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.

Cutibacterium acnes biofilm forming clinical isolates modify the formation and structure of Staphylococcus aureus biofilms, increasing their susceptibility to antibiotics.

Cutibacterium acnes (formally Propionibacterium acnes) is frequently identified within surgical device related infections. It is often co-isolated from infection sites with other opportunistic pathogens. Recent studies have demonstrated that C. acnes is able to form biofilms and when co-cultured with Staphylococcus spp. both inhibitory and stimulatory effects have been reported across several studies. Here, we investigated the biofilm-forming ability of 100 clinical C. acnes isolates from various infection sites in human patients, both deep tissue and superficial, followed by an investigation of how the supernatants of C. acnes cultures influenced the attachment and maturation of Staphylococcusaureus NCTC 6571 biofilms. All of the C. acnes isolates were able to form biofilms in vitro, although biofilm biomass varied between isolates. Nineteen isolates were weakly adherent, 33 were moderately adherent and the majority (48) showed strong adherence. The presence of C. acnes sterile supernatants reduced the biomass of S. aureus cultures, with a > 90% reduction observed in the presence of several of the C. acnes isolates. We observed that this decrease was not due to C. acnes affecting S. aureus viability, nor due to the presence of propionic acid. Biofilm maturation was however delayed over a 24-h period as was biofilm surface structure, although initial (up to 8 h) surface attachment was not affected. We hypothesis that this defective biofilm maturation is the cause of the observed biomass decrease. In turn, these altered biofilms showed a greater susceptibility to antibiotic treatments. In contrast the presence of C. acnes supernatant in planktonic (defined as a free moving, non-surface attached population within the liquid column) S. aureus cultures increased antibiotic tolerance, via a currently undefined mechanism. This study suggests that complex interactions between C. acnes and other opportunistic pathogens are likely to exist during colonisation and infection events. Further investigation of these interactions may lead to increased treatment options and a better prognosis for patients.

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.

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.

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.

WGS to determine the extent of Clostridioides difficile transmission in a high incidence setting in North Wales in 2015.

OBJECTIVES: Rates of Clostridioides (Clostridium) difficile infection (CDI) are higher in North Wales than elsewhere in the UK. We used WGS to investigate if this is due to increased healthcare-associated transmission from other cases. METHODS: Healthcare and community C. difficile isolates from patients across North Wales (February-July 2015) from glutamate dehydrogenase (GDH)-positive faecal samples underwent WGS. Data from patient records, hospital management systems and national antimicrobial use surveillance were used. RESULTS: Of the 499 GDH-positive samples, 338 (68%) were sequenced and 299 distinct infections/colonizations were identified, 229/299 (77%) with toxin genes. Only 39/229 (17%) toxigenic isolates were related within ≤2 SNPs to ≥1 infections/colonizations from a previously sampled patient, i.e. demonstrated evidence of possible transmission. Independent predictors of possible transmission included healthcare exposure in the last 12 weeks (P = 0.002, with rates varying by hospital), infection with MLST types ST-1 (ribotype 027) and ST-11 (predominantly ribotype 078) compared with all other toxigenic STs (P < 0.001), and cephalosporin exposure in the potential transmission recipient (P = 0.02). Adjusting for all these factors, there was no additional effect of ward workload (P = 0.54) or failure to meet cleaning targets (P = 0.25). Use of antimicrobials is higher in North Wales compared with England and the rest of Wales. CONCLUSIONS: Levels of transmission detected by WGS were comparable to previously described rates in endemic settings; other explanations, such as variations in antimicrobial use, are required to explain the high levels of CDI. Cephalosporins are a risk factor for infection with C. difficile from another infected or colonized case.

Comparing identification of clinically relevant Prevotella species by VITEK MS and MALDI biotyper.

In this multicenter study, we aimed to evaluate the performance of MALDI Biotyper and VITEK MS, for identification of Prevotella species. Three hundred and fourteen clinical isolates, collected in eight European countries between January 2014 and April 2016, were identified at the collecting sites by MALDI Biotyper (versions 3.0 and 3.1) and then reidentified by VITEK MS (version 3.0) in the central laboratory. 16S rRNA gene sequencing was used as a standard method. According to sequence analysis, the 314 Prevotella strains belonged to 19 species. MALDI Biotyper correctly identified 281 (89.5%) isolates to the species level and 33 (10.5%) only at the genus level. VITEK MS correctly identified 253 (80.6%) isolates at the species level and 276 (87.9%) isolates at the genus level. Thirty-three isolates belonging to P. bergensis, P. conceptionensis, P. corporis, P. histicola, and P. nanciensis, unavailable in the VITEK MS 3.0 database, were resulted in genus level or no identification. Six Prevotella strains, belonged to P. veroralis, P. timonensis, and P. conceptionensis not represented in the MALDI Biotyper system database, were misidentified at the genus level. In conclusion, both VITEK MS and MALDI Biotyper provided reliable and rapid identification. However, the permanent extension of the databases is needed.

Performance of mass spectrometric identification of clinical Prevotella species using the VITEK MS system: A prospective multi-center study.

Prevotella species, members of the human microbiota, can cause opportunistic infections. Rapid and accurate identification of Prevotella isolates plays a critical role in successful treatment, especially since the antibiotic susceptibility profile differs between species. Studies, mostly carried out using the Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Biotyper system, showed that MALDI-TOF MS is an accurate, rapid and satisfactory method for the identification of clinically important anaerobes. In this multi-center study, we assessed the performance of the MALDI-TOF MS VITEK MS system for the identification of clinical Prevotella isolates. A total of 508 Prevotella isolates, representing 19 different species, collected from 11 European countries, Kuwait and Turkey between January 2014 and April 2016, were identified using VITEK MS (v3.0). The reliability of the identification was assessed by 16S rRNA gene sequencing. Using VITEK MS, 422 (83.1%) of the 508 isolates were identified on the species level, 459 (90.4%) on the genus level. A total of 49 (9.6%) isolates were not identified correctly. 16S rRNA gene sequencing results showed that this was partly due to the fact that several species were not represented in the database. However, some species that were represented in the database were also not identified. Five Prevotella strains were misidentified at the genus level, 2 of these strains belonged to a species not represented in the database. In general, the VITEK MS offers a reliable and rapid identification of Prevotella species, however the databases needs to be expanded.

A multicenter survey of antimicrobial susceptibility of Prevotella species as determined by Etest methodology.

Knowledge about the antimicrobial susceptibility patterns of different Prevotella species is limited. The aim of this study was to determine the current antimicrobial susceptibility of clinical isolates of Prevotella species from different parts of Europe, Kuwait and Turkey. Activity of 12 antimicrobials against 508 Prevotella isolates, representing 19 species, were tested according to Etest methodology. EUCAST, CLSI and FDA guidelines were used for susceptibility interpretations. All Prevotella species were susceptible to piperacillin/tazobactam, imipenem, meropenem, tigecycline and metronidazole. Ampicillin/sulbactam and cefoxitin also showed good activity. Ampicillin, clindamycin, tetracycline and moxifloxacin were less active; 51.2%, 33.7%, 36.8% and 18.3% of isolates were non-susceptible, respectively. A total of 49 (9.6%) isolates were resistant to three or more antimicrobials. Prevotella bivia was the most prevalent species (n = 118) and accounted for most of the multidrug-resistant isolates. In conclusion, the level of non-susceptibility to antimicrobials, which may be used for treatment of infections involving Prevotella species, are a cause of concern. This data emphasizes the need for species level identification of clinical Prevotella isolates and periodic monitoring of their susceptibility to guide empirical treatment.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted.

A new chromogenic medium for isolation of Bacteroides fragilis suitable for screening for strains with antimicrobial resistance.

There have been an increasing number of reports describing the acquisition of antimicrobial resistance by Bacteroides fragilis including the occurrence of strains with resistance to multiple antimicrobials that are relied upon for treatment of infections. The aim of this study was to design a chromogenic selective medium for isolation of B. fragilis that could be adapted for specific isolation of antimicrobial-resistant strains. Bacteroides chromogenic agar (BCA) was the result of this endeavour and allowed growth of Bacteroides spp. as black colonies and the efficient inhibition of almost all other genera tested. The medium also allowed some differentiation of B. fragilis from other members of the B. fragilis group. When compared with an adaptation of Bacteroides bile-esculin agar (BBE) for the isolation of B. fragilis from 100 stool samples, 30 isolates of B. fragilis were recovered on BCA compared with 19 isolates recovered on BBE (P = 0.022). When supplemented with meropenem (4 µg/ml) or metronidazole (2 µg/ml), BCA could be used to select for the growth of B. fragilis isolates with resistance to these agents. We conclude that BCA is a useful research tool for surveillance studies to assess the prevalence of B. fragilis and, in particular, the occurrence of antimicrobial-resistant strains.

Rapid, Point-of-Care Microwave Lysis and Electrochemical Detection of Clostridioides difficile Directly from Stool Samples.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 µg/mL to 250 µg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

Antimicrobial resistance surveillance of Bacteroides fragilis isolated from blood cultures, Europe, 2022 (ReSuBacfrag).

OBJECTIVES: Bacteroides fragilis is the most frequent cause of anaerobic bacteraemia. Although recent data suggest a rise in antimicrobial resistance (AMR) of this and other anaerobic bacteria, surveillance remains limited due to a lack of both data availability and comparability. However, a newly introduced standardised method for antimicrobial susceptibility testing (AST) of anaerobic bacteria has made larger scale surveillance possible for the first time. The aim of this study was to investigate phenotypic AMR of Bacteroides fragilis isolates from bacteraemia across Europe in 2022. METHODS: In a multicentre approach, clinical microbiology laboratories in Europe were invited to contribute results of AST for Bacteroides fragilis blood culture isolates (including only the first isolate per patient and year). AST of a selection of four antibiotics was performed locally by participating laboratories in a prospective or retrospective manner, using the new EUCAST disc diffusion method on fastidious anaerobe agar (FAA-HB). RESULTS: A total of 16 European countries reported antimicrobial susceptibilities in 449 unique isolates of Bacteroides fragilis from blood cultures in 2022. Clindamycin demonstrated the highest resistance rates (20.9%, range 0 - 63.6%), followed by piperacillin-tazobactam (11.1%, 0-54.5%), meropenem (13.4%, 0-45.5%), and metronidazole (1.8%, 0-20.0%), all with wide variation between countries. CONCLUSION: Considering that the mean resistance rates across Europe were higher than expected for three of the four anti-anaerobic antibiotics under surveillance, both local AST of clinically relevant isolates of Bacteroides fragilis and continued surveillance on an international level is warranted.

The European committee on antimicrobial susceptibility testing disc diffusion susceptibility testing method for frequently isolated anaerobic bacteria.

OBJECTIVES: Antimicrobial resistance in anaerobic bacteria is increasing and there is a link between inappropriate antimicrobial therapy and poor clinical outcome in the treatment of infections caused by anaerobic bacteria. Accurate and timely antimicrobial susceptibility testing of anaerobic bacteria is therefore of critical importance. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently described a disc diffusion susceptibility testing method for anaerobic bacteria using fastidious anaerobe agar (FAA) supplemented with 5% defibrinated horse blood (HB). This method was previously validated for Bacteroides spp. only. The aim of this study was to determine the suitability of FAA-HB for disc diffusion and also for frequently isolated anaerobic bacteria. METHODS: Clinical isolates, including 54 Bacteroides/Phocaeicola/Parabacteroides spp., 49 Prevotella spp., 51 Fusobacterium necrophorum, 58 Clostridium perfringens, and 54 Cutibacterium acnes were evaluated against six antimicrobial agents. MICs were determined by agar dilution following Clinical and Laboratory Standards Institute methodology, modified to use FAA-HB as recommended by EUCAST, instead of supplemented Brucella agar, and disc diffusion was performed on FAA-HB following EUCAST methodology. RESULTS: Results for quality control strains were reproducible, with 99.3% of zones within range. Disc diffusion by EUCAST methodology was able to distinguish between susceptible and resistant isolates of anaerobic bacteria for benzylpenicillin, piperacillin-tazobactam, meropenem, clindamycin, and metronidazole (98.7% correct categorization). No isolates resistant to vancomycin were tested, but zone diameters correctly categorized the susceptible isolates, and there was a logical relationship between MICs and inhibition zones. DISCUSSION: The recently published EUCAST method for disc diffusion for anaerobic bacteria based on FAA-HB is a reproducible and accurate method for susceptibility testing of frequently isolated anaerobic bacteria.

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.

Unusual neurological presentation of Fusobacterium necrophorum disease.

A 2-year-old girl presented to hospital, with reduced consciousness and fever. She had a 4-week history of fever treated with two courses of amoxicillin for tonsillitis diagnosed in primary care. Neuroimaging revealed multiple cerebral abscesses and subdural empyema. Pus aspirated from the intracranial collections grew Fusobacterium necrophorum and meropenem was started. Following neurosurgery, the patient continued to be agitated with fluctuating fever. She underwent close monitoring with regular neuroimaging. To control the progression of intracranial infection, she underwent three separate neurosurgical procedures following which she made a good recovery. This case demonstrates how an organism rarely associated with childhood illnesses presented atypically and progressed into a complex potentially fatal intracranial infection requiring a high degree of neurosurgical intervention. Awareness of this organism is important. The combination of source control together with appropriate antibiotic use was crucial in controlling the infection.

Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile.

The aim of this study was to determine whether alternative resistance mechanisms, other than mutation in the quinolone resistance-determining region (QRDR) of DNA gyrase, could confer fluoroquinolone resistance in Clostridium difficile. An in vitro-generated C. difficile mutant exhibiting increased fluoroquinolone resistance was isolated through antibiotic selection on ciprofloxacin. The QRDR of this mutant was investigated by chain-termination sequencing and was found to be devoid of mutation. To determine the nature of the non-QRDR resistance mechanism in this strain, the genomes of the mutant and wild-type strains were sequenced. The gyrBA region from a collection of clinical isolates exhibiting variable fluoroquinolone resistance levels was also sequenced and was compared with that present in 918 publicly available C. difficile genomic data sets. Whole-genome sequence analysis of the fluoroquinolone-resistant mutant revealed a single non-synonymous substitution (Ala384Asp) at the predicted primary dimer interface of GyrA, far beyond the classically defined QRDR. This novel mutation caused increased resistance to ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin while conferring hypersusceptibility to novobiocin. Several novel extra-QRDR polymorphisms in C. difficile DNA gyrase were identified among clinical isolates, whilst observed fluoroquinolone resistance in strains devoid of gyrBA mutations confirmed the existence of DNA gyrase-independent resistance mechanisms in this species. In conclusion, we report the first non-QRDR mutation to confer fluoroquinolone resistance in C. difficile. Although the Ala384Asp substitution was not detected in clinical isolates, this study revealed a diversity of alternative extra-QRDR polymorphisms in DNA gyrase whose association with fluoroquinolone resistance warrants further investigation.

Surface bacteriology of venous leg ulcers and healing outcome.

AIM: Bacteria can be cultured from all venous leg ulcers (VLUs) regardless of healing status, and the significance of a positive swab result in non-clinically infected ulcers is unknown. The aim of this study was to characterise the bacteriological flora of VLUs by routine culture to determine whether the data generated had prognostic value. METHODS: The ulcers of 178 patients were sampled weekly for 12 weeks and healing outcome monitored while the limb was treated with graduated compression. Wound bacteriology was assessed using culture methodology standardised to ensure data reproducibility. RESULTS: 153 individual bacterial species were identified. The species most frequently found were Staphylococcus aureus (64.3% of assessments), Corynebacterium striatum (60.6%), Pseudomonas aeruginosa (32.6%), Helcococcus kunzii (22.0%), Finegoldia magna (21.4%) and Proteus mirabilis (16.1%). No single species or the presence of anaerobes and increasing diversity of bacterial species, previously thought to be predictive of impaired healing, was shown to be associated with healing outcome. The presence of C striatum was associated with healing outcome but not after adjusting for the known prognostic factors of wound area and duration. CONCLUSION: Routine bacteriological culture analysis of the VLU wound surface may be used to identify diverse flora in all ulcers. However, the data generated are of no additional value as a prognostic indicator of healing outcome. The presence of C striatum may represent colonisation of non-healing VLU by normal skin flora.