Strains Of Bacteroides Fragilis Group Research Articles

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

ObjectivesThis study screened the prevalence of rare β-lactamase genes in Bacteroides fragilis group strains from clinical specimens and normal microbiota and examined the genetic properties of the strains carrying these genes. MethodsblaHGD1, 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. ResultsAll 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. ConclusionsBased on these observations, β-lactamases produced by rare β-lactamase genes in B. fragilis group strains should not be overlooked because they could encode important resistance phenotypes.

First Report of Antibiotic Resistance Markers cfiA and nim Among Bacteroides fragilis Group Strains in Ecuadorian Patients.

In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.

An improved selective/differential medium for culturing the Bacteroides fragilis group from wastewater.

The aim of this study was to develop an effective selective/differential medium for culturing environmental strains of the Bacteroides fragilis group (BFG). This goal was achieved by modifying standard commercial Bacteroides Bile Esculin Agar (BBE Agar). Bacteroides Bile Esculin Agar was combined with substances that inhibit the growth of non-BFG bacteria, mostly Klebsiella pneumoniae and Fusobacterium mortiferum. The strains isolated from standard and modified BBE Agar were identified as BFG strains by PCR and 16S rRNA gene sequencing. The supplementation of standard BBE Agar with colistin (40 mg L-1), kanamycin (400 mg L-1) and vancomycin (7.5 mg L-1) increases the effectiveness of BFG bacteria isolation from <10% to 35%, and additional Gram staining improves the effectiveness of bacterial isolation five-fold relative to standard BBE Agar. The results of the present study also suggest that the presence of the bfr gene is not a reliable indicator for the identification of BFG strains.

Comparison of Phenotypic and Genotypic Patterns of Antimicrobial-Resistant Bacteroides fragilis Group Isolated from Healthy Individuals in Vietnam and Japan.

PurposeNormal non-pathogenic flora can harm the host by acting as a reservoir of resistance determinants that are potentially transferable to human pathogens. This study aimed to assess the phenotypic and genotypic antimicrobial susceptibility patterns of the Bacteroides fragilis group (BFG) isolated from healthy individuals in Vietnam and Japan in order to elucidate the prevalence of antimicrobial resistance in human flora in the two economically and geographically different countries.Materials and MethodsBFG was isolated from fecal samples of 80 healthy individuals in Vietnam (n=51) and Japan (n=29). Isolated strains were identified using MALDI-TOF MS, and the minimum inhibitory concentration (MIC) of 18 antibiotics was determined using the agar dilution method. Additionally, 20 antimicrobial resistance genes were detected using standard PCR.ResultsA total of 139 BFG strains belonging to 11 BFG species were isolated from the two countries, with diversity in the prevalence of each species. B. fragilis was not the predominant species. Isolations from Vietnam and Japan showed some similarities in terms of MIC50 values, MIC90 values, and the percentage of resistant strains. However, isolations from Vietnam showed significantly higher resistance to piperacillin, cefmetazole, clindamycin, tetracycline, and minocycline. ErmB, tet36, tetM, nim, catA, and qnrA were not found in either country. CepA was more common in B. fragilis than in non-fragilis Bacteroides. In contrast, cfiA, ermG, mefA, msrSA, tetX, tetX1, bexA, qnrB, and qnrS were found only in non-fragilis Bacteroides. There were differences in the prevalence of ermG, linA, mefA, msrSA, and qnrS between isolates from Vietnam and Japan.ConclusionThis study is the first report on the antimicrobial susceptibility patterns in the BFG isolated from healthy individuals in Vietnam and Japan. Compared to isolations from Japan, isolations from Vietnam showed significantly higher resistance to antimicrobial agents. The distribution of various antibiotic resistance genes also differed between the two countries.

Bacteroides fragilis Grubu İzolatların Klindamisin, Tetrasiklin ve Tigesikline Duyarlılıkları ve Dirençten Sorumlu tet ve ermF Genlerini Barındırma Durumları

Objective: We aimed to determine the resistance of Bacteroides fragilis group (BFG) bacteria to clindamycin, tetracycline and tigecycline and establish the distribution of related resistance genes. Method: In total 82 BFG strains, isolated from different clinical samples between January 2017 and December 2018, were identified by MALDI-TOF MS. Their antimicrobial sensitivities to were determined using agar dilution methodology (CLSI; M11-A7). The tetM, tetQ, tetX, tetX1, tet36, and ermF genes were investigated by PCR. Results: Eighty-two strains of BFG bacteria, isolated from intra-abdominal abscess (n=36), tissue biopsy (n=16), blood (n=14) and other sterile body fluids (n=12), were identified as Bacteroides fragilis (n=48), Bacteroides thetaiotaomicron (n=17), Bacteroides vulgatus (n=5), Bacteroides ovatus (n=4), Bacteroides caccae (n=1), Bacteroides uniformis (n=1) and Parabacteroides distasonis (n=6). The resistance rates to clindamycin, tetracycline and tigecycline were 54.9%, 84.1%, 4.9%, respectively. Non-B. fragilis isolates were more resistant than B. fragilis strains. In total, 57.3% of the isolates were ermF gene positive, while B. thetaiotaomicron had the highest rate (70.6%). The tet gene positivity ranged from 18.8% to 66.7% among species. The tetQ gene positivity was higher than other tet genes. The 92.7% of the isolates were resistant to at least one antibiotic, while 94% had at least one resistance gene. Conclusion: This study provided data on antimicrobial resistance of our BFG isolates to clindamycin, tetracycline and tigecycline and the related resistance genes. However, our information obtained could also be a starting point for further investigation of the antibiotic resistance mechanisms of Bacteroides species, as well as, resistance transfer among BFG isolates and other bacteria.

Agarda Dilüsyon ve Deneme Aşamasındaki EUCAST Disk Difüzyon Yöntemi ile Saptanan Bacteroides fragilis Grubu İzolatlarına Ait Antimikrobiyal Duyarlılık Sonuçlarının Karşılaştırılması

Objective: In this study it was aimed to determine the antimicrobial susceptibility of Bacteroides fragilis group (BFG) bacteria using recently developed European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion method and agar dilution method recommended by Clinical Laboratory Standart Institude (CLSI) for anaerobes and to investigate the agreement of the results of two tests. Method: The antimicrobial susceptibilities of a total of 56 BFG strains isolated from clinical samples and identified by MALDI-TOF MS analysis between January 2017 and December 2018, were tested to ampicillin, ampicillin/sulbactam, cefoxitin, imipenem, clindamycin, tigecycline, moxifloxacin and metronidazole MICs were determined by agar dilution method using sheep blood supplemented Brucella agar and disk diffusion test using host blood supplemented Fastidius Anaerobic Agar (FAA). Results: Six different BFG species consisting mostly strains of Bacteroides fragilis (n=34, 61%) and Bacteroides thetaiotaomicron (n=11, 20%) isolated from various clinical samples such as intraabdominal abscess (n= 24), blood (n=10) and tissue biopsy samples (n=11).were identified. Imipenem and metronidazole were the most effective antimicrobials with 98.2% susceptibility rates, followed by tigecycline, ampicillin/sulbactam, moxifloxacin and clindamycin with susceptibility rates of 89.3%, 66.1%, 57.1% and 46.4%, respectively. Most concordant results were obtained with metronidazole (100%), imipenem (89.8%) and tigecycline (89.8%). Acceptable compliance rates were not found for other antimicrobials. Conclusion: We can say that disc diffusion method is a fast, easy-to-apply, and reliable method used in clinical microbiology laboratories to determine the susceptibility of BFG bacteria to metronidazole, imipenem and tigecycline. However, to evolve a standard method especially for other antimicrobials, the experimental conditions should be optimized with studies dome with greater number of isolates.

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 β-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.

Isolation of anaerobic bacteria of the Bacteroides fragilis group from environmental samples

The aim of this study was to analyze the effectiveness of identification of Bacteroides fragilis group (BFG) strains isolated from human and rat feces, hospital wastewater, and untreated and treated sewage from a wastewater treatment plant (WWTP). BFG strains were plated on Bacteroides Bile Esculin (BBE) agar. Characteristic colonies were isolated from the culture medium, tested for antibiotic resistance and identified by PCR and MALDI-TOF MS. A total of 319 strains that formed characteristic colonies were isolated from BBE agar, of which 250 were resistant to kanamycin, colistin and vancomycin. PCR revealed that only 135 strains harbored the bfr gene specific to the BFG. In MALDI-TOF MS analysis, only 123 isolates were classified as members of the BFG. The most frequently isolated species was Parabacteroides distasonis (51.22% of strains). B. fragilis, B. ovatus and B. thetaiotaomicron were less frequently encountered in the analyzed environmental samples (30.01%, 8.13% and 5.69%, respectively). The strains isolated from human and rat feces on BBE agar were most reliably identified, and 100% of the isolated strains were classified as members of the BFG. The effectiveness of isolation of BFG strains from hospital wastewater and untreated and treated sewage from the WWTP was relatively low (20%, 19%, 40%, respectively). The results of this study suggest that the method for isolating BFG strains from environmental samples on BBE agar requires modification. Additional methods, such as PCR and MALDI-TOF MS, can be used to more effectively identify BFG strains isolated from different environmental samples.

Lactate dehydrogenase activity in Bacteroides fragilis group strains with induced resistance to metronidazole.

The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8mg/L to 96mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1U/mg and 222.0U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity.

Analysis of Romanian Bacteroides isolates for antibiotic resistance levels and the corresponding antibiotic resistance genes

As part of an ESCMID Study Group on Anaerobic Infections (ESGAI) project, a study was conducted to measure the antibiotic susceptibilities and corresponding gene contents of 53 Bacteroides fragilis group strains isolated in Romania. The antibiotic resistance data was comparable with the data found for other East-European countries. Here, no resistant isolate was found for imipenem, metronidazole and tigecycline. An increasing role of the cepA, cfxA and cfiA genes was observed in their corresponding antibiotic resistances. Moreover, no isolate was found that harbored the cfiA gene with a possible activating IS element. Clindamycin resistance was low, similarly to that the rate for the ermF gene. However, we did find some isolates with nimB, ermB, msrSA, linA, satG, tetX, tetM and bexA genes. This study was the first to provide antibiotic resistance data for clinical Bacteroides strains from Romania.

The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries

From the 2008–2009 European Bacteroides antibiotic resistance survey, we selected 161 strains for detection of antibiotic resistance genes (cepA, cfxA, cfiA, nim, ermB, ermF, ermG, linA, mefA, msrSA, tetM, tetQ, tetX, tetX1, tet36 and bexA). To facilitate the throughput, the genes were detected by Real-Time PCR. The presence of the genes was correlated with the known MIC data of the strains for the appropriate antibiotics. For the β-lactams, the cepA gene was found in 70.8% of the tested strains (all resistant to ampicillin), but its presence did not correlate with the ampicillin MIC values. The cepA gene occurred at different frequencies among Bacteroides fragilis and non-fragilis Bacteroides strains. The cfxA gene was not a major factor in determining cefoxitin resistance and it was found with higher prevalence in non-fragilis Bacteroides strains than in B. fragilis. Among the five possible clindamycin resistance genes, ermF was the most common and had the highest effect on clindamycin resistance after linA. The ermG-mefA-msrSA combination was found in a set of strains and their linked occurrence implied that they were harbored by the conjugative transposon CTnGERM1. All strains tested were susceptible to metronidazole and none of them harbored nim genes. TetQ was prevalent among both the B. fragilis and non-fragilis Bacteroides strains (78.9 and 84.8%, respectively) and no gene could be clearly linked to tigecycline resistance other than tetQ. BexA, which codes for the fluoroquinolone efflux pump, was found in 7.5% of strains and occurred at different frequencies among B. fragilis and non-fragilis Bacteroides strains, but was represented only in a minor proportion of moxifloxacin-resistant strains.

Isolation, identification and antimicrobial susceptibility of Bacteroides fragilis group strains recovered from broiler faeces

1. The objective was to evaluate the occurrence of cultivable components of the Bacteroides fragilis group in faeces of broiler chickens and their antimicrobial susceptibility patterns. 2. Faecal samples of 36 × 45-d-old Cobb broilers of both sexes from 15 different flocks on one farm were diluted 10-fold and plated on to Bacteroides-bile-esculin agar for colony count and isolation. Identification was by molecular methods and antimicrobial susceptibility in the agar dilution assay. 3. A total of 236 isolates was recovered from a mean population of 3·32 × 107 colony-forming units/g of faeces. B. fragilis was shown to be the predominant Bacteroides species (45·3%), followed by B. distasonis (35·6%), B. vulgatus (8·9%), B. ovatus (2·5%) and B. stercoris (1·3%). 4. Among 204 bacterial isolates tested, high resistance to ampicillin (98·5%), norfloxacin (95·1%) and tetracycline (88·2%) were observed. High (89·7%) multi-drug resistance was observed to 3–7 of the tested drugs. 5. Components of the B. fragilis group were sub-dominant in broiler faecal microbiota, with a different species pattern compared with human and high antimicrobial multi-drug resistance.

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs < 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC > 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of Bacteroides fragilis group in diarrheic and non-diarrheic feces from Brazilian infants

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0–5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

In Vitro susceptibility to selected antibiotics in bacteria of the Bacteroides fragilis group

Susceptibility of strains of Bacteroides fragilis group (1284 isolates from cancerous and noncancerous patients in 1994-2004) showed an increase in resistance toward some antibiotics (by 9 % toward penicillin and 8 % toward clindamycin) compared with the resistance level of 10 years ago. The increase in resistance was not detected in the case of ampicillin + sulbactam and metronidazole.

Antimicrobial susceptibility of anaerobic bacteria in Belgium as determined by E-test methodology

The objective was to collect recent data on the antibiotic susceptibility of clinically significant anaerobes in Belgium. A total of 333 anaerobic clinical isolates from various body sites were prospectively collected between 2005 and 2007 at two tertiary care hospitals in Belgium. The minimal inhibitory concentrations (MICs) were determined using the E-test method for nine anti-anaerobic antibiotics. Sixty-one percent of the isolates were beta-lactamase producers, which explains the poor activity of penicillin. Amoxicillin/clavulanic acid, piperacillin/tazobactam, metronidazole and meropenem were very active against most anaerobes, but around 10% of the Bacteroides fragilis group strains were non-susceptible to the two beta-lactam/beta-lactamase inhibitors. No resistance was observed to metronidazole, while 3% of the Bacteroides spp. had decreased susceptibility to meropenem (MIC > or = 4 mg/L). Cefoxitin, clindamycin and moxifloxacin were less active, with 33%, 52% and 57% of the B. fragilis group being non-susceptible respectively. Tigecycline showed consistently good activity against most anaerobes with MIC(50) and MIC(90) of 0.25 and 2 mg/L. Metronidazole, amoxicillin/clavulanate, piperacillin/tazobactam and meropenem remain good empirical choices when anaerobes are expected in our setting. Because of the occurrence of resistance to most classes of current anti-anaerobic antibiotics, it is recommended that the antimicrobial resistance patterns be monitored regularly in order to guide empirical therapy.

Third Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria

To collect recent data on the susceptibility of anaerobes and to compare them with results from previous studies. Four hundred and forty-three anaerobic clinical isolates from various body sites were prospectively collected from October 2003 to February 2005 in nine Belgian hospitals. MICs were determined for nine anti-anaerobic and three recently developed antibiotics. Most gram-negative bacilli except Fusobacterium spp. were resistant to penicillin. Piperacillin/tazobactam, metronidazole, chloramphenicol, meropenem and amoxicillin/clavulanic acid were very active against all groups, but only 86% of Bacteroides fragilis group strains were susceptible to the latter. Cefoxitin, cefotetan and clindamycin were less active. In particular, only 62%, 52% and 48% of B. fragilis group strains were susceptible, respectively. Clindamycin shows a continuing decrease in activity, as 83% were still susceptible in 1987 and 66% in 1993-94. Anti-anaerobic activity of the new antibiotics is interesting, with MIC50 and MIC90 of 1 and >32 mg/L for moxifloxacin, 2 and 4 mg/L for linezolid and 0.5 and 8 mg/L for tigecycline. The susceptibility of anaerobic bacteria remains stable in Belgium, except for clindamycin, which shows a continuous decrease in activity. However, for each of the tested antibiotics, at least a few resistant organisms were detected. Consequently, for severe infections involving anaerobic bacteria, it could be advisable to perform microbiological testing instead of relying on known susceptibility profiles. Periodically monitoring background susceptibility remains necessary to guide empirical therapy.

In vitro activity of Bulgarian propolis against 94 clinical isolates of anaerobic bacteria

The aim was to evaluate the effect of 30% ethanolic extract of Bulgarian propolis on 94 clinical anaerobic strains. The strains were tested by both agar-well diffusion (wells, 7 mm diameter) and disk-diffusion methods. Only 15% of Clostridium-, 3.3% of other Gram-positive- and 9.1% of Gram-negative anaerobic strains were not inhibited by 30 μL propolis extract per well. Propolis extract was more active than the ethanol ( P < 0.001 ). By 30 μL extract per well, mean inhibitory diameters of the clostridia, other Gram-positive- and Gram-negative anaerobes were 11.5, 13.1, and 11.3 mm, and those by 90 μL were 16, 18.1 and 15.4 mm, respectively. Mean inhibitory diameters of all strains by 30 and 90 μL ethanol were only 8.4 and 9.5 mm. By 30 μL propolis extract per well, inhibitory diameters of 15 mm or more were more common in Gram-positive (32%) than in Gram-negative bacteria (13.6%, P < 0.05 ). Moist propolis disks inhibited more strains (89.4%) than dried disks (68.1%, P < 0.001 ). Most (81.8%) Bacteroides fragilis group strains and 75% of clostridial strains were inhibited by moist EEP disks. Conclusion: Bulgarian propolis was active against most anaerobic strains of different genera. In addition to oral pathogens, an activity of propolis against Clostridium, Bacteroides and Propionibacterium species was observed. The results could motivate a higher medical interest and further trials for evaluating the use of bee glue for prophylaxis or treatment of some anaerobic infections such as oral, skin and wound diseases.

Inducible Metronidazole Resistance in nim–Positive and nim–Negative Bacteroides fragilis Group Strains after Several Passages Metronidazole Containing Columbia Agar Plates

Recent data show an emergence of resistance in the Bacteroides fragilis group against several antimicrobial agents and inducible resistance against metronidazole in nim-positive strains. The aim of the present study was to investigate inducible metronidazole resistance in nim-positive as well as in nim-negative B. fragilis group strains. Of 18 B. fragilis strains (including four nim-positive reference strains and one ATCC strain), two Bacteroides ovatus strains, and one Bacteroides thetaiotaomicron DSM strain minimum inhibitory concentration (MIC) values for metronidazole were determined by Etest and analyzed for nim genes (nimA to -G) by PCR. For this purpose bacterial suspensions were incubated on supplemented Columbia agar plates containing metronidazole at twice the MIC value of the specific strain and incubated under anaerobic conditions for 48 hours. After incubation, growing bacteria were harvested and thereafter incubated at four times the original MIC. This procedure was repeated with increasing antibiotic concentrations. The resulting MIC values were confirmed by Etest. The MIC values for metronidazole of the four nim-positive reference strains ranged from 3 to 8 mg/l. The B. fragilis ATCC 25285 strain and the B. thetaiotaomicron DSM 2255 strain were nim negative with MIC values of 0.19 mg/l and 0.75 mg/l, respectively. Three clinical isolates of B. fragilis strains showed MIC values of > 256 mg/l. In all three strains, nim genes were detected by PCR. The other clinical isolates were nim negative. In these strains, MIC values ranged from 0.19 to 0.75 mg/l. After several passages on metronidazole containing agar, all B. fragilis group strains exhibited MIC values of > 256 mg/l determined by Etest. Metronidazole resistance can be selected not only in nim-positive strains but also in nim-negative strains, suggesting that mechanisms other than nim genes are involved. These findings and the emerging resistance of the B. fragilis group against several antimicrobial agents underscore the importance of susceptibility testing of anaerobes even in routine laboratories.

Mechanism of quinolone resistance in anaerobic bacteria

Several recently developed quinolones have excellent activity against a broad range of aerobic and anaerobic bacteria and are thus potential drugs for the treatment of serious anaerobic and mixed infections. Resistance to quinolones is increasing worldwide, but is still relatively infrequent among anaerobes. Two main mechanisms, alteration of target enzymes (gyrase and topoisomerase IV) caused by chromosomal mutations in encoding genes, or reduced intracellular accumulation due to increased efflux of the drug, are associated with quinolone resistance. These mechanisms have also been found in anaerobic species. High-level resistance to the newer broad-spectrum quinolones often requires stepwise mutations in target genes. The increasing emergence of resistance among anaerobes may be a consequence of previous widespread use of quinolones, which may have enriched first-step mutants in the intestinal tract. Quinolone resistance in the Bacteroides fragilis group strains is strongly correlated with amino acid substitutions at positions 82 and 86 in GyrA (equivalent to positions 83 and 87 of Escherichia coli). Several studies have indicated that B. fragilis group strains possess efflux pump systems that actively expel quinolones, leading to resistance. DNA gyrase seems also to be the primary target for quinolones in Clostridium difficile, since amino acid substitutions in GyrA and GyrB have been detected in resistant strains. To what extent other mechanisms, such as mutational events in other target genes or alterations in outer-membrane proteins, contribute to resistance among anaerobes needs to be further investigated.