Detection of the antibiotic resistance genes content of intestinal Bacteroides, Parabacteroides and Phocaeicola isolates from healthy and carbapenem-treated patients from European countries.

BACKGROUND: Bacteroides fragilis group (BFG) species are the most significant anaerobic pathogens and are also the most antibiotic-resistant anaerobic species. Therefore, surveying their antimicrobial resistance levels and investigating their antibiotic resistance mechanisms is recommended. Since their infections are endogenous and they are important constituents of the intestinal microbiota, the properties of the intestinal strains are also important to follow. The aim of this study was to investigate the main antibiotic gene content of microbiota isolates from healthy people and compare them with the gene carriage of strains isolated from infections. RESULTS: We detected 13, mainly antibiotic resistance determinants of 184 intestinal BFG strains that were isolated in 5 European countries (Belgium, Germany, Hungary, Slovenia and Turkey) and compared these with values obtained earlier for European clinical strains. Differences were found between the values of this study and an earlier one for antibiotic resistance genes that are considered to be mobile, with higher degrees for cfxA, erm(F) and tet(Q) and with lower degrees for msrSA, erm(B) and erm(G). In addition, a different gene prevalence was found depending on the taxonomical groups, e.g., B. fragilis and NBFB. Some strains with both the cepA and cfiA ß-lactamase genes were also detected, which is thought to be exceptional since until now, the B. fragilis genetic divisions were defined by the mutual exclusion of these two genes. CONCLUSIONS: Our study detected the prevalences of a series of antibiotic resistance genes in intestinal Bacteroides strains which is a novelty. In addition, based on the current and some previous data we hypothesized that prevalence of some antibiotic resistance genes detected in the clinical and intestinal BFG strains were different, which could be accounted with the differential composition of the Bacteroides microbiota and/or the MGE mobilities at the luminal vs. mucosal sites of the intestine.

Evaluation of Automated Disk Diffusion Antimicrobial Susceptibility Testing Using Radian® In-Line Carousel.

Antimicrobial susceptibility testing (AST) by disk diffusion provides an accurate image of bacterial growth, enabling the detection of culture purity, heterogeneous growth, and antibiotic interactions. However, this manual method is time-consuming and visual interpretation is prone to errors. To overcome these disadvantages, the Radian® In-Line Carousel (Copan, Brescia, Italy) was launched, which is a WASPLab® module dedicated to full automation of (pre)-analytical steps as well as interpretation of disk diffusion AST. However, until now, no evaluation of Radian® against manual disk diffusion has been performed. We assessed the categorical agreement (CA) between standardized disk diffusion (reference method) and Radian® using EUCAST 2021 breakpoints. We tested 135 non-duplicate strains, selected from the National EUCAST challenge panel, clinical strains, and external quality controls. The strains included Enterobacterales (n = 63), Enterococcus faecalis (n = 3), Enterococcus faecium (n = 10), Pseudomonas aeruginosa (n = 16), Staphylococcus aureus (n = 19), coagulase-negative staphylococci (n = 4), and Streptococcus spp. (n = 20). Furthermore, we explored antibiotic disk thermolability in the WASP Radian® carousel by testing 10 ATCC® strains up to 7 days. The observed CA was 95.3%, 96.3%, 93.8%, 97.3% and 98.0% for Enterobacterales, Enterococcus spp., P. aeruginosa, Staphylococcus spp. and Streptococcus spp., respectively, resulting in an acceptable overall CA for all groups. (Very) major error rates were ≤ 5% for all antibiotics. Antibiotic disk thermostability was confirmed up to 4 days in the WASP Radian® In-Line Carousel. The Radian® In-Line Carousel provides a fully automated solution for accurate disk diffusion AST, reducing workload and improving standardization and traceability. In addition, our study demonstrated the thermostability of antibiotic disks up to 4 days in the WASP Radian® In-Line Carousel.

In Vitro Susceptibility of Achromobacter Species Isolated from Cystic Fibrosis Patients: a 6-Year Survey.

We conducted in vitro antimicrobial susceptibility testing of 267 Achromobacter isolates for 16 antibiotics from 2017 to 2022. The highest susceptibility was found for piperacillin-tazobactam (70%) and ceftazidime-avibactam (62%). Between 30% and 49% of strains were susceptible to tigecycline, ceftazidime, and meropenem. We applied species-specific Achromobacter xylosoxidans breakpoints for piperacillin-tazobactam, meropenem, and trimethoprim-sulfamethoxazole and EUCAST pharmacokinetic/pharmacodynamic (PK/PD) breakpoints for the others. A. xylosoxidans was the most frequently isolated species, followed by Achromobacter insuavis and Achromobacter ruhlandii.

Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept.

It is generally accepted that microorganisms can colonize a non-pathological endometrium. However, in a clinical setting, endometrial samples are always collected by passing through the vaginal-cervical route. As such, the vaginal and cervical microbiomes can easily cross-contaminate endometrial samples, resulting in a biased representation of the endometrial microbiome. This makes it difficult to demonstrate that the endometrial microbiome is not merely a reflection of contamination originating from sampling. Therefore, we investigated to what extent the endometrial microbiome corresponds to that of the vagina, applying culturomics on paired vaginal and endometrial samples. Culturomics could give novel insights into the microbiome of the female genital tract, as it overcomes sequencing-related bias. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy were included. An additional vaginal swab was taken from each participant right before hysteroscopy. Both endometrial biopsies and vaginal swabs were analyzed using our previously described WASPLab-assisted culturomics protocol. In total, 101 bacterial and two fungal species were identified among these 10 patients. Fifty-six species were found in endometrial biopsies and 90 were found in vaginal swabs. On average, 28 % of species were found in both the endometrial biopsy and vaginal swab of a given patient. Of the 56 species found in the endometrial biopsies, 13 were not found in the vaginal swabs. Of the 90 species found in vaginal swabs, 47 were not found in the endometrium. Our culturomics-based approach sheds a different light on the current understanding of the endometrial microbiome. The data suggest the potential existence of a unique endometrial microbiome that is not merely a presentation of cross-contamination derived from sampling. However, we cannot exclude cross-contamination completely. In addition, we observe that the microbiome of the vagina is richer in species than that of the endometrium, which contradicts the current sequence-based literature.

Validation of the Colibrí Instrument for Automated Preparation of MALDI-TOF MS Targets for Yeast Identification.

Recently, Copan (Italy) introduced the Colibrí instrument for automated colony picking and preparation of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) target plates. Our study aimed to validate this system for yeasts as such testing has not been performed yet and is a missing link needed to implement the system for routine use. Fifty-five Candida strains were selected to evaluate the accuracy of Colibrí. For each strain, a sheep blood agar plate supplemented with X and V factors (HEM) and a Sabouraud agar plate (SAB) were inoculated and incubated using the WASPlab specimen processing system (Copan). After 18 h and 36 h of incubation, the isolates were spotted in parallel using Colibrí and manually onto MALDI-TOF target plates with the addition of formic acid and identified using MALDI-TOF mass spectrometry. The reproducibility was evaluated using ATCC reference and clinical isolate-derived strains. The cumulative percentage of acceptable identification scores (IDs) after 36 h was 91% for strains cultured on HEM plates using both Colibrí and the manual method. The SAB plates showed inferior results for both Colibrí (76%) and the manual method (78%). We observed an overall agreement of 92% at 18 h for identification of the strains on the HEM plates between Colibrí and the manual method and 94% after 36 h. For the SAB plates, the agreement was 78% after 18 h and 84% after 36 h. Apart from Candida dubliniensis and Candida tropicalis, all Candida species were identified with 100% accuracy using Colibrí on HEM plates. We observed good agreement between Colibrí and the manual reference method. These results demonstrate that Colibrí is a reliable system for MALDI-TOF target preparation for yeast identification, allowing increased standardization and less hands-on time.

Validation of Rapid Antimicrobial Susceptibility Testing directly from blood cultures using WASPLab®, including Colibrí™ and Radian® in-Line Carousel.

With the increase in antimicrobial resistance, fast reporting of antimicrobial susceptibility testing (AST) results is becoming increasingly important. EUCAST developed a method for rapid AST (RAST) directly from the broth of positive blood cultures (BC). Inhibition zones are read after 4, 6, and 8 h, with specific breakpoints per time point. We evaluated the RAST method based on EUCAST disk diffusion methodology with inoculation of BC broth using WASPLab® (inclusive Colibrí™ and Radian®). Forty-nine non-duplicate strains were tested: Escherichia coli n = 17, Klebsiella pneumoniae n = 7, Pseudomonas aeruginosa n = 4, Acinetobacter baumannii n = 2, Staphylococcus aureus n = 10, Enterococcus faecalis n = 6, and Enterococcus faecium n = 3. Results were compared to direct AST and standardized AST. Good categorical agreement was obtained at all time points for all groups, except P. aeruginosa. RAST cut-offs for extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales enabled the detection of all included ESBL isolates (n = 5) at all time points, except for 1 E. coli ESBL after 4 h. RAST cut-offs for carbapenemase-producing Enterobacterales enabled the detection of only one carbapenemase after 6 h. However, all carbapenemases (n = 3) were correctly detected after 8 h. Two methicillin-resistant S. aureus were included; both were correctly categorized as cefoxitin-resistant at 6 and 8 h. At 4 h, there was insufficient growth for inhibition zone interpretation. EUCAST RAST is a fast supplementary tool for direct AST of positive BC. WASPLab® provides a significant advantage as pictures are made automatically implicating that we are not strictly bound to the time points for inhibition zone interpretation.

A comparison of the antimicrobial resistance of fecal Bacteroides isolates and assessment of the composition of the intestinal microbiotas of carbapenem-treated and non-treated persons from Belgium and Hungary.

The antimicrobial susceptibilities of Bacteroides strains isolated from the feces of imipenem-treated patients from Belgium and Hungary were compared with those isolated from the normal microbiota from these two and five other European countries and assessed. Of the 10 antibiotics tested, highly significant differences were found with cefoxitin (decrease for Belgium and for this two and the five countries from the previous study), clindamycin (decrease for Belgium and for this two and the five countries from the previous study) and moxifloxacin (increase for Belgium and for this two and the five countries from the previous study) relative to normal microbiota strains reported earlier. Imipenem treatment brought about modest, but notable differences in the compositions of the microbiomes where there was less diversity in the treated group relative to the non-treated group.

Culturomics to Investigate the Endometrial Microbiome: Proof-of-Concept.

The microbiome of the reproductive tract has been associated with (sub)fertility and it has been suggested that dysbiosis reduces success rates and pregnancy outcomes. The endometrial microbiome is of particular interest given the potential impact on the embryo implantation. To date, all endometrial microbiome studies have applied a metagenomics approach. A sequencing-based technique, however, has its limitations, more specifically in adequately exploring low-biomass settings, such as intra-uterine/endometrial samples. In this proof-of-concept study, we demonstrate the applicability of culturomics, a high-throughput culturing approach, to investigate the endometrial microbiome. Ten subfertile women undergoing diagnostic hysteroscopy and endometrial biopsy, as part of their routine work-up at Brussels IVF, were included after their informed consent. Biopsies were used to culture microbiota for up to 30 days in multiple aerobic and anaerobic conditions. Subsequent WASPLab®-assisted culturomics enabled a standardized methodology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA sequencing was applied to identify all of bacterial and fungal isolates. Eighty-three bacterial and two fungal species were identified. The detected species were in concordance with previously published metagenomics-based endometrial microbiota analyses as 77 (91%) of them belonged to previously described genera. Nevertheless, highlighting the added value of culturomics to identify most isolates at the species level, 53 (62.4%) of the identified species were described in the endometrial microbiota for the first time. This study shows the applicability and added value of WASPLab®-assisted culturomics to investigate the low biomass endometrial microbiome at a species level.

Antimicrobial susceptibility testing of Eggerthella lenta blood culture isolates at a university hospital in Belgium from 2004 to 2018.

OBJECTIVES: Eggerthella lenta is a Gram-positive anaerobic bacillus that is an important cause of bloodstream infections. This study aims to test the susceptibility of Eggerthella lenta blood culture isolates to commonly used antibiotics for the empirical treatment of anaerobic infections. METHODS: In total, 49 positive blood cultures for Eggerthella lenta were retrospectively included from patients hospitalised at the Universitair Ziekenhuis Brussel, Belgium, between 2004 and 2018. Identification was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Antimicrobial susceptibility testing was performed using the reference agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines with Brucella agar supplemented with 5 µg/mL hemin, 1 µg/mL vitamin K1 and 5% laked sheep blood. The minimal inhibitory concentrations were interpreted using the EUCAST breakpoints. Clinical characteristics were collected by reviewing the patient's medical records. RESULTS: All isolates were susceptible to amoxicillin-clavulanate, metronidazole and meropenem. Eighty-eight % of them were susceptible to clindamycin and 94% (20% S, 74% I) were susceptible to piperacillin-tazobactam. The mean age of the patients was 64 (±20) and they showed a 30-day mortality of 27%. The source of infection was in 65.3% of the cases abdominal, 20.4% were sacral pressure ulcers and 14.3% were unknown causes. While all isolates were fully susceptible at standard dosing regimen to amoxicillin-clavulanate, most were only susceptible at increased exposure or resistant to piperacillin-tazobactam. CONCLUSIONS: Our results suggest to be careful with the use of piperacillin-tazobactam and clindamycin in the empirical treatment of Eggerthella lenta infections.

A Europe-wide assessment of antibiotic resistance rates in Bacteroides and Parabacteroides isolates from intestinal microbiota of healthy subjects.

Here, we sought to assess the levels of antibiotic resistance among intestinal Bacteroides and Parabacteroides strains collected between 2014 and 2016 in Europe and also attempted to compare resistance levels between clinical and commensal isolates. Bacteroides and Parabacteroides isolates were recovered from faecal samples via the novel Bacteroides Chromogenic Agar (BCA) method. Antibiotic susceptibilities were determined by agar dilution for ten antibiotics. The values obtained were then statistically evaluated. Altogether 202 Bacteroides/Parabacteroides isolates (of which 24, 11.9%, were B. fragilis) were isolated from the faecal specimens of individuals taken from five European countries. The percentage values of isolates resistant to ampicillin, amoxicillin/clavulanate, cefoxitin, imipenem, clindamycin, moxifloxacin, metronidazole, tetracycline, tigecycline and chloramphenicol were 96.6, 4.5, 14.9, 2.0, 47.3, 11.4, 0, 66.2, 1.5 and 0%, respectively. These values are close to those reported in the previous European clinical Bacteroides antibiotic susceptibility survey except for amoxicillin/clavulanate and clindamycin, where the former was lower and the latter was higher in normal microbiota isolates. To account for these latter findings and to assess temporal effects we compared the data specific for Hungary for the same period (2014-2016), and we found differences in the resistance rates for cefoxitin, moxifloxacin and tetracycline.

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.

Case series of patients with Fusobacterium nucleatum bacteremia with emphasis on the presence of cancer.

Fusobacterium nucleatum is anaerobic oral microbiota that might be associated with cancer. We reported 22 consecutive cases of patients (mean age of 63.8 years (range 34-89), 59.1% male) with F. nucleatum bacteremia that were admitted to a university hospital over a 10-year period. In 17 (77.2%) of these patients, F. nucleatum was the sole possible pathogen. Seven of the 22 patients (31.8%) had active cancer: esophagus carcinoma (n = 3), hematologic malignancies (n = 1), gastrointestinal stromal tumor (n = 1), melanoma (n = 1), and breast cancer (n = 1). In six out of seven patients (85.7%), the F. nucleatum was found within six months of the diagnosis of cancer. Four of seven (57.1%), patients with cancer were on chemotherapy. Three of 22 patients (13.4%) died within 1 month of F. nucleatum bacteremia due to cancer. In conclusion, F. nucleatum bacteremia occurs rarely and when it is found, it is often in patients with cancer, especially those with a recent diagnosis.

Anaerobic bacteraemia: a 10-year retrospective epidemiological survey.

In order to identify current trends in anaerobic bacteraemia, a 10-year retrospective study was performed in the University Hospital Brussel, Belgium. All clinically relevant bacteraemia detected from 2004 until 2013 were included. Medical records were reviewed in an attempt to define clinical parameters that might be associated with the occurrence of anaerobic bacteraemia. 437 of the isolated organisms causing anaerobic bacteraemia were thawed, subcultured and reanalyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). There were an average of 33 cases of anaerobic bacteraemia per year during 2004-2008 compared to an average of 27 cases per year during 2009-2013 (P = 0.017), corresponding to a decrease by 19% between the first and the latter period. Also, the total number of cases of anaerobic bacteraemia per 100,000 patient days decreased from 17.3 in the period from 2004 to 2008 to 13.7 in the period 2009 to 2013 (P = 0.023). Additionally, the mean incidence of anaerobic bacteraemia decreased during the study period (1.27/1000 patients in 2004 vs. 0.94/1000 patients in 2013; P = 0.008). In contrast, the proportion of isolated anaerobic bacteraemia compared to the number of all bacteraemia remained stable at 5%. Bacteroides spp. and Parabacteroides spp. accounted for 47.1% of the anaerobes, followed by 14.4% Clostridium spp., 12.6% non-spore-forming Gram-positive rods, 10.5% anaerobic cocci, 8.2% Prevotella spp. and other Gram-negative rods and 7.1% Fusobacterium spp. The lower gastrointestinal tract (47%) and wound infections (10%) were the two most frequent sources for bacteraemia, with the origin remaining unknown in 62 cases (21%). The overall mortality rate was 14%. Further studies focusing on the antimicrobial susceptibility and demographic background of patients are needed to further objectify the currently observed trends.

Performance of BacT/Alert resin-based FN plus bottles compared with BacT/Alert charcoal-based FN bottles for the detection of anaerobes in experimentally seeded blood cultures.

Recently new resin-based BacT/Alert FAN Plus bottles containing antibiotic-binding polymeric beads, were introduced as an improvement of the charcoal-based FAN bottles for the recovery of bacteria and fungi. To assess the performance of the novel anaerobic FN plus bottles in the detection of anaerobic organisms, we compared the detection rate and the time to detection (TTD) in spiked resin-based FN Plus bottles and charcoal-based FN bottles. The bottles were experimentally seeded with reference strains or clinical strains collected from positive blood cultures. Five reference strains and fifty-five clinically significant anaerobic isolates were investigated, of which 91% (61/67) showed growth in both types of bottles within an incubation time of 5 days. A significant prolonged median TTD of 45 h for anaerobic microorganisms was observed in the resin-based bottles versus 29 h in the charcoal-based bottles (P < 0.0001). Bacteroides spp., associated with higher virulence and higher mortality rates in bloodstream infections, were detected faster in the charcoal-based bottles as compared to the resin-based bottles. In conclusion recently improved resin FN bottles showed a significantly increased median TTD for the recovery of anaerobic isolates.

Fusobacterium necrophorum and other Fusobacterium spp. isolated from head and neck infections: A 10-year epidemiology study in an academic hospital.

BACKGROUND: Fusobacterium spp. from clinical specimens are increasingly reported. We sought to describe the epidemiology, the microbiological, and the clinical characteristics of head and neck infections caused by Fusobacterium necrophorum and other Fusobacterium spp. MATERIALS AND METHODS: Retrospective cohort study between October 1st, 2004 and September 30(th), 2014 performed in an academic hospital. Electronic patient charts and the laboratory information system were reviewed for demographic and microbiological data. The number and percentages of specific diagnosis and treatment among patients with positive Fusobacterium spp. culture were calculated. The incidence was calculated based on the number of specimens investigated each year. RESULTS: Included were 230 cultures of 230 patients (median age of 28 years, 61.7% men). F. necrophorum was often found in young patients with high C-reactive protein (CRP) and high number of leukocytes in blood. Other Fusobacterium spp. were often found in middle aged patients with relatively high CRP and slightly increased leukocytes. Three major causes of the isolation of F. necrophorum and other Fusobacterium spp. were acute tonsillitis (n = 18, incidence of 0.2%), peritonsillar abscess (n = 39, 0.5%) and acute otitis (n = 45, 0.1%). While F. necrophorum was found in majority (37/57) of patients with acute tonsillitis or peritonsillar abscess, Fusobacterium spp. other than F. necrophorum were found in the majority (35/45) of patients with acute otitis. Isolated fusobacteria were susceptible to beta-lactam antibiotics, clindamycin and metronidazole. The outcomes of patients with Fusobacterium spp. were good. CONCLUSION: F. necrophorum and other Fusobacterium spp. are rare cause of head and neck infections. The infections are well treated by combination of antibiotics and surgery.

Fourth Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria.

OBJECTIVES: To collect recent data on the susceptibility of anaerobes to antimicrobial agents with known activity against anaerobes, and to compare them with results from previous Belgian multicentre studies. METHODS: Four hundred and three strict anaerobic clinical isolates were prospectively collected from February 2011 to April 2012 in eight Belgian university hospitals. MICs were determined by one central laboratory for 11 antimicrobial agents using Etest methodology. RESULTS: According to EUCAST breakpoints, >90% of isolates were susceptible to amoxicillin/clavulanate (94%), piperacillin/tazobactam (91%), meropenem (96%), metronidazole (92%) and chloramphenicol (98%), but only 70% and 40% to clindamycin and penicillin, respectively. At CLSI recommended breakpoints, only 71% were susceptible to moxifloxacin and 79% to cefoxitin. MIC50/MIC90 values for linezolid and for tigecycline were 1/4 and 0.5/4 mg/L, respectively. When compared with survey data from 2004, no major differences in susceptibility profiles were noticed. However, the susceptibility of Prevotella spp. and other Gram-negative bacilli to clindamycin decreased from 91% in 1993-94 and 82% in 2004 to 69% in this survey. Furthermore, the susceptibility of clostridia to moxifloxacin decreased from 88% in 2004 to 66% in 2011-12 and that of fusobacteria from 90% to 71%. CONCLUSIONS: Compared with previous surveys, little evolution was seen in susceptibility, except a decline in activity of clindamycin against Prevotella spp. and other Gram-negative bacteria, and of moxifloxacin against clostridia. Since resistance was detected to all antibiotics, susceptibility testing of anaerobic isolates is indicated in severe infections to confirm appropriateness of antimicrobial therapy.

Evaluation of two automated real-time PCR-based quantification methods for whole blood Epstein-Barr viral load.

Quantification of EBV DNA is important in transplantation settings for the diagnosis of post-transplantation. We evaluated the performance of the AltoStar® EBV PCR Kit 1.5 on whole blood specimens: limit of detection, linearity, accuracy, and precision were determined using the WHO NIBSC 09/260 international standard. Results of 69 clinical samples were compared between the AltoStar® EBV PCR Kit 1.5 (altona Diagnostics) and the RealTime EBV assay (Abbott). The LoD of the AltoStar® Kit was 148 IU/mL and linearity was between 375 and 500000. A high concordance was found between nominal value of the NIBSC dilutions and the AltoStar EBV result. The total variation ranged from 2.2% to 9.6%. Out of 69 clinical samples tested, there was a high concordance between the 22 paired results within the overlapping linear ranges of both tests. The AltoStar® EBV assay is reliable and accurate for EBV viral load determination on whole blood samples.