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.

Comparative evaluation of culture results and composition of microbiome of removed tonsils due to distant focal disease or other reasons. A prospective pilot study.

The aim of this prospective pilot study was to compare culture and microbiome results of the removed tonsils of patients with assumed distant focal disease (11 patients) and those who underwent a tonsillectomy, due to other reasons, such as recurrent tonsillitis, tonsil stones or snoring (nine patients). Aerobic culture was carried out for samples taken from the surface of the tonsils by swabs before tonsillectomy for all 20 patients. The squeezed detritus and the tissue samples of removed tonsils, taken separately for the right and left tonsils, were incubated aerobically and anaerobically. The microbiome composition of tissue samples of removed tonsils was also evaluated. Based on the culture results of the deep samples Staphylococcus aureus was the dominating pathogen, besides a great variety of anaerobic and facultative anaerobic bacteria present in the oral microbiota in those patients who underwent tonsillectomy due to distant focal diseases. Microbiome study of the core tissue samples showed a great diversity on genus and species level among patients of the two groups however, S. aureus and Prevotella nigrescens were present in higher proportion in those, whose tonsils were removed due to distant focal diseases. Our results may support previous findings about the possible triggering role of S. aureus and P. nigrescens leading to distant focal diseases. Samples taken by squeezing the tonsils could give more information about the possible pathogenic/triggering bacteria than the surface samples cultured only aerobically.

Novel and rare ß-lactamase genes of Bacteroides fragilis group species: Detection of the genes and characterization of their genetic backgrounds.

OBJECTIVES: This study screened the prevalence of rare ß-lactamase genes in Bacteroides fragilis group strains from clinical specimens and normal microbiota and examined the genetic properties of the strains carrying these genes. METHODS: blaHGD1, blaOXA347, cblA, crxA, and pbbA were detected by real-time polymerase chain reaction in collections of Bacteroides strains from clinical (n = 406) and fecal (n = 184) samples. To examine the genetic backgrounds of the samples, end-point PCR, FT-IR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used. RESULTS: All B. uniformis isolates were positive for cblA in both collections. Although crxA was B. xylanisolvens-specific and associated with carbapenem resistance, it was only found in six fecal and three clinical B. xylanisolvens strains. Moreover, the crxA-positive strains were not clonal among B. xylanisolvens (contrary to cfiA in B. fragilis), implicating a rate of mobility or emergence by independent evolutionary events. The Phocaeicola (B.) vulgatus/P. dorei-specific gene blaHGD1 was detected among all P. vulgatus/P. dorei isolates from fecal (n = 36) and clinical (n = 26) samples. No blaOXA347-carrying isolate was found from European collections, but all US samples (n = 6) were positive. For three clinical isolates belonging to B. thetaiotaomicron (n = 2) and B. ovatus (n = 1), pbbA was detected on mobile genetic elements, and pbbA-positive strains displayed non-susceptibility to piperacillin or piperacillin/tazobactam phenotypically. CONCLUSIONS: Based on these observations, ß-lactamases produced by rare ß-lactamase genes in B. fragilis group strains should not be overlooked because they could encode important resistance phenotypes.

A novel Bacteroides metallo-ß-lactamase (MBL) and its gene (crxA) in Bacteroides xylanisolvens revealed by genomic sequencing and functional analysis.

OBJECTIVES: We sought to characterize the carbapenem resistance mechanism of Bacteroides xylanisolvens 14880, an imipenem-resistant strain from Germany, and assess its prevalence. METHODS: Antimicrobial susceptibilities were determined using agar dilution or Etest methodology and specific imipenemase activity was detected. The genomic sequence of B. xylanisolvens 14880 was determined and analysed for antibiotic resistance genes and genomic islands. We also used gene transfer to a carbapenem susceptible host, along with 5'-RACE, conventional PCR with capillary sequencing and RT-PCR-based screening. RESULTS: B. xylanisolvens 14880 displayed resistance to carbapenems and produced high specific imipenemase activity. Its genomic sequence was 6.1 Mbp and a class B1 ß-lactamase gene (termed crxA) was detected in it. crxA was carried on a putative genomic island with insertion sequence (IS) elements and a putative GNAT (Gcn5-like acetyltransferase) toxin gene. Promoter localization by 5'-RACE and gene targeting to an imipenem-susceptible Bacteroides host indicated that it is activated by an IS1380-like IS element and it can confer carbapenem resistance. The PCR screening of Bacteroides strains showed that crxA was specific to B. xylanisolvens with a carriage rate of 16.7%. CONCLUSIONS: B. xylanisolvens strains can harbour a carbapenem resistance gene, which has many similarities to the 'cfiA system': metallo-ß-lactamase (MBL), IS element activation, carriage of a GNAT toxin gene, specific for a unique Bacteroides species with a significant prevalence.

Molecular characterization of metronidazole resistant Bacteroides strains from Kuwait.

Eleven metronidazole resistant Bacteroides and one newly classified Phocaeicola dorei strain from Kuwait were investigated for their resistance mechanisms and the emergence of their resistant plasmids. All but one strain harbored nimE genes on differently sized plasmids. Of the 11 nimE genes, 9 were preceded by full copies of the prototype ISBf6 insertion sequence element, one carried a truncated ISBf6 and one was activated by an additional copy of IS612B. Nucleotide sequencing results showed that the nimE ISBf6 distances were constant and all five different plasmids shared a common region, suggesting that (i) the nimE-ISBf6 configuration was inserted into an undisclosed common genetic element, (ii) over time, this common element was mutated by insertions and deletions, spreading the resultant plasmids. Of the 10 B. fragilis strains in this collection, 6 were also cfiA-positive, one with full imipenem resistance, indicating a tendency for multidrug resistance (MDR) among such isolates. The significant number of metronidazole resistant Bacteroides spp. and P. dorei strains with the MDR phenotype warns of difficulties in treatment and suggests promoting adherence to antibiotic stewardship recommendations in Kuwait.

Detection of beta-lactamase production in clinical Prevotella species by MALDI-TOF MS method.

Penicillins, can be used in treatment of infections due to Prevotella species if they are susceptible to penicillin. Early and accurate preliminary detection of ß-lactamase-producing isolates is crucial for treatment of infection. The aim of this study was to determine ß-lactamase-producing Prevotella species by MALDI-TOF MS and screen them for the presence of cfxA gene, responsible for ß-lactamase production. A total of 500 clinically relevant Prevotella isolates, collected from 13 countries for the previous European antibiotic resistance surveillance study, were tested. Susceptibility testing was performed against ampicillin and ampicillin/sulbactam by Etest methodology. EUCAST guidelines were used for susceptibility interpretations; the isolates with MIC value ≤ 0.5 for ampicillin were considered susceptible and >2 resistant. All Prevotella isolates, were tested for detection of ß-lactamase activity by MALDI-TOF MS (Vitek® MS Research Use Only) system and the presence of the cfxA gene by PCR method. The susceptibility levels of the isolates to ampicillin/sulbactam and ampicillin were 99.6% and 43.4%, respectively. A total 59% of isolates presented ß-lactamase activity and 60.8% were cfxA gene positive. Both these tests were positive for isolates in the resistant category. Additionally, >95% of the isolates (n = 65) which ampicillin MIC values ranged from >0.5 µg/mL to 2 µg/ml displayed ß-lactamase activity. We also found that the MALDI-TOF MS-based ß-lactamase assay delivers results in 2 h. We found a high concordance between the MALDI-TOF MS ß-lactamase results in terms of cfxA ß-lactamase gene presence. MALDI-TOF MS may serve as a simple and efficient alternative method of the existing phenotypic and PCR-based methods.

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.

Point-of-care measurement of activated clotting time for cardiac surgery as measured by the Hemochron signature elite and the Abbott i-STAT: agreement, concordance, and clinical reliability.

BACKGROUND: Since inadequate heparin anticoagulation and insufficient reversal can result in complications during cardiopulmonary bypass (CPB) surgery, heparin anticoagulation monitoring by point-of-care (POC) activated clotting time (ACT) measurements is essential for CPB initiation, maintainance, and anticoagulant reversal. However, concerns exist regarding reproducibility of ACT assays and comparability of devices. METHODS: We evaluated the agreement of ACT assays using four parallel measurements performed on two commonly used devices each (i.e., two Hemochron Signature Elite (Hemochron) and two Abbott i-STAT (i-STAT) devices, respectively). Blood samples from 30 patients undergoing cardiac surgery on CPB were assayed at specified steps (baseline, after heparin administration, after protamine administration) with four parallel measurements (two of each device type) using commercial Kaolin activated assays provided by the respective manufactures. Measurements were compared between identical and different device types using linear regression, Bland-Altman analyses, and calculation of Cohen's kappa coefficient. RESULTS: Parallel i-STAT ACTs demonstrated a good linear correlation (r = 0.985). Bias, as determined by Bland-Altman analysis, was low (- 3.8 s; 95% limits of agreement (LOA): - 77.8 -70.2 s), and Cohen's Kappa demonstrated good agreement (kappa = 0.809). Hemochron derived ACTs demonstrated worse linear correlation (r = 0.782), larger bias with considerably broader LOA (- 13.14 s; 95%LOA:-316.3-290 s), and lesser concordance between parallel assays (kappa = 0.554). Although demonstrating a fair linear correlation (r = 0.815), parallel measurements on different ACT-devices showed large bias (-20s; 95% LOA: - 290-250 s) and little concordance (kappa = 0.368). Overall, disconcordant results according to clinically predefined target values were more frequent with the Hemochron than i-STAT. Furthermore, while discrepancies in ACT between two parallel iSTAT assays showed little or no clinical relevance, deviations from parallel Hemochron assays and iSTAT versus Hemochron measurements revealed marked and sometimes clinically critical deviations. CONCLUSION: Currently used ACT point-of-care devices cannot be used interchangeably. Furthermore, our data question the reliability of the Hemochron in assessing adequacy of heparin anticoagulation monitoring for CPB.

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.

What do we know about the diagnostics, treatment and epidemiology of Clostridioides (Clostridium) difficile infection in Europe?

Clostridium difficile, recently reclassified as Clostridioides difficile is responsible for a significant part of diarrheal diseases in the hospitals and in the community. Besides the main pathogenic factors, toxin A, toxin B and the binary toxin, several other putative virulence factors have been investigated. This manuscript summarize recent findings in Europe concerning source of infection, epidemiology of CDI, the changing pattern of PCR ribotypes of C. difficile strains in different European countries, recommendations for diagnosis and treatment of CDI.

Sample preparation method influences direct identification of anaerobic bacteria from positive blood culture bottles using MALDI-TOF MS.

Rapid detection and identification of anaerobic bacteria from blood is important to adjust antimicrobial therapy by including antibiotics with activity against anaerobic bacteria. Limited data is available about direct identification of anaerobes from positive blood culture bottles using MALDI-TOF mass spectrometry (MS). In this study, we evaluated the performance of two sample preparation protocols for direct identification of anaerobes from positive blood culture bottles, the MALDI Sepsityper kit (Sepsityper) and the in-house saponin (saponin) method. Additionally, we compared two blood culture bottle types designed to support the growth of anaerobic bacteria, the BacT/ALERT-FN Plus (FN Plus) and the BACTEC-Lytic (Lytic), and their influence on direct identification. A selection of 30 anaerobe strains belonging to 22 different anaerobic species (11 reference strains and 19 clinical isolates) were inoculated to 2 blood culture bottle types in duplicate. In total, 120 bottles were inoculated and 99.2% (n = 119) signalled growth within 5 days of incubation. The Sepsityper method correctly identified 56.3% (n = 67) of anaerobes, while the saponin method correctly identified 84.9% (n = 101) of anaerobes with at least log(score) ≥1.6 (low confidence correct identification), (p < 0.001). Gram negative anaerobes were better identified with the saponin method (100% vs. 46.5%; p < 0.001), while Gram positive anaerobes were better identified with the Sepsityper method (70.8% vs. 62.5%; p = 0.454). Average log(score) values among only those isolates that were correctly identified simultaneously by both sample preparation methods were 2.119 and 2.029 in favour of the Sepsityper method, (p = 0.019). The inoculated bottle type didn't influence the performance of the two sample preparation methods. We confirmed that direct identification from positive blood culture bottles with MALDI-TOF MS is reliable for anaerobic bacteria. However, the results are influenced by the sample preparation method used.

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.

Performance of two blood culture systems to detect anaerobic bacteria. Is there any difference?

We studied the performance characteristics of two blood culture (BC) bottles/systems, (i) BacT/ALERT-FN Plus/3D (bioMérieux, Marcy l'Étoile, France) and (ii) BACTEC-Lytic/9000 (Becton Dickinson, Sparks, USA) for detection of growth and time-to-positivity (TTP) against a balanced and diverse collection of anaerobic bacterial strains (n = 48) that included reference strains (n = 19) and clinical isolates (n = 29) of 32 species (15 Gram-negative and 17 Gram-positive). Standard suspension of bacteria was inoculated to each bottle in duplicates and incubated in the corresponding system. Overall, 62.5% (n = 30) of strains were detected by both BC bottle types. Comparing the two, 70.8% (n = 34) and 79.2% (n = 38) of strains were detected by BacT/ALERT-FN Plus and BACTEC-Lytic bottles, respectively (p = 0.38). Among Gram-negative anaerobes (n = 25) the detection rate was 76.0% (n = 19) vs. 92.0% (n = 23) (p = 0.22), respectively. Among Gram-positive anaerobes (n = 23) the detection rate was 65.2% (n = 15) in both bottles (p = 1). The average TTP per bottle was calculated only for the strains detected by both systems (n = 30) and was 40.85 h and 28.08 h for BacT/ALERT-FN Plus and BACTEC-Lytic, respectively (p < 0.001). The mean difference was 12.76 h (95% CI: 6.21-19-31 h). Six anaerobic strains were not detected by any system, including Gram-negative Porphyromonas gingivalis, and five Gram-positive strains: Finegoldia magna, Peptostreptococcus anaerobius, Propionibacterium acnes, Clostridium novyi and Clostridium clostridioforme. Furthermore, Eggerthella lenta and Prevotella bivia were detected only by BacT/ALERT-FN Plus, while Prevotella disiens and Prevotella intermedia were detected only by BACTEC-Lytic bottles. There were no major differences in detection rate among clinical and reference strains. Anaerobic bacteria represent a minority of BC isolates, however, far from ideal detection rate was observed in this study for both tested bottle/system combinations. Nevertheless, in those cases where both gave positive signal, BACTEC-Lytic was superior to BacT/ALERT FN Plus with 12.76 h shorter mean TTP. Improvements of media in blood culture bottles available for detection of anaerobes are warranted.

Emergence and evolution of an international cluster of MDR Bacteroides fragilis isolates.

OBJECTIVES: The aim of this study was to examine the antibiotic resistance profiles, antibiotic resistance mechanisms and possible 'clonal' nature of some MDR Bacteroides fragilis strains that simultaneously harboured cfiA, nimB, IS1186 and IS4351. METHODS: Antibiotic susceptibilities were determined by Etests and antibiotic resistance genes and different genetic elements were detected by applying PCR methods. The environments of the cfiA and nimB genes were also determined by sequencing. The transferability of the cfiA, nimB and tet(Q) genes was tested by conjugation. The genetic relatedness of the test strains was tested by ERIC-PCR or PFGE. The complete genome sequences of two strains (B. fragilis BF8 and O:21) were determined by next-generation sequencing. RESULTS: Most of the seven B. fragilis strains tested displayed multidrug resistance phenotypes; five strains were resistant to at least five types of antibiotics. Besides the common genetic constitution, ERIC-PCR implied high genetic relatedness. Similarities in some of the antibiotic resistance mechanisms [carbapenems (cfiA) and metronidazole (nimB)] also confirmed their common origin, but some other resistance mechanisms {MLSB [erm(F)] and tetracycline [tet(Q)]} and PFGE typing revealed differences. In B. fragilis BF8 and O:21, erm(F) and tet(X) genes were found with IS4351 borders, thus constituting Tn4351. All the strains were tet(Q) positive and transferred this gene in conjugation experiments, but not the cfiA and nimB genes. CONCLUSIONS: An international cluster of MDR B. fragilis strains has been identified and characterized. This 'clone' may have emerged early in the evolution of division II B. fragilis strains, which was suggested by the low-complexity ERIC profiles and differences in the PFGE patterns.

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.

Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates.

With the emergence of antibiotic resistance among Bacteroides fragilis group isolates the need of susceptibility testing in routine laboratories is increasing. The aims of the present study were to evaluate the disk diffusion method for susceptibility testing in case of different clinical isolates of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic susceptibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to amoxicillin/clavulanic acid, cefoxitin, clindamycin, imipenem, metronidazole, moxifloxacin, piperacillin/tazobactam, tigecycline by agar dilution method previously. The inhibition zones of the same antibiotics including meropenem disc were determined by the disc diffusion on Brucella blood agar supplemented with haemin and vitamin K1. Plates were incubated at 37 °C in an anaerobic atmosphere for 24 h. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement between the inhibition zone diameters and the MICs for imipenem, metronidazole, moxifloxacin and tigecyclin. The inhibition zone diameters of meropenem also separated clearly the isolates, which can be considered wild-type isolates. In case of amoxicillin/clavulanic acid and piperacillin/tazobactam intermediate and susceptible isolates according to the MIC determination, overlap during the zone diameter determination. Isolates with an inhibition zone <23 mm for amoxicillin/clavulanic acid and <25 mm for piperacillin/tazobactam should be retested by a MIC determination method. The 10 µg clindamycin disc clearly separated the resistant and the susceptible population of B. fragilis group strains. In the case of cefoxitin only resistant population could be separated with an inhibition zone <17 mm, intermediate and susceptible isolates overlap. In conclusion, we suggest that disk diffusion can be an option for susceptibility testing of B. fragilis group isolates for most relevant antibiotics in routine laboratories.

Investigation of the MICs of fidaxomicin and other antibiotics against Hungarian Clostridium difficile isolates.

The aim of this study was to investigate in vitro activities of fidaxomicin and other antibiotics against 188 Clostridium difficile strains collected from different centers of Hungary. C. difficile isolates showed minimum inhibitory concentration (MIC) range for fidaxomicin of ≤0.008-0.5 mg/L, with a MIC90 of 0.125 mg/L. Only four isolates (2.1%) had 0.5 mg/L MIC to fidaxomicin. The obtained MICs showed identical distribution to those found in the EUCAST database for wild-type strains.

A study on Nim expression in Bacteroides fragilis.

Members of the genus Bacteroides, mainly Bacteroides fragilis, can cause severe disease in man, especially after intestinal perforation in the course of abdominal surgery. Treatment is based on a small number of antibiotics, including metronidazole, which has proved to be highly reliable throughout the last 40 to 50 years. Nevertheless, metronidazole resistance does occur in Bacteroides and has been mainly attributed to Nim proteins, a class of proteins with a suggested nitroreductase function. Despite the potentially high importance of Nim proteins for human health, information on the expression of nim genes in B. fragilis is still lacking. It was the aim of this study to demonstrate expression of nim genes in B. fragilis at the protein level and, furthermore, to correlate Nim levels with the magnitude of metronidazole resistance. By the application of 2D gel electrophoresis, Nim proteins could be readily identified in nim-positive strains, but their levels were not elevated to a relevant extent after induction of resistance with high doses of metronidazole. Thus, the data herein do not provide evidence for Nim proteins acting as nitroreductases using metronidazole as a substrate, because no correlation between Nim levels and levels of metronidazole resistance could be observed. Furthermore, no evidence was found that Nim proteins protect B. fragilis from metronidazole by sequestering the activated antibiotic.