Upregulation of the cfiA carbapenemase gene in a Bacteroides fragilis strain by the novel integrative and conjugative element Tn7563.

We describe Tn7563, a 31,844-bp integrative and conjugative element (ICE) carrying promoters upregulating the cfiA carbapenemase gene in Bacteroides fragilis strain Tbg-22. Excision and circularization of Tn7563 was demonstrated by PCR. Previously, only insertion sequences (IS) have been shown to carry mobile promoters for cfiA.

Antimicrobial susceptibility testing of Bacteroides species by disk diffusion: The NordicAST Bacteroides study.

OBJECTIVES: Antimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp. METHODS: A panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole. RESULTS: A total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80-0.93) for piperacillin-tazobactam, 0.95 (0.91-0.97) for meropenem and 0.89 (0.83-0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of <28 mm in B. fragilis indicated presence of cfiA. Piperacillin-tazobactam had the most false susceptible results. Categorical errors for this antimicrobial were particularly prevalent in cfiA-positive strains, and piperacillin-tazobactam had the highest number of comments describing zone reading difficulties. CONCLUSIONS: Inter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted.

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.

Fatal primary amoebic meningoencephalitis in a Norwegian tourist returning from Thailand.

INTRODUCTION: Primary amoebic meningoencephalitis (PAM) is a rare disease caused by the free-living amoeba Naegleria fowleri. Infection occurs by insufflation of water containing amoebae into the nasal cavity, and is usually associated with bathing in freshwater. Nasal irrigation is a more rarely reported route of infection. CASE PRESENTATION: A fatal case of PAM in a previously healthy Norwegian woman, acquired during a holiday trip to Thailand, is described. Clinical findings were consistent with rapidly progressing meningoencephalitis. The cause of infection was discovered by chance, owing to the unexpected detection of N. fowleri DNA by a PCR assay targeting fungi. A conclusive diagnosis was established based on sequencing of N. fowleri DNA from brain biopsies, supported by histopathological findings. Nasal irrigation using contaminated tap water is suspected as the source of infection. CONCLUSION: The clinical presentation of PAM is very similar to severe bacterial meningitis. This case is a reminder that when standard investigations fail to identify a cause of infection in severe meningoencephalitis, it is of crucial importance to continue a broad search for a conclusive diagnosis. PAM should be considered as a diagnosis in patients with symptoms of severe meningoencephalitis returning from endemic areas.