Resistance profile of Bacteroides fragilis isolated in Brazil. Do they shelter the cfiA gene?

Wilenes das Graças Silva e Souza, Katia Eliane Santos Avelar, Luis Caetano Martha Antunes, Leandro Araujo Lobo, Regina Maria Cavalcanti Pilotto Domingues and Maria Candida de Souza Ferreira*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The epidemiology of antimicrobial resistance of clinical isolates and human intestinal strains of Bacteroides fragilis has assumed great importance in the last few years since this microorganism, like other members of the B. fragilis group, can be responsible for the spread of resistance determinants. It is possible that the presence of B. fragilis in polluted aquatic environments might contribute to the spread of resistance. The antimicrobial resistance profile of 44 clinical B. fragilis strains isolated from 1981–1988 and 1991–1998 from the University hospital of Rio de Janeiro, and of 17 faecal and 17 polluted aquatic environmental B. fragilis strains isolated between 1991 and 1998 was determined. The susceptibility tests against penicillin, cefoxitin, imipenem, meropenem, clindamycin, chloramphenicol and metronidazole were performed by Etest in Wilkins-Chalgren agar enriched with 5% sheep blood. Motivated by some high MIC values for cefoxitin and meropenem, the cfiA gene, which codes for a metallo-ß-lactamase, was investigated among all strains, using PCR amplification. The resistance to penicillin was high in the samples from 1981 to 1988 (92.9%) and also in those from 1991 to 1998 (100%), although the MIC90 decreased from 256 mg/L to 24 mg/L. An increase in the resistance level to clindamycin and cefoxitin was seen from one decade to the other, the MIC90 values changing from 4 mg/L to 12 mg/L and from 8 mg/L to 32 mg/L, respectively. The susceptibility profile for metronidazole, chloramphenicol, imipenem and meropenem remained stable, although two clinical strains showed MICs of 6 mg/L and 8 mg/L against meropenem. Almost all human intestinal strains were resistant to penicillin and all of them were susceptible to imipenem, meropenem, chloramphenicol and metronidazole. The MICs of meropenem against two strains isolated from a polluted aquatic environment were 6 mg/L and 32 mg/L. The cfiA gene was detected in five strains, two of which were isolated from clinical specimens against which the MIC values of cefoxitin were high and three from an aquatic environment, whose susceptibility to both cefoxitin and meropenem ranged from sensitive to resistant.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies on the susceptibility of anaerobic bacteria have been performed in many countries with the aim of monitoring the development of resistance in a specific organism. Bacteroides fragilis represents one of the most important anaerobic pathogens in humans for its frequent involvement in serious infections1 and also for its ability to acquire and to transfer antimicrobial resistance genes.2 The detection of antimicrobial resistance in normal human intestinal strains is important as such strains are potential endogenous pathogens. An increase in the level of antibiotic resistance among strains isolated from polluted aquatic environments, particularly in developing countries, might indicate an overuse or misuse of antimicrobial agents or other non-antibiotic agents. Bacteroides spp., mainly the B. fragilis group, have been recognized as the anaerobic bacteria most resistant to antimicrobial agents, particularly to ß-lactams, since the 1970s.3 The majority of clinically isolated B. fragilis are ß-lactamase producers. Most of the enzymes are chromosomally mediated cephalosporinases, with activities against many narrow- and broad-spectrum penicillins and cephalosporins. These enzymes are inhibited by ß-lactamase inhibitors and were assigned to group 2e and molecular class A,4 being encoded by chromosomal5 or rarely plasmid6 cepA gene. So, the preferred ß-lactams for treatment of B. fragilis-associated infections include cefoxitin, ß-lactams/ß-lactamase inhibitor combinations and carbapenems. However, more recently an enzyme encoded by cfxA gene,7 which confers resistance to cefoxitin among strains of the B. fragilis group has been described, but its expression seems to be rare. Moreover, the production of a metallo-ß-lactamase by B. fragilis strains has compromised the clinical use of cefoxitin and more recently of carbapenems. This enzyme, encoded by the chromosomal8 or plasmid9 cfiA gene, and belonging to molecular class B and functional group 3,4 hydrolyses a broad spectrum of ß-lactams, including cephamycins and carbapenems. The cfiA-type metallo-ß-lactamase has only been reported in B. fragilis strains isolated from clinical specimens in the USA,10 France11 and the UK,12 maybe because it has been demonstrated that a small number of B. fragilis strains (2.4%) carry a silent metallo-ß-lactamase gene,13 which requires migration of an insertion sequence upstream of the ß-lactamase gene for its expression.14 In addition, the widespread use of imipenem and, more recently, of meropenem can be an important selective pressure.15 The purpose of the present study was to determine the antimicrobial resistance profile of B. fragilis strains isolated from clinical specimens in two periods (1981–1988 and 1991–1998) in a Brazilian University hospital, so that trends in the emergence of resistance could be detected. Strains from human intestinal microflora and polluted aquatic environments were also included in this study as an indicator of the use of antibiotics by the community. Finally, the presence of the metallo-ß-lactamase gene, cfiA, has also been investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 (1981–1988 and 1991–1998) from patients at the University hospital (Federal University of Rio de Janeiro-HU-UFRJ) in Brazil. The strains from the 1981–1988 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 Wilkins–Chalgren (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 Tris–HCl (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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of the susceptibility tests of clinical, intestinal and environmental strains are summarized in Tables I, II and IIIGoGoGo, respectively. In the two periods (1981–1988 and 1991–1998) a high rate of resistance to penicillin among clinical isolates was observed (Table IGo). However, the MIC90 decreased significantly from the first to the second period. In contrast, an increased rate of resistance to cefoxitin was seen from the first period to the second among the clinical isolates. All the strains studied were ß-lactamase producers confirming the universal presence of this enzyme in B. fragilis. All clinical isolates were susceptible to imipenem and meropenem. However, two strains, from intra-abdominal infections, showed intermediate resistance to meropenem (MIC 6 mg/L and 8 mg/L).


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Table I. Resistance profile of Bacteroides fragilis strains isolated in a Brazilian university hospital in two periods (1981–1988 and 1991–1998)
 

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Table II. Susceptibility of Bacteroides fragilis strains (17) isolated from human intestinal flora to seven antimicrobial agents
 

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Table III. Susceptibility of Bacteroides fragilis strains (17) isolated from polluted aquatic environments to seven antimicrobial agents
 
The cfiA investigation by PCR generated a fragment corresponding in size to the cfiA gene in the clinical strains 1033 and 1386-4 (Figure 1Go), which presented relatively high levels of resistance against cefoxitin (32 mg/L and 64 mg/L), penicillin (6 mg/L and 12 mg/L) and clindamycin (8 mg/L and >256 mg/L). An increase was seen in the rate of resistance to clindamycin from the first period analysed (7.1%) to the second period (26.7%). Strains resistant to chloramphenicol were not found during the two periods analysed. Also, there were no strains resistant to metronidazole, with MIC90 in both periods much lower than the breakpoint as established by NCCLS.20



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Figure 1. Detection of cfiA gene in B. fragilis strains from clinical specimens by analysis of PCR products in 1% agarose gels. A band (arrow) was detected in two strains, but not found in the other strains studied. Lanes A: molecular weight standard (Gibco-1 kb); B: ATCC 25285; C: strain 1032; D: 1033; E: 1058.4; F: 1070-b; G: 1077; H: 1108-3b and I: 1386-4.

 
Strains from human intestinal microbiota and polluted aquatic environments (Tables II and IIIGoGo) showed high resistance rates to penicillin and clindamycin, moderate rates of resistance to cefoxitin and no resistance to imipenem, meropenem, chloramphenicol and metronidazole. The exceptions were one strain from a polluted river (SPA-2) against which the MIC of cefoxitin was 64 mg/L and that of meropenem was >32 mg/L, and another strain from an artesian well (AA10), against which the MIC meropenem was 6 mg/L (considered ‘intermediate’ by NCCLS20) The cfiA gene was detected (Figure 2Go) by PCR in these two strains and another strain susceptible to meropenem (AA7b) from a polluted river.



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Figure 2. Detection of cfiA gene in B. fragilis strains from polluted aquatic environments and intestinal microbiota by analysis of PCR products in 1% agarose gels. A band (arrow) was detected in three strains from polluted aquatic environments, not found in the other strains studied. Lanes A: molecular weight standard (Gibco-1 kb); B: strains AA10; C: AA7b; D: AA10a; E: AA10c; F: FT-2; G: SPA02; H: F36-6; I: EC 01-2; J: FD 19 A and K: ES 08.1.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Brazil, susceptibility testing of anaerobic bacteria is rarely done. This report investigated the trends in resistance to the main antimicrobial agents traditionally used in the management of anaerobic infections, since different rates among medical institutions have been reported as a consequence of differing use of antibiotics.24,25 Nowadays, clindamycin and metronidazole are extensively used as empirical therapy in surgical wards at the HU–UFRJ. This may explain the increase in the rate of resistance to clindamycin among clinical isolates observed from the first period to the second analysed, although the difference found is not significant. Dubreuil et al.26 have shown similar results, in a retrospective survey in France (1977 to 1992), with a range from 1% to a peak of 19% up to 1988. Recently, Breuil and co-workers27 have asserted that similar rates to clindamycin and tetracycline can be detected in different geographical regions. These results need to be interpreted with caution, as different methodology has been used, particularly for clindamycin, where differences can occur between the conventional agar dilution test and the Etest.19

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 (1981–1988) 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 HU–UFRJ, 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.


    Acknowledgments
 
We thank Joaquim Santos Filho for his technical assistance. This work was supported by grants from the following Brazilian institutions: CNPq, FINEP-BID, PRONEX, CEPG and FUJB.


    Notes
 
* Corresponding author. Tel/Fax: +55-21-560-8028/560-8344; E-mail; immmcan{at}microbio.ufrj.br Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 4 May 1999; accepted 20 September 1999