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.

Molecular study on metronidazole resistance in Bacteroides fragilis group isolates from a South Indian tertiary care center.

OBJECTIVE: Bacteroides species are an important part of human intestinal microbiota. They can cause infections of significant mortality and morbidity when moved out of their niche in the gut. The cornerstone drug for prophylaxis and therapy, metronidazole, is exhibiting signs of resistance, which are frequently attributed to nitroimidazole (nim) resistance genes. The aim of this study was to use Epsilometer test (E-test) to assess the metronidazole susceptibility and conventional PCR methodology to map the distribution of nim genes in Bacteroides fragilis group (BFG) isolates. METHODS: MALDI-TOF MS was used to identify BFG isolates. Using the E-test methodology, metronidazole minimum inhibitory concentrations (MICs) were determined. The presence of nim genes in these isolates were checked by conventional PCR methodology. Sequencing was done on selected amplicons for determining the nim gene types. RESULTS: Bacteroides fragilis accounted for 55.3% of the total 273 BFG members identified. Of these, 196 (71.8%) were susceptible, 43 (15.8%) intermediate and 34 (12.5%) resistant to metronidazole as determined by the E-test. nim gene was present in 101 (37%) of the total 273 isolates. Out of the 34 phenotypically resistant isolates (MIC ≥32 µg/ml), 29 harboured nim gene (Chi-square test, p < 0.0000001) but nim gene was absent in 5 (14.7%) isolates. Also, nim gene was detected in 72 (30.1%) of the 239 isolates with susceptible and intermediate metronidazole MIC. Sequencing of 20 amplicons gave a nimE gene type. CONCLUSIONS: In view of the rising metronidazole resistance among BFG and its close association with nim genes, there is a need for implementing routine metronidazole susceptibility testing and more researches are needed to find the molecular basis of these nim genes.

Comparative evaluation of agar dilution and broth microdilution by commercial and in-house plates for Bacteroides fragilis group: An economical and expeditious approach for resource-limited settings.

OBJECTIVE: To compare the performance of agar dilution and broth microdilution by commercial and in-house prepared plates for the Bacteroides fragilis group. The cost analysis was performed to demonstrate that in-house prepared BMD plates were a suitable alternative to agar dilution given the high cost and low feasibility of incorporating commercial BMD plates in routine, particularly in the tertiary care institutes of many low- and middle-income countries. METHODS: Thirty B. fragilis group isolates were tested against six antibiotics, frequently used as empirical therapy for anaerobic infections including metronidazole, clindamycin, imipenem, piperacillin-tazobactam, cefoxitin, and chloramphenicol. The running consumable expenditure for all methodologies was calculated. RESULTS: The results demonstrated essential and categorical agreement of >90% for all antibiotics except cefoxitin, which showed <90% categorical agreement. No major or very major errors were observed. We observed a high agreement and strong concordance for MIC values between both methods and inter-rate reliability of >0.9 by Cohen's kappa analysis, indicating almost perfect agreement between both methods using either of the plates. In contrast to agar dilution, a 20.5 fold cost reduction was seen in BMD using in-house plates and a 5.8 fold reduction using commercial plates to test a single isolate. However, when testing 30 isolates concurrently the cost significantly increased for commercial BMD plates by 8.4 folds, and only 1.03 fold cost reduction was seen with in-house BMD plates. CONCLUSION: BMD gives comparable results to agar dilution and can be considered a method of choice to test a small number of samples. The technique is an economical option when plates are standardized in-house and could be employed for susceptibility testing of the B. fragilis group.

Change in antimicrobial susceptibility of pathogens isolated from surgical site infections over the past decade in Japanese nation-wide surveillance study.

Inappropriate antimicrobial therapy for surgical site infections (SSIs) can lead to poor outcomes and an increased risk of antibiotic resistance. A nationwide survey was conducted in Japan from 2018 to 2019 to investigate the antimicrobial susceptibility of pathogens isolated from SSIs. The data were compared with those obtained in 2010 and 2014-2015 surveillance studies. Although the rate of detection of extended-spectrum ß-lactamase producing strains of Escherichia coli was increased from 9.5% in 2010 to 23% in 2014-2015, the incidence decreased to 8.7% in 2018-2019. Although high susceptibility rates were detected to piperacillin/tazobactam (TAZ), the geometric mean MICs were substantially higher than to meropenem (2.67 vs 0.08 µg/mL). By contrast, relatively low geometric mean MICs (0.397 µg/mL) were demonstrated for ceftolozane/TAZ. Although the MRSA incidence rate decreased from 72% in the first surveillance to 53% in the second, no further decrease was detected in 2018-2019. For the Bacteroides fragilis group species, low levels of susceptibility were observed for moxifloxacin (65.3%), cefoxitin (65.3%), and clindamycin (CLDM) (38.9%). In particular, low susceptibility against cefoxitin was demonstrated in non-fragilis Bacteroides, especially B. thetaiotaomicron. By contrast, low susceptibility rates against CLDM were demonstrated in both B. fragilis and non-fragilis Bacteroides species, and a steady decrease in susceptibility throughout was observed (59.3% in 2010, 46.9% in 2014-2015, and 38.9% in 2018-2019). In conclusion, Japanese surveillance data revealed no significant lowering of antibiotic susceptibility over the past decade in organisms commonly associated from SSIs, with the exception of the B. fragilis group.

First Case of Bacteraemia Due to Carbapenem-Resistant Bacteroides faecis.

Multidrug resistant (MDR) bacteria are increasingly observed in nosocomial and community-acquired settings. Anaerobes are no exception to this rule, but there are fewer reports of MDR in the scientific literature on anaerobes than there are for other bacteria. In this short case report, we describe the first case of bacteraemia caused by a multidrug-resistant Bacteroides faecis, which produces a carbapenemase encoded by the blaCfiA gene. This bacteraemia followed a digestive surgery operation. Surprisingly, these findings did not lead to a change in antibiotic therapy, probably because the patient's clinical state had improved. Nevertheless this report calls for better knowledge of anaerobic bacteria and for a systematic antimicrobial stewardship procedure following bacteraemia.

Frequency and associated factors for carbapenem-non-susceptible Bacteroides fragilis group bacteria colonization in hospitalized patients: Case control study in a university hospital in Turkey.

PURPUSE: The carbapenem-resistant Bacteroides fragilis group (CR-BFG) bacteria have been reported in several countries recently with increasing global attention. The high incidence of CR-BFG isolated from our hospitalized patients has become an important problem. Therefore, we aimed to determine the frequency and associated factors for intestinal colonization by carbapenem-non-susceptible BFG (CNS-BFG) among adult patients hospitalized at intensive care units, neurosurgery and internal medicine wards in our hospital. METHODS: Rectal swabs (n = 1200), collected from 766 patients between February 2014 and March 2015, were inoculated onto kanamycin-vancomycin-leaked blood agar containing 0.125 mg/L meropenem. The isolates were identified by MALDI-TOF MS. Susceptibility testing was performed by agar dilution method. The carbapenemase gene (cfiA) was detected by PCR. Logistic regression analysis was used to evaluate the associated factors for intestinal colonization by CNS-BFG. RESULTS: A total 180 non-duplicate BFG isolates were obtained from 164 patients. Ten different species, including Parabacteroides distasonis (n = 46, 25.6%), and Bacteroides fragilis (n = 30; 16.6%), were identified. Twenty-five percent of the isolates were non-susceptible to meropenem (MIC >2 mg/L). The highest prevalence of meropenem resistant strains (MIC >8 mg/L) was detected among B. fragilis (n = 12), followed by Parabacteroides spp. (n = 4). All but one B. fragilis strains were cfiA gene positive. Hospital admission, increasing Charlson score, use of antibiotics; including carbapenems in past three months, colonization with other accompanying carbapenem-resistant Gram negative bacteria (Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa), and having undergone surgical operations were significantly associated with RCS- BFG colonization. CONCLUSIONS: The high carriage rate of CNS-BFG in hospitalized patients may lead to worse clinical outcomes, such as serious infections and mortality, and deserves attention.

Markers Specific to Bacteroides fragilis Group Bacteria as Indicators of Anthropogenic Pollution of Surface Waters.

The aim of this study was to evaluate the applicability of markers specific to Bacteroides fragilis group (BFG) bacteria as indicators of anthropogenic pollution of surface waters. In addition, the impact of wastewater treatment plants (WWTPs) on the spread of genes specific to fecal indicator bacteria and genes encoding antimicrobial resistance in water bodies was also determined. Samples of hospital wastewater (HWW), untreated wastewater (UWW), and treated wastewater (TWW) evacuated from a WWTP were collected, and samples of river water were taken upstream (URW) and downstream (DRW) from the wastewater discharge point to determine, by qPCR, the presence of genes specific to BFG, Escherichia coli and Enterococcus faecalis, and the abundance of 11 antibiotic resistance genes (ARGs) and two integrase genes. The total number of bacterial cells (TCN) in the examined samples was determined by fluorescence in situ hybridization (FISH). Genes specific to BFG predominated among the analyzed indicator microorganisms in HWW, and their copy numbers were similar to those of genes specific to E. coli and E. faecalis in the remaining samples. The abundance of genes specific to BFG was highly correlated with the abundance of genes characteristic of E. coli and E. faecalis, all analyzed ARGs and intI genes. The results of this study indicate that genes specific to BFG can be used in analyses of human fecal pollution, and as indicators of environmental contamination with ARGs. A significant increase in the copy numbers of genes specific to BFG, E. coli, and seven out of the 11 analyzed ARGs was noted in samples of river water collected downstream from the wastewater discharge point, which suggests that WWTPs are an important source of these genes in riparian environments.

A Soluble Fiber Diet Increases Bacteroides fragilis Group Abundance and Immunoglobulin A Production in the Gut.

Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Previous studies have shown that Bacteroidetes, the major phylum of gut microbiota together with Firmicutes, impact IgA production. However, the relative abundances of species of Bacteroidetes responsible for IgA production were not well understood. In the present study, we identified some specific Bacteroidetes species that were associated with gut IgA induction by hsp60-based profiling of species distribution among Bacteroidetes The levels of IgA and the expression of the gene encoding activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were increased in soluble high-fiber diet (sHFD)-fed mice. We found that Bacteroides acidifaciens was the most abundant Bacteroidetes species in both sHFD- and normal diet-fed mice. In addition, the gut IgA levels were associated with the relative abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciens Conversely, the ratio of B. acidifaciens to other Bacteroidetes species was reduced in insoluble high-fiber diet fed- and no-fiber diet-fed mice. To investigate whether B. acidifaciens increases IgA production, we generated B. acidifaciens monoassociated mice and found increased gut IgA production and AID expression. Collectively, soluble dietary fiber increases the ratio of gut Bacteroides fragilis group, such as B. acidifaciens, and IgA production. This might improve gut immune function, thereby protecting against bowel pathogens and reducing the incidence of inflammatory bowel diseases.IMPORTANCE Immunoglobulin A (IgA) is essential for defense of the intestinal mucosa against harmful pathogens. Gut microbiota impact IgA production, but the specific species responsible for IgA production remain largely elusive. Previous studies have shown that IgA and Bacteroidetes, the major phyla of gut microbiota, were increased in soluble high-fiber diet-fed mice. We show here that the levels of IgA in the gut and the expression of activation-induced cytidine deaminase (AID) in the large intestine lamina propria, which is crucial for class switch recombination from IgM to IgA, were correlated with the abundance of Bacteroides fragilis group species such as Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciensB. acidifaciens monoassociated mice increased gut IgA production and AID expression. Soluble dietary fiber may improve gut immune function, thereby protecting against bowel pathogens and reducing inflammatory bowel diseases.

Environmental fate of Bacteroidetes, with particular emphasis on Bacteroides fragilis group bacteria and their specific antibiotic resistance genes, in activated sludge wastewater treatment plants.

The aim of this study was to determine the effect of the activated sludge process on the abundance of anaerobic bacteria of the phylum Bacteroidetes, with special emphasis on Bacteroides fragilis group (BFG) bacteria, in twelve full-scale wastewater treatment plants. The composition of bacterial phyla and classes in wastewater samples were analyzed by next-generation sequencing. The presence of specific to BFG bacteria genes and the abundance of ARGs and genes encoding class 1 integrase in wastewater samples were determined by qPCR. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were dominant bacterial phyla in wastewater samples. Next-generation sequencing revealed similar proportions of Bacteroidia (<1.0-8.2 % of all bacteria) in wastewater influents and effluents, which suggest that these microorganisms are not completely eliminated in the activated sludge process. The average copy numbers of specific to BFG bacteria gene, were 106, and 104 copies in 1 mL of wastewater influents and effluents, respectively. The results revealed a correlation between the abundance of BFG bacteria and BFG-specific genes encoding resistance to antibiotics. The observed changes in the prevalence of BFG-specific genes and ARGs in untreated and treated wastewater indicate that the activated sludge process decreases the number of gene copies in the effluent evacuated to the environment.

Antimicrobial Resistance Profile and Nim Gene Detection among Bacteroides fragilis Group Isolates in a University Hospital in South India.

INTRODUCTION: Members of Bacteroides fragilis group are the most frequently isolated anaerobic pathogens in the clinical laboratory from diverse infection sites. The objective of this study was to characterize B. fragilis isolates from various clinical specimens, to analyze their susceptibility profile toward most common anti-anaerobic antimicrobials, and to study the frequency of nim gene determining resistance to nitroimidazoles. METHODS: Specimens processed for anaerobic culture between January 2013 and December 2015 were analyzed. Isolates of B. fragilis group were identified and speciated by mass spectrometry. ß-lactamase production was detected using nitrocefin disks. Agar dilution and antimicrobial gradient diffusion methods were performed to study their susceptibility profile. The isolates were screened for nim gene by conventional gel-based polymerase chain reaction. RESULTS: A total of 57 isolates of B. fragilis group were studied. The commonly isolated species was B. fragilis (73.7%), followed by Bacteroides thetaiotaomicron (8.8%), Bacteroides vulgatus (8.8%), and others. Most of the isolates were recovered from deep-seated abscesses (47.4%). All isolates were found to be ß-lactamase producers. Metronidazole (Mtz) resistance was observed in 4 (7%) isolates. Higher rate of resistance was observed toward clindamycin (31.6%). None of the isolates tested were found resistant to chloramphenicol, piperacillin-tazobactam, and meropenem. nim genes were present in 4 (11.4%) B. fragilis isolates (n = 35). CONCLUSIONS: Resistance to the most commonly used empirical anti-anaerobic drugs including Mtz was noted in the isolates of B. fragilis group. Routine anaerobic cultures when indicated and continual surveillance of antimicrobial resistance among the anaerobic bacterial pathogens is essential.

Evaluation of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card for identification of Bacteroides fragilis group isolates and antimicrobial susceptibilities of these isolates in a Chinese university hospital.

BACKGROUND AND PURPOSE: Bacteroides fragilis group isolates are most frequently isolated anaerobic pathogens. This study aimed to evaluate the accuracy of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card on the identification of clinical B. fragilis group isolates, as well as to determine their antimicrobial susceptibilities. METHODS: A total of 138 isolates of B. fragilis group isolates were identified with the three MALDI-TOF MS systems and VITEK 2 ANC cards. 16S rRNA gene sequencing was used as the reference identification method for comparison. Antimicrobial susceptibilities were determined by agar dilution method to 19 antimicrobial agents recommended by Clinical and Laboratory Standards Institute (CLSI). RESULTS: Hundred thirty three isolates of Bacteroides spp. and 5 isolates of Parabacteroides spp. were identified by 16S rRNA sequencing. The rates of accurate identification at species level of VITEK MS, Clin-ToF-II MS, Autof MS 1000 and VITEK 2 ANC card were 94.2%, 94.2%, 98.6% and 94.9%, respectively, while that at genus level were 99.3%, 100%, 100% and 97.8%, respectively. Metronidazole and chloramphenicol were the most susceptible agents (99.3% and 92.8%, respectively), followed by meropenem, ertapenem, imipenem and piperacillin/tazobactam to which the susceptible rates ranged from 76.8% to 79.0%. The susceptible rates to carbapenems decreased 12.4-15.3% from 2010-2013 to 2014-2017. CONCLUSION: All the four systems provided high accurate rate on the identification of B. fragilis group isolates. Metronidazole showed highest activity against these isolates. Attention should be paid to the higher resistant rates to carbapenems, clindamycin, moxifloxacin and tigecycline than the other countries.

The emergence of antimicrobial resistance in environmental strains of the Bacteroides fragilis group.

Anaerobic bacteria of the genus Bacteroides are a large group of commensal microorganisms that colonize the human and animal digestive tract. The genus Bacteroides and the closely related genus Parabacteroides include the Bacteroides fragilis group (BFG) of potentially pathogenic bacteria which are frequently isolated from patients with anaerobic infections. The aim of this study was to assess the antimicrobial resistance of environmental strains of the Bacteroides fragilis group. Strains were isolated from human feces, hospital wastewater, influent (UWW) and effluent (TWW) wastewater from a wastewater treatment plant (WWTP), and from the feces of lab rats as a negative control to monitor the entire route of transmission of BFG strains from humans to the environment. The resistance of 123 environmental BFG strains to six antibiotic groups was analyzed with the use of culture-dependent methods. Additionally, the presence of 25 genes encoding antibiotic resistance was determined by PCR. The analyzed environmental BFG strains were highly resistant to the tested antibiotics. The percentage of resistant strains differed between the analyzed antibiotics and was determined at 97.56% for ciprofloxacin, 49.59% for erythromycin, 44.71% for ampicillin, 35.77% for tetracycline, 32.52% for amoxicillin/clavulanic acid, 26.83% for chloramphenicol, 26.01% for clindamycin, 11.38% for moxifloxacin, and 8.94% for metronidazole. The highest drug-resistance levels were observed in the strains isolated from UWW and TWW samples. The mechanisms of antibiotic-resistance were determined in phenotypically resistant strains of BFG. Research has demonstrated the widespread presence of genes encoding resistance to chloramphenicol (100% of all chloramphenicol-resistant strains), tetracyclines (97.78% of all tetracycline-resistant strains), macrolides, lincosamides and streptogramins (81.97% of all erythromycin-resistant strains). Genes encoding resistance to ß-lactams and fluoroquinolones were less prevalent. None of the metronidazole-resistant strains harbored the gene encoding resistance to nitroimidazoles. BFG strains isolated from UWW and TWW samples were characterized by the highest diversity of antibiotic-resistance genes and were most often drug-resistant and multidrug-resistant. The present study examines the potential negative consequences of drug-resistant and multidrug-resistant BFG strains that are evacuated with treated wastewater into the environment. The transmission of these bacteria to surface water bodies can pose potential health threats for humans and animals; therefore, the quality of treated wastewater should be strictly monitored.

Antimicrobial susceptibility of common pathogens isolated from postoperative intra-abdominal infections in Japan.

The principle of empirical therapy for patients with intra-abdominal infections (IAI) should include antibiotics with activity against Enterobacteriaceae and Bacteroides fragilis group species. Coverage of Pseudomonas aeruginosa, Enterobacter cloacae, and Enterococcus faecalis is also recommended for hospital-associated IAI. A nationwide survey was conducted to investigate the antimicrobial susceptibility of pathogens isolated from postoperative IAI. All 504 isolates were collected at 26 institutions and referred to a central laboratory for susceptibility testing. Lower susceptibility rates to ciprofloxacin and cefepime were demonstrated in Escherichia coli. Among E. coli, 24.1% of strains produced extended-spectrum ß-lactamase (ESBL). Carbapenems, piperacillin/tazobactam, cephamycins/oxacephem, aminoglycosides, and tigecycline had high activity against E. coli, including ESBL-producing isolates. Among E. cloacae, low susceptibility rates to ceftazidime were demonstrated, whereas cefepime retained its activity. P. aeruginosa revealed high susceptibility rates to all antimicrobials tested except for imipenem. Among B. fragilis group species, low levels of susceptibility were observed for cefoxitin, moxifloxacin, and clindamycin, and high susceptibility rates were observed for piperacillin/tazobactam, meropenem, and metronidazole. Ampicillin, piperacillin, and glycopeptides had good activity against E. faecalis. Imipenem had the highest activity against E. faecalis among carbapenems. In conclusion, we suggested the empirical use of antimicrobials with the specific intent of covering the main organisms isolated from postoperative IAI. Piperacillin/tazobactam, meropenem, or doripenem, are appropriate in critically ill patients. Combination therapy of cefepime (aztreonam in patients with ß-lactam allergy) plus metronidazole plus glycopeptides, imipenem/cilastatin or cephamycins/oxacephem plus ciprofloxacin plus metronidazole are potential therapeutic options.

MALDI-TOF MS versus 16S rRNA sequencing: Minor discrepancy between tools in identification of Bacteroides isolates.

Members of the genus Bacteroides are important components of the normal microbiota of gastrointestinal tract; however, as opportunistic pathogens are also associated with severe or even life-threatening infections with significant mortality. Various species within Bacteroides fragilis group are phenotypically very similar; thus, their identifications with traditional-automated biochemical methods are frequently inaccurate. The identification of the newly discovered or reclassified bacteria can be doubtful because of the lack of biochemical profile in the database of these tests. The aim of this study was to determine the accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method by testing of 400 Hungarian Bacteroides clinical isolates. Inaccurate identification results with MALDI-TOF MS were confirmed by 16S rRNA gene sequencing and findings were compared with traditional-automated biochemical test rapid ID 32A method as well.

Identification and Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Rubik's Cube of Clinical Microbiology?

Anaerobic bacteria have pivotal roles in the microbiota of humans and they are significant infectious agents involved in many pathological processes, both in immunocompetent and immunocompromised individuals. Their isolation, cultivation and correct identification differs significantly from the workup of aerobic species, although the use of new technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, whole genome sequencing) changed anaerobic diagnostics dramatically. In the past, antimicrobial susceptibility of these microorganisms showed predictable patterns and empirical therapy could be safely administered but recently a steady and clear increase in the resistance for several important drugs (ß-lactams, clindamycin) has been observed worldwide. For this reason, antimicrobial susceptibility testing of anaerobic isolates for surveillance purposes or otherwise is of paramount importance but the availability of these testing methods is usually limited. In this present review, our aim was to give an overview of the methods currently available for the identification (using phenotypic characteristics, biochemical testing, gas-liquid chromatography, MALDI-TOF MS and WGS) and antimicrobial susceptibility testing (agar dilution, broth microdilution, disk diffusion, gradient tests, automated systems, phenotypic and molecular resistance detection techniques) of anaerobes, when should these methods be used and what are the recent developments in resistance patterns of anaerobic bacteria.

Prevalence of antimicrobial resistance and the cfiA resistance gene in Danish Bacteroides fragilis group isolates since 1973.

OBJECTIVES: The purpose of this study was to determine the prevalence of resistance and the cfiA carbapenemase-producing gene in historical Bacteroides fragilis group isolates. METHODS: Danish clinical B. fragilis group isolates (n = 444) from 1973 to 2015 were identified with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) on the Biotyper platform. Antimicrobial resistance was determined using a disk diffusion screening method and commercial antibiotic gradient strips. Division I (cfiA-negative) and division II (cfiA-positive) B. fragilis strains were differentiated using MALDI-TOF MS and real-time polymerase chain reaction (PCR). RESULTS: From 1973-1980 to 2010-2015 the prevalence of antimicrobial resistance rose from 0% to 21.2%, 2.5%, and 1% for clindamycin, meropenem, and metronidazole, respectively. MALDI-TOF MS and real-time PCR identified 16 of 266 (6.0%) B. fragilis strains as division II, of which 4 strains, isolated between 2010 and 2015, were resistant to meropenem. CONCLUSIONS: Substantial increases in resistance were found throughout this study. This supports the general perception that antimicrobial resistance in the B. fragilis group has been established in the recent decades in Europe. Resistance to meropenem, facilitated by expression of the cfiA resistance gene, seems to be increasing; therefore, it is imperative to monitor the occurrence of this gene, e.g. using MALDI-TOF MS.

Antimicrobial resistance in the Bacteroides fragilis group in faecal microbiota from healthy Danish children.

The Bacteroides fragilis group constitute a significant portion of the human gut microbiota and comprise a major proportion of anaerobic bacteria isolated in human infections. We established a baseline of antimicrobial susceptibility rates in the B. fragilis group in the intestinal tract of relatively antibiotic-naive healthy Danish children. From 174 faecal samples collected from children attending day care, 359 non-duplicate isolates were screened for antimicrobial susceptibility. Of these, 0.0%, 1.9%, 5.0% and 21.2% of isolates were intermediate-susceptible or resistant to metronidazole, meropenem, piperacillin/tazobactam and clindamycin, respectively. Eighteen additional studies reporting susceptibility rates in the B. fragilis group bacteria were identified by conducting a literature search. Heterogeneity among results from studies of B. fragilis group antimicrobial susceptibility rates in faecal microbiota exists.

Antimicrobial resistance in the Bacteroides fragilis group in faecal samples from patients receiving broad-spectrum antibiotics.

Members of the Bacteroides fragilis group are opportunistic pathogens and cause severe infections including bacteraemia. As increased levels of antimicrobial resistance in B. fragilis group bacteria can be detected years after administration of specific antibiotics, monitoring antimicrobial susceptibility in the gut microbiota could be important. The objectives of this study were to 1) investigate the distribution of species and the occurrence of reduced antimicrobial susceptibility in the B. fragilis group from patients treated at departments with a high level of antibiotic use, 2) to determine the prevalence of the carbapenem resistance gene cfiA in B. fragilis in this patient group, and 3) to determine the association between previous antibiotic treatment and reduced susceptibility to clindamycin, meropenem, metronidazole, and piperacillin-tazobactam. Consecutive faecal samples (n = 197) were collected from patients at the departments of haematology, oncology, and infectious diseases at Odense University Hospital, Denmark. Three colonies from each sample were identified by Matrix Assisted Lazer Desorption Ionization Time of Flight Mass Spectrometry and isolates were screened for resistance to clindamycin, meropenem, metronidazole, and piperacillin-tazobactam. B. fragilis isolates were tested for the cfiA metallo-beta-lactamase gene. Fisher's Exact test was used to test for correlation between antimicrobial exposure and reduced susceptibility. A total of 359 isolates were tested for reduced susceptibility. Of these 28%, 5%, <1%, and 11% were intermediate susceptible or resistant to clindamycin, meropenem, metronidazole, and piperacillin-tazobactam respectively. Three metronidazole resistant Bacteroides spp. were isolated. The proportion of B. fragilis belonging to division II (cfiA+) was 5.3%. Previous exposure to meropenem was associated with reduced susceptibility to meropenem (p= 0.001). In conclusion, antimicrobial resistance is prevalent and the distribution of species appears to be affected in the B. fragilis group from patients receiving broad-spectrum antibiotics, with meropenem exposure being associated with meropenem resistance.