameters. All B. fragilis-group isolates displayed confluent growth after 16-20 hours. Metronidazole inhibition zones correlated well with the reference MICs. There was an area of poorer separation for meropenem and piperacillin-tazobactam between 19-27 and 14-23 mm respectively. Prolonged incubation (40-44 h) of clindamycin resulted in better separation and the area of overlap was reduced from 13 to 8 mm compared with 16-20 hours' incubation. CONCLUSION: FAA is a suitable medium for disk diffusion of these rapidly growing anaerobic bacteria.

Impact of the introduction of nucleic acid amplification testing on Clostridioides difficile detection and ribotype distribution in Wales.

OBJECTIVES: To determine the impact of the 2018 introduction of nucleic acid amplification tests (NAATs) for C. difficile detection on the laboratory diagnosis of C. difficile infection (CDI), and the distribution of C. difficile ribotypes. METHODS: A retrospective analysis of five years (2015-2019) of C. difficile diagnostic laboratory and PCR ribotyping test results. RESULTS: A total of 255,104 diagnostic results, from 136,353 patients were analysed: 199,794 samples where glutamate dehydrogenase (GDH) was used as the primary screen; and 55,310 where NAATs were employed. An overall decrease in frontline positivity from 2015 to 2019 (10.3% [n = 5017] to 6% [n = 3190] - p < 0.0001) was observed, despite an increase in the number of samples tested (48,778 to 52,839). NAAT positivity was lower than GDH (p < 0.0001) for the two years where it was implemented. The variance was accounted for by increased overall C. difficile isolation and reduced toxin negative strain culture from NAAT positive samples (p < 0.0001). Ribotype distribution (6546 samples) remained stable with decreasing RT27 isolation in each year except 2017 (p < 0.0001). RT78 was associated with toxin A/B EIA positivity (p < 0.0001). CONCLUSIONS: Use of NAAT for the detection of C. difficile, as part of a 2-step algorithm, has not led to an increase in CDI laboratory diagnostic test positivity. In spite of ribotype distribution being comparable for screening in toxin A/B positive samples, there is a significantly greater correlation between NAAT positivity and culture of toxigenic strains compared to GDH.

Ceftriaxone treatment of spondylodiscitis and other serious infections with Cutibacterium acnes.

OBJECTIVES: To establish testing and treatment recommendations for a ceftriaxone once-daily dose regimen for systemic infections with Cutibacterium acnes. METHODS: A review of the literature and a retrospective evaluation of patients diagnosed with C. acnes spondylodiscitis and treated with ceftriaxone were performed. Ceftriaxone and penicillin MICs were determined for C. acnes isolates from blood and biopsies and the epidemiological cut-off (ECOFF) was determined with surveillance data from the UK Anaerobe Reference Laboratory in Cardiff. RESULTS: Limited clinical data exist from endocarditis and prosthetic joint infections using treatment with ceftriaxone 2 g once daily for C. acnes with ceftriaxone MICs ≤0.5 mg/L. In this case study, five patients were successfully treated with ceftriaxone as part of the treatment for spondylodiscitis with C. acnes. Ceftriaxone and penicillin MICs of the C. acnes isolates from the patients were 0.016-0.125 mg/L and 0.012-0.032 mg/L, respectively. The ceftriaxone ECOFF was 0.5 mg/L and the penicillin ECOFF was 0.25 mg/L based on available surveillance data. CONCLUSIONS: From the data presented in this study it would be acceptable to consider treatment with a once-daily dose of ceftriaxone 2 g for systemic infections, including endocarditis, spondylodiscitis and prosthetic joint infections with C. acnes using a clinical breakpoint of ≤0.5 mg/L (the ECOFF). However, clinical data are still limited and the response of patients treated with ceftriaxone for serious infections with C. acnes should be monitored closely.