Departamento de Microbiologia Médica, Instituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CEP 21941-590, Rio de Janeiro, Brazil
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Abstract |
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Introduction |
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Materials and methods |
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A total of 78 strains of B. fragilis, including 44 clinical strains, 17 strains isolated from human intestinal microflora and 17 strains from polluted aquatic environments were analysed. The clinical specimens were collected in two periods (19811988 and 19911998) from patients at the University hospital (Federal University of Rio de Janeiro-HU-UFRJ) in Brazil. The strains from the 19811988 period were stored at 70°C in 20% skimmed milk (BBL Microbiology Systems, Cockeysville, MD, USA). Human intestinal B. fragilis strains were isolated from normal individuals who agreed to the use of their excrement, which was collected in PYG-bile,16 between 1991 and 1998. The environmental strains were collected from polluted aquatic environments at sewage plants, polluted rivers and an artesian well during the same period. Strains from polluted aquatic environments were isolated after sample collection (300 mL) in sterile flasks containing 0.05% sodium thioglycolate (Difco Laboratories, Detroit, MI, USA) and 0.15% cysteine hydrochloride (Sigma Chemical Co, St Louis, MO, USA), centrifuged at 6000g for 60 min and the sediment inoculated on PYG-bile with gentamicin (Schering-Plough, Rio de Janeiro, RJ, Brazil) 0.2 g/L (37°C/24 h) for B. fragilis enrichment. All specimens were plated on to Bacteroides bile aesculin agar-BBE.17 Suspect organisms were transferred to brain heart infusion broth (Difco) (pre-reduced anaerobically sterilized, BHI-PRAS)16 and were identified by established methodology.18 The strains were stored at 70°C in skimmed milk.
Antimicrobial susceptibility testing
Antimicrobial susceptibility tests against penicillin, cefoxitin, imipenem, meropenem, clindamycin, chloramphenicol and metronidazole were performed by Etest method (AB Biodisk, Dalvägen, Solna, Sweden) according to the manufacturer's instructions. The inoculum (108 cfu/mL) in Brucella broth (BBL) was applied with a sterile cotton swab on WilkinsChalgren (Difco) enriched with 5% defibrinated sheep blood.19 NCCLS breakpoints were used for susceptibility categorization.20 Nitrocefin discs (BBL) were inoculated with a small portion of growth from culture on blood agar plates (Oxoid Ltd, Basingstoke, UK), supplemented with haemin (Sigma) and menadione (Sigma) and any colour change from yellow to red in 15 to 30 min at 37°C observed. Reference strain ATCC 25285 of B. fragilis (American Type Culture Collection, Rockville, MD, USA) was included in both experiments to assess the reliability of the methods.
Statistical analysis
Fisher's exact test21 was used to compare the results obtained with the B. fragilis strains analysed. All significant differences were reported at the 95% CI (P < 0.05).
Polymerase chain reaction investigation of cfiA
For DNA extraction, cells from broth cultures (5 mL) in the logarithmic phase were harvested by centrifugation at 13000g for 1 min. The discarded and individual cell pellets was stored at 20°C without additional treatment until DNA isolation. Pure genomic DNA from all strains tested was obtained by a standard miniprep procedure22 to which a ribonuclease A (Sigma) treatment was added.23 The concentration of DNA in the samples was determined spectrophotometrically using GenQuant apparatus (Pharmacia Biotech, Björkgata, Uppsala, Sweden). For the cfiA investigation the primers used had the sequence of nucleotides 558 to 578 (5'-CCATGCTTTTCCCTGTCGCAG-3') and the complementary sequence of nucleotides 1266 to 1285 (5'-GGGCTATGGCTTTGAAGTGC-3')11 of the cfiA gene.8 These primers were obtained from Gibco BRL (Grand Island, NY, USA). Fifty nanograms of DNA template were used per reaction. The amplification was carried out in a 25 µL volume containing 80 pmol of each primer, 200 µM deoxynucleoside triphosphates mixture (Gibco BRL), PCR buffer (Gibco BRL) (200 mM TrisHCl (pH 8.4), 500 mM KCl), 50 mM MgCl2 (Gibco BRL), and 1.25 U Taq DNA polymerase (Gibco BRL). The PCR mixtures were overlaid with mineral oil (M 3516; Sigma). Temperature cycling was controlled in a model PTC-100 (Peltier-Effect Cycling, Watertown, MA, USA) and programmed as following: 30 cycles of 1 min at 92°C, 2 min at 62°C, 2 min at 72°C with a final extension time of 5 min at 72°C. The samples (10 µL) were analysed by electrophoresis in 1% agarose gels (Gibco, BRL) in Tris-acetate buffer (Gibco, BRL) (0.04 M Tris-acetate, 0.002 M EDTA, pH 8.5), stained with ethidium bromide (Sigma) and photographed on a UV light transilluminator using the Polaroid MP4 system (St Albans, UK). A molecular weight standard (1 kb DNA ladder, Gibco BRL) was included.
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Results |
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Discussion |
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Metronidazole had consistently good activity, ratifying the concept that a uniform sensitivity of B. fragilis to this antimicrobial agent is expected,28,29 although a few reports of resistant strains can be found in the literature.30,31 Chloramphenicol showed results as good as metronidazole, in spite of the fact that it was frequently used during the first period (19811988) to treat patients with anaerobic infections in the University hospital (personal communication, 1998). The resistance rate observed for cefoxitin in the last period (6.7%) can be attributed to the relatively low use of this specific agent in the HUUFRJ, although the MIC90 increased four-fold from the first to the second period. Lower rates (1.9%) for cefoxitin had been detected by our group in bacterial isolates from another different medical centre.29 Such data reinforce the possibility of variation in susceptibility profiles between institutions, and emphasize the importance of monitoring, especially in countries where the treatment of anaerobic infections is empirical. In this study, the resistance to penicillin and clindamycin among intestinal and environmental strains seems to be linked to the oral form of administration of these drugs. On the other hand, as cefoxitin is marketed only in intravenous form, its use is more restricted to the hospital environment. So, these results also seem to ratify the correlation between drug consumption and the emergence of bacterial drug resistance among colonic bacteria.
Bacteroides as well as Gram-positive bacteria are numerically predominant in the human colon and may act as important reservoirs for resistance genes, potentially transferable directly or indirectly to human pathogens.2 In addition, transfer of antibiotic resistance genes in the natural environment can occur between phylogenetically distant bacteria, in particular between Gram-positive and Gram-negative bacteria.32,33 Therefore, it would be essential to monitor the intensity of antibiotic-mediated selection not only in clinical strains but also in intestinal and environmental strains, which are, in turn, potential exogenous pathogens.34 Unfortunately, there has been little data on the natural frequency of antibiotic resistance genes in the normal anaerobic flora in Brazil.35 The same applies to environmental strains whose analysis the present study reports for the first time. The strains with reduced susceptibility to meropenem, found in this study, appear to produce the cfiA-type metallo-ß-lactamase. According to the literature, carbapenem resistance is related to this enzyme812 which requires two tightly bound Zn2+ ions for full activity.36 This metal ion active site seems to be responsible for both the decreased susceptibility to ß-lactamase inhibitors and the capacity to hydrolyse a broad range of ß-lactams. Also, such enzyme was detected even before the widespread use of carbapenems.37 As the cfiA gene has been detected in some clinical and environmental strains, and has also been found in transmissible determinants9 the spread of antimicrobial resistance represents a threat, mainly in regions where policies on antibiotic use are not very strict. Although the ß-lactamase activities or alterations of membrane permeability have not been investigated, the screening of isolates by PCR with the primers synthesized from cfiA sequence represents a practical methodology which allows an estimation of the potential for the development of carbapenem resistance among B. fragilis strains.
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Acknowledgments |
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Notes |
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References |
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Received 4 May 1999; accepted 20 September 1999