In-vitro susceptibilities of species of the Bacteroides fragilis group to newer ß -lactam agents

Carmen Betriu*, Ana Sánchez, Mara Gómez, M. Luisa Palau and Juan J. Picazo

Servicio de Microbiología Clínica, Hospital Clínico San Carlos, Plaza Cristo Rey s/n, 28040 Madrid, Spain

Abstract

The in-vitro activities of imipenem and four ß-lactam–ß-lactamase inhibitor combinations were tested against 816 strains of the Bacteroides fragilis group, and compared with other anti-anaerobic agents. None of the strains was resistant to metronidazole, and only one was resistant to chloramphenicol. Mezlocillin and piperacillin were moderately active, while clindamycin was the least active. Rates of resistance varied between various species. The new ß-lactam agents tested showed excellent activity; piperacillin–tazobactam and imipenem were the most active. The emergence of strains that are resistant to these agents, observed in this study, suggests there is a need to perform periodic antimicrobial susceptibility tests.

Introduction

Of the anaerobic bacteria, members of the Bacteroides fragilis group are the ones most frequently isolated from human infections. In recent years, reports of increasing resistance to several of the traditionally used antimicrobial agents, and to some of the newer ß-lactam agents, have been published.1 2 ,3 ,4 ,5 Differences in the rates of resistance from one medical centre to another, and from one geographic location to another, have also been reported.6 ,7 In the present study, we compared the in-vitro activities of imipenem and the currently available combinations of aß-lactam agent with a ß-lactamase inhibitor (amoxycillin– clavulanate, ampicillin–sulbactam, ticarcillin–clavulanate and piperacillin–tazobactam) with those of cefoxitin, ceftizoxime, piperacillin, mezlocillin, metronidazole, chloramphenicol and clindamycin.

Materials and methods

Organisms were all clinical strains isolated from patients in this hospital from 1990 to 1995. They were identified using the rapid ID32 A system (bioMérieux, Marcy l'Etoile, France). A total of 816 strains of the Bacteroides fragilis group were tested, comprising 439 Bacteroides fragilis, 108 Bacteroides thetaiotaomicron, 67 Bacteroides ovatus, 61 Bacteroides uniformis, 44 Bacteroides caccae, 43Bacter oides vulgatus, 41 Bacteroides distasonis, eight Bacteroides merdae and five Bacteroides stercoris. The sources of these isolates included intra-abdominal abscesses (57%), skin and soft tissues (27.8%), blood (8.7%), female genital tract (2%), body fluids (1.5%), respiratory tract (1.1%), hepatic abscess (1.1%) and miscellaneous (0.9%). Quality control strains (B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741) were included.

Standard laboratory powders were supplied as follows: metronidazole, Rhône–Poulenc Rorer, Madrid, Spain; chloramphenicol, Zyma Farmacéutica, Barcelona, Spain; clindamycin, Pharmacia & Upjohn, Barcelona, Spain; cefoxitin and imipenem, Merck Sharp & Dohme, West Point, PA, USA; mezlocillin, Bayer, Barcelona, Spain; ceftizoxime, amoxycillin, ticarcillin and clavulanate, SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA; ampicillin and sulbactam, Pfizer, New York, NY, USA; and piperacillin and tazobactam, Wyeth Lederle, Pearl River, NY, USA.

Agar dilution susceptibility testing was performed by the method recommended by the NCCLS8 using Wilkins-Chalgren agar. Antibiotic concentrations ranged from 256 to 0.06 mg/L. The agar plates were inoculated with a Steers replicator. The inoculum was prepared by growing organisms in supplemented thioglycolate broth overnight at 35°C and diluted so that the turbidity was equal to that of a 0.5 MacFarland standard. The final inoculum was approximately 105 cfu/spot. The plates were incubated at 35°C for 48 h in an anaerobic chamber (Forma Scientific, Marietta, OH, USA) containing 85% N2, 5% H2 and 10% CO2. The MIC was defined as the lowest concentration of each antimicrobial agent at which there was no growth, one discrete colony, a barely visible haze or any distinct change from the growth control. MICs were determined for the group as a whole, as well as for individual species. For the ß-lactam–ß-lactamase inhibitor combinations, the MICs were expressed in terms of the concentrations of the ß-lactam component. The breakpoints used were those recommended by the NCCLS.8

Results and discussion

The results of susceptibility testing are presented in Tables I and II. As can be seen from Table I, the most active of the single agents tested were metronidazole, chloramphenicol and imipenem. All strains were susceptible to metronidazole (MIC90 5 1 mg/L). Although in most studies no resistance to metronidazole has been reported, resistance to this antibiotic has been reported in Spain by Pélaez et al.1 We detected one chloramphenicol-resistant strain (MIC 5 32 mg/L). Chloramphenicol resistance in the B. fragilis group has rarely been reported.7 Of the test strains, 26.2% were resistant to clindamycin (breakpoint 4 mg/L); this rate of resistance is similar to that reported by our group in previous studies 2 and by other researchers in Spain. 6 Resistance to clindamycin (MIC > 4 mg/L) reported in different studies vary, with rates of 4%, 9 10–14% 4 ,5 ,7 and >=20%.1 ,6


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Table I. Antimicrobial agents against 816 strains of the Bacteroides fragilis group
 

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Table II. Incidence of resistance in species of the Bacteroides fragilis group
 
Cefoxitin at 32 mg/L inhibited 92.2% of the strains tested. The incidence of resistance to cefoxitin was com parable to the rates of 6–9% reported from some studies, 4 ,6 but lower than the 45% described by other investigators in Spain.1 Ceftizoxime was slightly less active than cefoxitin. For ceftizoxime the incidence of resistance (MIC >64 mg/L) of 10.8% found in this study is similar to that reported by Aldridge et al.4

During the past decade, the incidence of resistance to mezlocillin (MIC > 64 mg/L) has increased significantly (P < 0.01) in this hospital, from 6.7% in 19892 to 13.8% in the present study. The incidence of resistance to ceftizoxime increased between 1989 2 and 1991 from 5.2 to 12.5% (P < 0.05) and declined notably in 1994 from 18% to 7% (P <0.005). The patterns of susceptibility to the remaining antimicrobials tested did not change significantly over the 5 years of the study.

All combinations of ß-lactam–ß-lactamase inhibitors evaluated in this study displayed excellent activity against strains of the B. fragilis group, with >98% of the strains being susceptible. Piperacillin–tazobactam was the most active agent. The addition of tazobactam to piperacillin lowered the MIC90 from 128 to 16 mg/L, with 99.3% of the strains being susceptible (breakpoint 64 mg/L). The MIC50 and MIC90 of imipenem were 0.12 and 0.5 mg/L, respectively. Six strains were highly resistant to imipenem (MIC >256 mg/L) and to all other ß-lactam agents tested, and one strain showed reduced susceptibility to imipenem (MIC = 16 mg/L). These strains have been shown to produce metallo-ß-lactamases.10 Resistance to imipenem and ß-lactamase inhibitor combinations was first detected by our group in 1989; 2 in this hospital the incidence of imipenem resistance in the B. fragilis group has remained low (0.5–1%) since then. Elsewhere, low rates of resistance (0.1–2%) to imipenem have been reported by several investigators, 1 ,4 ,5 but rates in Japan have increased, from 2% in 1987 to 5.9% in 1992. 3

As expected, different species in the B. fragilis group varied in their resistance to many of the antimicrobials tested (Table II). As reported by others, 6 ,7 the non-B. fragilis species were significantly more resistant to the ß-lactam drugs than were strains of B. fragilis, with the exception of imipenem and the ß-lactamase inhibitor combinations. Piperacillin showed the greatest degree of variability in resistance patterns (breakpoint 64 mg/L). Of the strains tested, those of B. fragilis were the least resistant to piperacillin (8.2%) while strains of B. distasonis were the most resistant (34.1%).

These results indicate that the new ß-lactam agents that were tested can be used for the treatment of infections with the B. fragilis group, and that metronidazole and chloramphenicol continued to be the most active non-ß-lactam agents. The changing susceptibility rates over time, the variability among clinical centres of rates of antimicrobial resistance, and the emergence of strains that are resistant to new ß-lactam agents, suggest the need to perform periodic susceptibility testing in each hospital to ensure that empirical antimicrobial therapy of infections caused by these organisms is appropriate.

Acknowledgments

This research was presented at the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 28 September to 1 October, 1997.

Notes

* Tel: +34-1-3303486; Fax +34-1-3303478; E-mail: cbetriu{at}efd.edu Back

References

1 . Pélaez, M. T., Cercenado, E., Rodrguez-Creixems, M. & Bouza, E. (1991). Resistance of anaerobic bacteria to antimicrobial agents. Reviews of Infectious Diseases13 , 183.

2 . Betriu, C., Cabronero, C., Gómez, M. & Picazo, J. J. (1992). Changes in the susceptibility of Bacteroides fragilis group organisms to various antimicrobial agents 1979–1989. European Journal of Clinical Microbiology and Infectious Diseases 11, 352–6.[ISI][Medline]

3 . Bandoh, K., Ueno, K., Watanabe, K. & Kato, N. (1993). Susceptibility patterns and resistance to imipenem in the Bacteroides fragilis group species in Japan: a 4-year study. Clinical Infectious Diseases 16, Suppl. 4, S382–6.[ISI][Medline]

4 . Aldridge, K. E., Gelfand, M., Reller, L. B., Ayers, L. W., Pierson, C. L., Schoenknecth, F. et al. (1994). A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States. Diagnostic Microbiology and Infectious Diseases 18, 235–41.[ISI][Medline]

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7 . Phillips, I., King, A., Nord, C. E. & Hoffstedt, B. (1992). Antibiotic sensitivity of the Bacteroides fragilis group in Europe. European Journal of Clinical Microbiology and Infectious Diseases 11, 292–304.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (1993). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Third Edition: Approved Standard. M11-A3. NCCLS, Villanova, PA.

9 . Mastrantonio, P., Spigaglia, P. & Sebastianelli, A. (1994). Susceptibility patterns and characterization of beta-lactamases in clinical isolates of Bacteroides fragilis. European Journal of Clinical Microbiology and Infectious Diseases 13,475 –80.[ISI][Medline]

10 . Payne, D. J., Betriu, C., Kushi, T., Hoyle, C., Reading, C. & Knowles, D. (1995). Imipenem hydrolysing ß-lactamases from 6 strains of B. fragilis. In Program and Abstracts of the 95th General Meeting of the American Society for Microbiology, Washington, DC, 1995. Abstract A75, p. 156. American Society for Microbiology, Washington, DC.

Received 10 March 1998; returned 6 July 1998; revised 6 July 1998; accepted 28 August 1998