Susceptibility to antimicrobial drugs of enterotoxin producing strains of Bacteroides fragilis isolated from different countries

Twenty two Bacteroides fragilis strains isolated from clinical samples in different countries (England, France, the Netherlands, Poland and USA) were used in the experiments. In all strains the presence of enterotoxin (fragilysin) gene was found by PCR with primers 404/407. Drug susceptibility of B. fragilis strains was determined with Etest (MICs for penicillin G, ceftriaxone, amoxicillin/clavulanic acid, imipenem, clindamycin and metronidazole). MICs were estimated in accordance to the NCCLS recommendations (1997). All tested strains were susceptible to imipenem and metronidazole. Twenty one strains were susceptible and one was intermediate susceptible to amoxicillin/clavulanic acid. Fourteen strains were resistant to ceftriaxone and five were found highly resistant to clindamycin. All examined strains were resistant to penicillin G. Four tested strains were simultaneously resistant to penicillin G, ceftriaxone and clindamycin (three French human strains isolated from postoperative wound, peritoneal fluid and bone inflammation, and one strain isolated from a pig).     

Bacterioides fragilis: production and sensitivity to bacteriocins

Bacteriocin production and sensitivity to bacteriocins have been successfully applied as an epidemiological tool in several species of bacteria. However, little work has been carried out on the bacteriocins produced by Bacteroides fragilis, which is the most frequently isolated anaerobe species from clinical specimens. Thirty two clinical isolates of B. fragilis grown anaerobically on a 0.22 microm membrane filter spotted on an agar plate, were tested for bacteriocin production and used in a screen for bacteriocin sensitivity. Sensitivity to at least one bacteriocin was found in 94% of the strains, 62.5% were sensitive to two bacteriocins, whereas 34.4% were sensitive to three or more and finally one strain was found sensitive to 17 bacteriocins. Of the strains studied, 94% inhibited at least one strain, 66% inhibited two strains, and 30% inhibited at least three strains or more. Finally, one strain was extremely active by inhibiting the growth of 17 strains. Bacteriocin types are characterised by geographic variation, and their epidemiological investigation by a simple method could be promoted.     

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.     

ESBL-positive strains of the Bacteroides fragilis group isolated from patients at the regional hospital center in Płock (Poland)

The aim of this study was to confirm a presumptive qualification of clinical B. fragilis group strains isolated in P?ock as ESBL-positive strains and to determine some properties of these strains. Twenty four clinical strains belonging to the B. fragilis group, isolated first of all from surgical patients, were received for testing. Identification of strains was performed in the automatic ATB Expression system (bioMerieux sa, France) using biochemical API 20 A strips. Strains were tested for the production of catalase (ID Color Catalase test, bioMerieux sa) and beta-lactamase (Cefinase, BBL, Becton Dickinson, USA). Susceptibility of strains to four antimicrobial agents: clindamycin, metronidazole, amoxicillin/clavulanic acid and imipenem was determined by Etest (AB Biodisk, Sweden). ESBLs were detected with the use of two disc diffusion methods: the double-disc synergy test (DDST) according to Jarlier et al. and the diagnostic disc (DD) test according to Appleton. Seventeen of examined strains belonged to the species Bacteroides fragilis, three--to B. ovatus/thetaiotaomicron, two--to B. distasonis, one--to B. uniformis and one--to B. stercoris/eggerthii. One strain (B. uniformis) did not produce catalase, whereas all strains produced beta-lactamases. Examined strains were susceptible in vitro to metronidazole, amoxicillin/clavulanic acid and imipenem. One clindamycin-resistant strain was detected (B. fragilis). Occurrence of ESBL-type enzymes was confirmed in 22 strains of following species: B. fragilis (17 strains), B. ovatus/thetaiotaomicron (3), B. distasonis (1) and B. uniformis (1). Clinical strains of the B. fragilis group with a new mechanism of resistance to beta-lactam antibiotics appeared during last years in Poland. They produce extended-spectrum beta-lactamases (ESBLs), so they are resistant to penicillins, cephalosporins and monobactams. Monitoring of infections caused by these threatening strains in hospital patients is very important.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

A comparative study of three bacteriocins of Bacteroides fragilis

Three different bacteriocins produced by strains of Bacteroides fragilis were compared in terms of their production kinetics, physico-chemical nature, and action on macromolecular synthesis in a common indicator strain. Bacteriocin 78/438 was produced during the logarithmic growth phase, was thermolabile and stable between pH 5 and 9. It was susceptible to trypsin and pepsin, and affected DNA, RNA and protein syntheses in susceptible cells. Bacteriocin A49 was produced during the stationary growth phase, was thermolabile and stable between pH 7 and 9. This bacteriocin was also susceptible to trypsin and pepsin, but only RNA synthesis was affected in the indicator strain. Bacteriocin A55 differed markedly from both 78/438 and A49, and was found to be predominantly cell-bound, resistant to inactivation by high temperatures and stable over a wide pH range of 2 to 12. It was susceptible to trypsin but resistant to pepsin. A55 had a delayed effect on macromolecular synthesis with DNA synthesis being inhibited after 60 min. With all three bacteriocins, killing of the indicator strain followed single hit kinetics with the interaction of bacteriocin and target cell occurring in two stages. Killing by bacteriocin A55 was much slower than the other two and this may be related to its effect on macromolecular synthesis. The killing action of all three bacteriocins was dependent on the growth phase of the susceptible cells.     

Bacteroides fragilis Group: Trends in Resistance

Representing the major part of the human colon microflora, members of the Bacteroides fragilis group are frequently involved in mixed aerobic and anaerobic infections. Recent studies show an increased resistance of the B. fragilis group against several antimicrobial agents. The aim of the present study was to determine the susceptibility of 87 B. fragilis group strains isolated in 2003/2004 in Western Austria against eight antimicrobial agents by Etest. Furthermore, the resistance patterns were compared with those of 45 B. fragilis group strains isolated in 1992 and referred to the world wide trend towards increased resistance. In 1992 as well as in 2003/2004, all strains were susceptible against metronidazole and imipenem. However, comparing the MIC-values of the B. fragilis group strains collected 1992 with data from 2003/2004, a significant increase in resistance was found for clindamycin (p<0.01). Regarding cefoxitin, a similar trend could be observed. However, this difference was not yet significant (p=0.144). Our findings underline the emerging resistance of the B. fragilis group against antimicrobial agents and underscore the importance of susceptibility testing of anaerobes even in routine laboratories.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G.

A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M. S. Strom, M. W. Eklund, and F. T. Poysky, Appl. Environ. Microbiol. 48:956-963, 1984). In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid. The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains. In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin. This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C. botulinum is mediated by a plasmid.     

Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.     

Analysis of the genetic region encoding a novel rhizobiocin from Rhizobium leguminosarum bv. viciae strain 306

Cross-testing of a number of strains of Rhizobium leguminosarum for bacteriocin production revealed that strain 306 produced at least two distinct bacteriocins. Further analysis involving plasmid transfer to Agrobacterium and other hosts demonstrated that there were bacteriocin determinants on plasmids pRle306b and pRle306c, as well as a third bacteriocin. The bacteriocin encoded by pRle306b was indistinguishable from the bacteriocin encoded by strain 248, whereas the bacteriocin encoded by plasmid pRle306c had a distinctive spectrum of activity against susceptible strains, as well as different physical properties from other bacteriocins that we have studied in our lab. Two mutants altered in production of the pRle306c bacteriocin were generated by transposon Tn5 mutagenesis, and the DNA flanking the transposon inserts in these mutants was cloned and characterized. DNA sequence analysis suggested that the pRle306c bacteriocin was a large protein belonging to the RTX family, and that a type I secretion system involving an ABC type transporter was required for export of the bacteriocin. A mutant unable to produce this bacteriocin was unaltered in its competitive properties, both in broth and in nodulation assays, suggesting that the bacteriocin may not play a major role in determining the ecological success of this strain.     

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL105

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis , one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.     

Plasmid-mediated bacteriocin production by Shigella flexneri isolated from dysenteric diarrhoea and their transformation into Escherichia coli

AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. Significance and IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.     

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.     

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.     

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators. pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS 257 was not involved in the recombination events which gave rise to pRJ29.     

Identification of Bacteroides fragilisby a modified immuno polymerase chain reaction (mIPCR), using a specific monoclonal antibody

Bacteroides fragilis is the anaerobic species most commonly isolated from human clinical specimens, and is resistant to many antimicrobial agents. A monoclonal antibody, mAb4H8 (IgG3), reacting with a specific epitope in the lipopolysaccharide (LPS) isolated from most of the B. fragilis strains, was produced and employed with modified Immuno Polymerase Chain Reaction (mIPCR) for identification of B. fragilis with a detection limit of 10(4) cfu/mL bacterial suspension. A number of bacterial strains were examined, including B. fragilis, Bacteroides spp. other than B. fragilis and other genera. All the B. fragilis strains with the immunodominant (beta1,6-linked D-galactosyl chain) epitope were positive. None of the other strains showed the positive reaction. The results indicate that mIPCR assay with mAb4H8 has a high specificity and high sensitivity.     

Isolation and identification of strains of Bacteroides fragilis group from the digestive tract of Callithrix penicillata marmosets

Thirty-five strains of the Bacteroides fragilis group were isolated from oral and intestinal samples from 5 wild caught, captive Callithrix penicillata. Nine oral strains of Bacteroides fragilis (7) and Bacteroides distasonis (2), and 26 intestinal strains of Bacteroides fragilis (14) and Bacteroides distasonis (12) were identified.     

Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate

The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.