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Survey of anaerobic susceptibility patterns: A French multicentre study
International Journal of Antimicrobial Agents 10 (1998) 229 – 236
Original article
Survey of anaerobic susceptibility patterns: A French multicentre study F. Mory a, A. Lozniewski a, S. Bland b, A. Sedallian b, G. Grollier c, F. Girard-Pipau d, M.F. Paris e, L. Dubreuil e,* a
Chu de Nancy, 29, a6. de Lattre de Tassigny, 54037 Nancy cedex, France Centre Hospitalier d’Annecy, a6. de Tresums, 74011 Annecy cedex, France c Hopitaux de la Mile´trie,350, a6. Jacques Coeur, 86021 Poitiers cedex, France d Hoˆpital Henri-Mondori 81, a6. du Mal de Lattre de Tassigny, 94000 Caete´il, France e Faculte´ de Pharmacie de Lille, 3, Rue Laguesse, BP83, 59006 Lille cedex, France b
Received 23 March 1998; received in revised form 1 May 1998; accepted 18 May 1998
Abstract In 1996, the in vitro antibiotic susceptibility of 463 anaerobes was measured in five hospitals using the reference agar dilution method. None of the 209 B. fragilis group strains showed resistance to imipenem or ticarcillin-clavulanic acid. High resistance rates (29%) were observed for cefotetan and clindamycin. b-Lactamase production was detected respectively in 64% of the Pre6otella and 7% of the Fusobacterium strains. Because the same standardized methods were used for many years, the authors were able to evaluate the evolution of antibiotic resistance. Clindamycin resistance had increased within the B. fragilis group (from 14% in 1992 to 29% in 1996) and also among strains of clostridia (32%), P. acnes (18%) and Peptostreptococcus (28%). In the B. fragilis group multidrug resistance was unlikely to occur. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Anaerobes; Antibiotic patterns; Resistance
1. Introduction Evolution of antibiotic resistance has been previously observed among anaerobes, in France [1,2]. New resistance mechanisms have emerged such as 5-nitroimidazole resistance among Bacteroides fragilis [3,4]. Penicillin resistance linked with b-lactamase production has been described for some Clostridium species [5], Pre6otella and Fusobacterium [6]. Some B. fragilis strains are now resistant to carbapenems [7,8]. The aim of our study was to evaluate the antibiotic susceptibility of anaerobic bacteria by determining the MICs of 11 anti-anaerobic drugs on a wide range of species. Thus, 463 anaerobes were collected in 1996 from five hospitals * Correponding author. Tel./fax: + 33 3 20 964008; e-mail: ldubreui @phare.univ-lille2.fr. 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0924-8579(98)00041-7
which are localized in different areas of our country: North, East, West, South-east and Center.
2. Materials and methods
2.1. Bacterial strains Anaerobic bacteria were isolated from human clinical samples during 1996. They were identified according to standard methods, then subcultured in a Rosenow medium (Diagnostics Pasteur, Marnes la Coquette, France). If they were not used immediately to determine MICs, the broth was kept frozen at− 20°C. Before being tested, the bacteria were checked for purity by subculturing on Colombia blood agar (bioMe´rieux, Marcy l’e´toile, France), either on laked blood-
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Table 1 Antimicrobial activities of 11 antibiotics against the Bacteroides fragilis group Resistance rates (%)a
Microorganism (N°)
MIC (mg/l)
Antibiotic
range
MIC 50%
MIC 90%
Bacteroides fragilis (109) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefotaxime Imipenem Clindamycin Metronidazole
0.12–\128 0.03–64 0.5–\128 0.03–8 0.12–\128 0.5– \128 0.015–2 0.015–\128 0.015–8
16.00 0.25 16.00 0.50 8.00 16.00 0.12 0.25 0.50
\128.0 4.0 128.0 4.0 128.0 128.0 0.5 64.0 2.0
41.0 2.0 15.0 0.0 13.0 20.0 0.0 22.0 1.8
B. thetaiotaomicron (28) and B. o6atus (9) Amoxicillin 4–\128 +Clavulanic acid 0.03–128 Ticarcillin 4–\128 +Clavulanic acid 0.25–16 Piperacillin 2– \128 Cefoxitin 2–128 Cefotetan 2–\128 Cefotaxime 32– \128 Imipenem 0.015–4 Clindamycin 0.03– \128 Metronidazole 0.25–4
32.0 0.5 32.0 1.0 32.0 16.0 64.0 64.0 0.12 1.0 0.5
\128.00 16.00 \128.00 16.00 \128.00 64.00 \128.00 \128.00 0.25 \128.00 2.00
86 10 18 0 32 15 61 53 0 39 0
Bacteroides 6ulgatus (33) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
64.00 0.50 64.00 0.50 32.00 8.00 32.00 32.00 0.12 0.50 0.50
\128.0 16.0 \128.0 8.0 \128.0 32.0 \128.0 \128.0 0.5 \128.0 2.0
67 6 42 0 36 9 26 50 0 29 6
Bacteroides distasonis – caccae -merdae group (21) Amoxicillin 0.5–\128 +Clavulanic acid 0.03–16 Ticarcillin 4– \128 +Clavulanic acid 0.12–32 Piperacillin 1– \128 Cefoxitin 1–64 Cefotetan 1–\128 Cefotaxime 1– \128 Imipenem 0.03–1 Clindamycin 0.03–\128 Metronidazole 0.03–2
32.00 2.00 32.00 4.00 16.00 16.00 64.00 4.00 0.25 0.50 0.25
\128 8 \128 16 \128 64 128 \128 1 \128 1
62 0 29 0 33 16 59 36 0 32 0
Bacteroides of the group fragilis (209) Amoxicillin 0.12– \128 +Clavulanic acid 0.03–128 Ticarcillin 0.5–\128 +Clavulanic acid 0.03–64 Piperacillin 0.12– \128 Cefoxitin 1–128 Cefotetan 0.25–\128 Cefotaxime 0.5– \128 Imipenem 0.012–4 Clindamycin 0.015– \128 Metronidazole 0.015–32
32.00 0.25 32.00 1.00 16.00 8.00 32.00 16.00 0.12 0.25 0.25
\128.0 8.0 \128.0 8.0 \128.0 32.0 128.0 128.0 0.5 \128.0 2.0
55.0 3.8 22.2 0.0 22.5 7.1 28.6 32.0 0.0 29.2 1.9
a
0.5–\128 0.03–128 2– \128 0.06–64 1– \128 2–64 1–\128 0.5– \128 0.03–1 0.03– \128 0.03–32
Resistance rates calculated using French breakpoints [14] described in the section material and methods.
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Table 2 Antimicrobial activities of 11 antibiotics against gram negative bacilli other than the B. fragilis group Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Porphyromonas (19) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–0.50 0.03–0.25 0.25–0.50 0.25–0.50 0.12 0.25–0.50 0.06–1.00 0.06–1.00 0.015–0.06 0.03–0.25 0.03–0.25
5]Pre6otella spp (56) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.06–\128.00 0.03–16.00 0.12–64.00 0.06–16.00 0.12–\128.00 0.06–4.00 0.03–16.00 0.06–64.00 0.015–0.12 0.03–0.06 0.015–2.00
Fusobacterium spp (27) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–8.00 0.03–1.00 0.06–4.00 0.06–2.00 0.06–2.00 0.06–2.00 0.03–32.00 0.06–4.00 0.015–0.25 0.03–8.00 0.03–0.25
Resistance rates(%) MIC 50%
MIC 90%
0.50 0.25 0.25 0.25 0.12 0.25 0.12 1.00 0.03 0.06 0.12
0.50 0.25 0.50 0.50 0.12 0.50 0.25 1.00 0.03 0.12 0.25
0 0 0 0 0 0 0 0 0 0 0
8.00 0.06 1.00 0.12 16.00 1.00 4.00 2.00 0.03 0.03 0.50
64.00 1.00 16.00 2.00 32.00 4.00 16.00 8.00 0.06 0.06 1.00
25 0 0 0 2 0 0 2 0 0 0
0.06 0.03 0.06 0.03 0.06 0.06 0.06 0.12 0.03 0.03 0.03
1.00 0.06 0.50 0.25 0.25 0.25 0.25 0.50 0.06 0.12 0.25
0 0 0 0 0 0 0 0 0 4 0
kanamycin-vancomycin plates (Serlabo,Bonneuil/ Marne,France) for Bacteroides spp. or josamycinnorfloxacin plates for Fusobacteria [9]. Purity was also checked by Gram staining. The Bacteroides fragilis group (209) included Bacteroides fragilis (109) B. thetaiotaomicron (28) B. o6atus (9) B. distasonis (16), B. caccae (3), B. merdae (2) B. uniformis (4) B. stercoris (3) B. eggerthii (2) B. 6ulgatus (33). Other strains were as follows: B. capillosus (1) B. spanchnicus (1) Anaerorhabdus furcosus (1) Dialister pneumosintes (1) Pre6otella intermedia /nigrescens (15) P. loescheii (1) P. melaninogenica (12) P. buccalis (1) P. buccae (7) P.oris (6), P. oralis (1) P. 6eroralis (1) P. bi6ia (10) P. disiens (2) Porphyromonas asaccharolytica (17) P. gingi6alis (2) Fusobacterium nucleatum (20) F. necrophorum (7) Bilophila wadsworthia (3) Veillonella par6ula (6) Clostridium difficile (37) C. perfringens (16) C. bifermentans (1) C. butyricum (1) C. clostridioforme (2), C. fallax (1), C. innocuum (4), C.
paraputrificum (1) C. ramosum (6) C. sphenoides (2), C. tertium (3), C. sp (4) Eubacterium lentum (17) E. biforme (1) E. 6entriosum (1) Pseudoramibacter alactolyticus (2) Propionibacterium acnes (8) P. granulosum (1), Bifidobacterium bifidum (5) Peptostreptococcus anaerobius (4) P. asaccharolyticus (4) P. indolyticus (1) P. magnus (10) P.micros (1) P. pre6otii (4) Ruminococcus gna6us (2). For quality control and assessment of reproducibility, four reference ATCC control strains were added to each batch of tests. The ATCC control strains, advocated by the M11 A3 Norma [10] of the National Committee for Clinical Laboratory Standards (NCCLS), were Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, Clostridium perfringens ATCC 13124 and Eubacterium lentum ATCC 43055. b-Lactamase production was assessed using a nitrocefin disk (cefinase® bioMe´rieux). As all strains of the B. fragilis group have a chromosomal cephalosporinase,
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Table 3 Antimicrobial activities of 11 antibiotics against gram positive bacilli Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Resistance rates (%) MIC 50%
MIC 90%
2 1 32 16 8 128 32 64 4 \128 1
0 0 0 0 0 89 3 95 0 54 0
C. difficile (37) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–2 0.06–2 0.5–32 0.5–32 0.12–8 32–128 8–\128 32–128 1–8 0.12–\128 0.03–4
0.50 0.50 8.00 8.00 4.00 64.00 16.00 64.00 4.00 4.00 0.25
Other clostridia (41) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–1 0.03–2 0.12–32 0.06–32 0.03–16 0.06– \128 0.03–\128 0.12–32 0.015–4 0.03–\128 0.03–2
0.06 0.06 4.00 0.50 0.06 1.00 4.00 2.00 0.06 0.25 0.25
1.0 0.5 16.0 16.0 8.0 32.0 \128.0 32.0 1.0 16.0 1.0
0 0 0 0 0 20 32 0 0 32 0
Non sporulated gram+ bacilli (35) Amoxicillin 0.03–2 +Clavulanic acid 0.03–2 Ticarcillin 0.25–64 +Clavulanic acid 0.06–64 Piperacillin 0.06–32 Cefoxitin 0.06–32 Cefotetan 0.03–64 Cefotaxime 0.06– \128 Imipenem 0.03–1 Clindamycin 0.03–16 Metronidazole 0.25–\128
0.50 0.50 4.00 1.00 1.00 8.00 8.00 1.00 0.12 0.03 0.50
2 1 32 2 16 16 64 \128 0.5 4 1
0 0 0 0 0 0 33 40 0 11 50
the test was applied only to gram negative bacilli other than B. fragilis and clostridia.
2.2. Antimicrobial agents Powders of known potency were supplied by the manufacturers or their French subsidiaries: metronidazole (Specia), amoxicillin, amoxicillin, ticarcillin, clavulanic acid (Smith Kline Beecham), piperacillin (Wyeth), cefoxitin, imipenem (Merck Sharp Dohme), cefotetan (Zeneca-Pharma),cefotaxime (Hoechst Marion Roussel), clindamycin (Upjohn) and metronidazole (Rhoˆne Poulenc-Rorer).
2.3. MIC determinations MICs were determined using a reference agar dilution method according to the Norma M11T [11] of the
NCCLS with further recommendations from the Norma M11 A3 [10]. Stock solutions of 512 mg/l of each antibiotic were prepared and two-fold dilutions were carried out in distilled water [12]. Metronidazole was first dissolved in 2 ml of methanol and then distilled water was added to the solution.Clavulanic acid was used in a fixed concentration of 2 mg/l when combined with amoxicillin or ticarcillin. Each antibiotic was incorporated into Wilkins Chalgren [13] agar (Oxoid-Unipath, Dardilly, France) with 5% added sterile defibrinated blood to provide adequate support for the growth of Fusobacteria, Peptostreptococcus spp, and Eubacterium spp. Plates contained serial doubling dilutions of antimicrobial agents (from 128 to 0.003 mg/l). All plates were used within 24 h of preparation. An actively growing culture in Rosenow medium was diluted in a Schaedler broth (BioMe´rieux) to reach and match the 0.5 point of a Mac Farland standard. The
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Table 4 Antimicrobial activities (MIC, in mg/l) of 11 antibiotics against anaerobic cocci Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Peptostreptococus spp (24) and Ruminococcus gna6us (2) Amoxicillin 0.03–16.0 +Clavulanic acid 0.03–0.5 Ticarcillin 0.06–8.0 +Clavulanic acid 0.03–8.0 Piperacillin 0.03–1.0 Cefoxitin 0.06–4.0 Cefotetan 0.06–32.0 Cefotaxime 0.12–4.0 Imipenem 0.015–4.0 Clindamycin 0.03–64.0 Metronidazole 0.03–2.0 Veillonella spp (6) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
Resistance rates (%) MIC 50%
MIC 90%
0.12 0.03 1.00 1.00 0.12 0.50 0.50 1.00 0.03 0.12 0.50
0.50 0.25 4.00 2.00 0.50 2.00 8.00 4.00 0.25 32.00 1.00
0.03–2.00 0.03–4.00 0.5–32.00 1–32.00 1–64.00 0.06–4.00 0.12–8.00 0.12–2.00 0.015–1.00 0.03–0.12 0.12–1.00
inocula were approximately 7.5×107 to 1×108 UFC/ ml. For fastidious strains, the following were added to the Schaedler broth: Hemin (5 mcg/l), menadione (0.1 mcg/l), sodium bicarbonate (1 g/l) and 1 ml of laked blood per tube containing 100 ml. The previous inocula (2–3 ml) were delivered by a Steers replicator (Mast Systems, London) and resulted in a final inocula of 105 CFU per spot of inoculation on the agar plates. At the end of each series of tests, two plates of Wilkins Chalgren agar were inoculated without antimicrobial agent. One plate was incubated anaerobically to determine the viability of the organisms and to serve as a control for growth comparison and the other aerobically to indicate possible aerobic contamination. Incubation of the tested plates containing the antibiotics was performed in an anaerobic chamber (Forma Scientific) at 35° – 36° C. Reading of the MICs was made after 48 h of incubation. The MIC for an organism was noted as the lowest concentration of an antimicrobial agent yielding no growth. Resistance rates were calculated in relation with French breakpoints (mg/l), which are identical for most antibiotics to those of the NCCLS with the exception of amoxicillin (\16), amoxicillin-clavulanate ( \ 16/2), metronidazole (\4),clindamycin (\2) [14].
0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0
3. Results Over all 463 anaerobes, no resistance could be detected for either imipenem or the ticarcillin clavulanic acid combination. MIC50 and MIC90 values for each antibiotic together with resistance rates and for each group of anaerobic bacteria are listed in the tables.
3.1. Bacteroides fragilis group (Table 1) Seven strains were resistant to amoxicillin-clavulanate but susceptible to ticarcillin- clavulanic acid and cefoxitin: including two B. fragilis, three B. thetaiotaomicron and two B. 6ulgatus strains. Among these seven strains, only the B. fragilis strains remained susceptible to cefotetan. Resistance to cefotetan was rare for B. fragilis, but frequent for other species such as B. thetaiotaomicron and B. distasonis. Half of the clindamycin resistant strains were susceptible to all other antibiotics with the exception of amoxicillin. Metronidazole resistance (CMI\4 mg/l) was observed for four strains but cross-resistance to clindamycin and metronidazole did not occur. Crossresistance to ticarcillin, piperacillin, cefoxitin, cefotetan, cefotaxime and clindamycin was observed for one of each strain of B. fragilis and B. uniformis.
AMC (\16/2) 3.8 0.0
0.0 1.8
AMX (\16)
55.0
7.4
0.0 28.7
Antibiotic (breakpoint in mg/l)
0.0 10.9
0.0
22.2
TIC (\64)
0 0
0
0
TCC (\64/2)
0.0 11.8
4.3
22.5
PIP (\64)
25.6 11.7
0.0
7.1
CFX (\32)
11.0 16.9
0.0
28.6
CTT (\32)
37.3 24.0
1.1
32.0
CTX (\32)
0 0
0
0
IMI (\8)
27.9 25.0
4.8
29.2
CLN (\2)
15.0 5.6
0.0
1.9
MOL (\4)
AMX, amoxicillin; AMC, amoxicillin-clavulanate; TIC, ticarcillin; TCC, ticarcillin+clavulanic acid; PIP, piperacillin; CFX, cefoxitin; CTT, cefotetan; CTI, cefotaximes; IMI, imipenem; CLN, clindamycin and MOL, metronidazole.
B. fragilis group Other gram negative bacilli Gram positive All
Anaerobes
Table 5 Antibiotic resistance rates (%) in anaerobes
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F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
3.2. Gram negati6e bacilli other than the B. fragilis group (Table 2) Porphyromonas strains were very susceptible (CMIB 1 mg/l) to all the antibiotics investigated. b-Lactamase production was never detected by the nitrocefin disk. In contrast b-lactamase production was frequent (84%) among Pre6otella species. Resistant strains were observed for amoxicillin, piperacillin (one strain of P. bi6ia) and cefotaxime (one strain of P. oris). Black pigmented Pre6otella were all inhibited by coamoxi-clav and ticarcillin at a concentration of 1mg/l whereas higher concentrations are needed to inhibit all the non-pigmented Prevotella. Most Fusobacterium species demonstrated low antibiotic MIC values and only one strain of F. necrophorum was resistant to clindamycin. 7.4% of the Fusobacterium strains produced a penicillinase. The three strains of Bilophila wadsworthia were resistant to amoxicillin and piperacillin (b-lactamase+) but suceptible to amoxicillin-clavulanate, ticarcillin, cefoxitin, cefotetan, imipenem, clindamycin and metronidazole. Considering all the 115 gram negative rods other than the B. fragilis group, no resistance could be detected for amoxicillin-clavulanate, ticarcillin either alone or combined with clavulanic acid, cefoxitin, cefotetan, imipenem and metronidazole.
3.3. Gram positi6e bacilli (Table 3) C. difficile strains were susceptible to all penicillins and to metronidazole. We observed intrinsic resistance to imipenem (low level) and to all cephalosporins. Resistance to clindamycin occurred for 54% of strains investigated. In contrast C. perfringens was susceptible to all antibiotics studied herein. Other clostridia were susceptible to penicillins, imipenem and metronidazole whereas resistance occurred with cefoxitin (20%), cefotetan and clindamycin (both 32%).Non sporulated gram positive bacilli were all inhibited by penicillins, cefoxitin and imipenem. Resistance to cefotaxime was common among strains of Eubacterium. Most strains of Bifidobacterium and all propionibacteria were resistant to metronidazole. Resistance to clindamycin was also common (22%) among P. acnes. The 113 gram positive bacilli were all inhibited by amoxicillin, ticarcillin, piperacillin and imipenem.
235
4. Discussion Assessment of any evolution in antibiotic resistance must be treated with great caution because the numerous methodological problems may result in invalid comparisons[15]. Change in antibiotic testing methodology may reflect spurious evolution in antibiotic resistance rates but we have used the same constant NCCLS methodology for the last twenty years in our outwork on possible antibiotic resistance. Previous French reports [1,2,16–20] demonstrated that resistance to imipenem and to metronidazole remained rare in the B. fragilis group. Decreased susceptibility [3,4] to metronidazole (MIC from 4 to 16 mg/l) was observed for less than 5% of the strains [17,19]. Resistance to most antibiotics is stable whereas resistance to clindamycin is still increasing in France. Clindamycin resistance rates increased from 14% in 1992 [1], to 20.6%, 1995 [20], reaching 29.2% in this study (Table 5). b-Lactamase production is common among Pre6otella and is becoming more frequent; in contrast it remains rare among strains of Fusobacterium.. In relation probably to the cephalosporinase activity of Pre6otella enzymes [6], ticarcillin had greater potency than piperacillin. Although we isolated few B. wadswortia our results are in agreement with those of other articles [21]. The penicillins were very effective against non sporulated gram positive anaerobes. Propionibacterium spp is naturally resistant to metronidazole (MIC= 32 mg/l). Higher clindamycin rates are observed among P. acnes and Peptostreptococcus strains. Gram positive cocci are generally known to be susceptible to metronidazole, the resistance rate being below 10% in France [16,17]. Resistance to clindamycin has increased among gram positive cocci from 9.8% in 1995 [20] to 28% at present. Over the whole range of anaerobes, higher resistance rates were observed in this study for clindamycin compared to previous results. Multidrug resistance to all b-lactams, clindamycin and metronidazole has not been observed at present among B. fragilis. Although resistance to some antibiotics is increasing, the lack of cross resistance to clindamycin and metronidazole is of interest. The good activity of imipenem and of the two combinations of clavulanic acid with either amoxicillin or ticarcillin offers some optimism for the future.
3.4. Anaerobic cocci (Table 4) References Peptostreptococci were inhibited by low concentrations of amoxicillin with the exception of one strain of P. micros (CMI=8 mg/l). No b-lactamase could be detected and MICs for ticarcillin and piperacillin were 2 and 1 mg/l, respectively. A high prevalence of resistance to clindamycin (28%) was observed. All Veillonella strains were inhibited by all the antibiotics investigated.
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[3] Dublanchet A, Caillon J, Edmond JP, et al. Isolation of Bacteroides strains with reduced sensitivity to 5-nitroimidazoles. Eur J. Clin Microbiol 1986;5:346–7. [4] Haggoud A, Reysset G, Sebald M. Cloning of a Bacteroides fragilis chromosomal determinant coding for 5-nitroimidazole resistance. FEMS Microbiol Lett 1992;95:1–6. [5] Appelbaum PC, Spangler SK, Pankuch G, et al. Properties of a b-lactamase from a strain of Clostridium clostridioforme. Program Abstr. 32th Intersci. Conf Antimicrob Agents Chemother Abstr 1273, 1992. [6] Appelbaum PC, Spangler SK, Jacobs MR. ß-Lactamase production and susceptibilities to amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate, cefoxitin, imipenem, and metronidazole of 320 non Bacteroides fragilis Bacteroides isolates and 129 fusobacteria from 28 U.S. Centers. Antimicrob Agents Chemother 1990;34:1546–50. [7] Cuchural GJ, Michael JR, Malamy M, et al. b-lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrob Agents Chemother 1986;30:645–8. [8] Heldberg M, Edlund C, Lindqvist L, et al. Purification and characterisation of an imipenem hydrolysing metallo-b-lactamase from Bacteroides fragilis. J Antimicrob Chemother 1992;29:105 – 13. [9] Brazier JS, Citron DM, Goldstein EJC. A selective medium for Fusobacterium spp. J. Appl Bacteriol. 1991;71:343–6. [10] National Committee for Clinical Laboratory Standards. Methods for antimicrobial testing of anaerobic bacteria-second edition; Approved standard, NCCLS publication M11-A3. National Committee for Clinical Laboratory Standards, Villanova Pa. 1993. [11] National Committee for Clinical Laboratory Standards. Reference dilution procedure for antimicrobial testing of anaerobic bacteria. Approved standard, NCCLS publication M-11T. National Committee for Clinical Laboratory Standards, Villanova Pa. 1979.
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[12] Ericsson HM, Sherris JC. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol Microbiol Scand 1971;27. [13] Wilkins TD, Chalgren S. Medium for use in antibiotic susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 1976;10:926 – 8. [14] Acar J, Carret G, Cavallo JD, et al. Communique´ de l’antibiogramme de la Socie´te´ Franc¸aise de microbiologie. Valeurs critiques pour l’antibiogramme. Path Biol 1997;45:1 – 20. [15] Wexler HM. Susceptibility testing of anaerobes bacteria:myth, magic, or method? Clin Microbiol Rew 1991;4:470 – 84. [16] Grollier G, Mory F, Quentin C, et al. Survey of anaerobic susceptibility patterns: A French multicentric study. Path Biol 1994;42:498 – 504. [17] Bland S, Sedallian A, Grollier G, et al. In vitro activity of three carbapenems biapenem, imipenem and meropenem and some other antibiotics against anaerobic bacteria. Path Biol 1995;43:289 – 93. [18] Patey O, Varon E, Podglajen I, et al. Multicentre survey in France of the antimicrobial susceptibilities of 416 blood culture isolates of the Bacteroides fragilis group. J Antimicrob Chemother 1994;33:1029 – 34. [19] Dubreuil L, Houcke I, Mouton Y, et al. In vitro activities of nitazoxanide and tizoxanide against anaerobes and aerobic organisms. Antimicrob Agents Chemother 1996;40:2266 – 70. [20] Dubreuil L, Houcke I, Singer E, et al. Comparative activity of Faropenem and other antimicrobial agents against 462 clinical anaerobes (abstract). International Congress of Antimicrobial Agents and Chemotherapy (ICAAC) Toronto, Ontario, Canada 1996;222. [21] Eley A, Harmas AE, Bennett KW. In vitro activity of twelve antibiotics against clinical isolates of Bilophila wadsworthia. J Antimicrob Chemother 1992;29:468 – 70.
Original article
Survey of anaerobic susceptibility patterns: A French multicentre study F. Mory a, A. Lozniewski a, S. Bland b, A. Sedallian b, G. Grollier c, F. Girard-Pipau d, M.F. Paris e, L. Dubreuil e,* a
Chu de Nancy, 29, a6. de Lattre de Tassigny, 54037 Nancy cedex, France Centre Hospitalier d’Annecy, a6. de Tresums, 74011 Annecy cedex, France c Hopitaux de la Mile´trie,350, a6. Jacques Coeur, 86021 Poitiers cedex, France d Hoˆpital Henri-Mondori 81, a6. du Mal de Lattre de Tassigny, 94000 Caete´il, France e Faculte´ de Pharmacie de Lille, 3, Rue Laguesse, BP83, 59006 Lille cedex, France b
Received 23 March 1998; received in revised form 1 May 1998; accepted 18 May 1998
Abstract In 1996, the in vitro antibiotic susceptibility of 463 anaerobes was measured in five hospitals using the reference agar dilution method. None of the 209 B. fragilis group strains showed resistance to imipenem or ticarcillin-clavulanic acid. High resistance rates (29%) were observed for cefotetan and clindamycin. b-Lactamase production was detected respectively in 64% of the Pre6otella and 7% of the Fusobacterium strains. Because the same standardized methods were used for many years, the authors were able to evaluate the evolution of antibiotic resistance. Clindamycin resistance had increased within the B. fragilis group (from 14% in 1992 to 29% in 1996) and also among strains of clostridia (32%), P. acnes (18%) and Peptostreptococcus (28%). In the B. fragilis group multidrug resistance was unlikely to occur. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Anaerobes; Antibiotic patterns; Resistance
1. Introduction Evolution of antibiotic resistance has been previously observed among anaerobes, in France [1,2]. New resistance mechanisms have emerged such as 5-nitroimidazole resistance among Bacteroides fragilis [3,4]. Penicillin resistance linked with b-lactamase production has been described for some Clostridium species [5], Pre6otella and Fusobacterium [6]. Some B. fragilis strains are now resistant to carbapenems [7,8]. The aim of our study was to evaluate the antibiotic susceptibility of anaerobic bacteria by determining the MICs of 11 anti-anaerobic drugs on a wide range of species. Thus, 463 anaerobes were collected in 1996 from five hospitals * Correponding author. Tel./fax: + 33 3 20 964008; e-mail: ldubreui @phare.univ-lille2.fr. 0924-8579/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0924-8579(98)00041-7
which are localized in different areas of our country: North, East, West, South-east and Center.
2. Materials and methods
2.1. Bacterial strains Anaerobic bacteria were isolated from human clinical samples during 1996. They were identified according to standard methods, then subcultured in a Rosenow medium (Diagnostics Pasteur, Marnes la Coquette, France). If they were not used immediately to determine MICs, the broth was kept frozen at− 20°C. Before being tested, the bacteria were checked for purity by subculturing on Colombia blood agar (bioMe´rieux, Marcy l’e´toile, France), either on laked blood-
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Table 1 Antimicrobial activities of 11 antibiotics against the Bacteroides fragilis group Resistance rates (%)a
Microorganism (N°)
MIC (mg/l)
Antibiotic
range
MIC 50%
MIC 90%
Bacteroides fragilis (109) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefotaxime Imipenem Clindamycin Metronidazole
0.12–\128 0.03–64 0.5–\128 0.03–8 0.12–\128 0.5– \128 0.015–2 0.015–\128 0.015–8
16.00 0.25 16.00 0.50 8.00 16.00 0.12 0.25 0.50
\128.0 4.0 128.0 4.0 128.0 128.0 0.5 64.0 2.0
41.0 2.0 15.0 0.0 13.0 20.0 0.0 22.0 1.8
B. thetaiotaomicron (28) and B. o6atus (9) Amoxicillin 4–\128 +Clavulanic acid 0.03–128 Ticarcillin 4–\128 +Clavulanic acid 0.25–16 Piperacillin 2– \128 Cefoxitin 2–128 Cefotetan 2–\128 Cefotaxime 32– \128 Imipenem 0.015–4 Clindamycin 0.03– \128 Metronidazole 0.25–4
32.0 0.5 32.0 1.0 32.0 16.0 64.0 64.0 0.12 1.0 0.5
\128.00 16.00 \128.00 16.00 \128.00 64.00 \128.00 \128.00 0.25 \128.00 2.00
86 10 18 0 32 15 61 53 0 39 0
Bacteroides 6ulgatus (33) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
64.00 0.50 64.00 0.50 32.00 8.00 32.00 32.00 0.12 0.50 0.50
\128.0 16.0 \128.0 8.0 \128.0 32.0 \128.0 \128.0 0.5 \128.0 2.0
67 6 42 0 36 9 26 50 0 29 6
Bacteroides distasonis – caccae -merdae group (21) Amoxicillin 0.5–\128 +Clavulanic acid 0.03–16 Ticarcillin 4– \128 +Clavulanic acid 0.12–32 Piperacillin 1– \128 Cefoxitin 1–64 Cefotetan 1–\128 Cefotaxime 1– \128 Imipenem 0.03–1 Clindamycin 0.03–\128 Metronidazole 0.03–2
32.00 2.00 32.00 4.00 16.00 16.00 64.00 4.00 0.25 0.50 0.25
\128 8 \128 16 \128 64 128 \128 1 \128 1
62 0 29 0 33 16 59 36 0 32 0
Bacteroides of the group fragilis (209) Amoxicillin 0.12– \128 +Clavulanic acid 0.03–128 Ticarcillin 0.5–\128 +Clavulanic acid 0.03–64 Piperacillin 0.12– \128 Cefoxitin 1–128 Cefotetan 0.25–\128 Cefotaxime 0.5– \128 Imipenem 0.012–4 Clindamycin 0.015– \128 Metronidazole 0.015–32
32.00 0.25 32.00 1.00 16.00 8.00 32.00 16.00 0.12 0.25 0.25
\128.0 8.0 \128.0 8.0 \128.0 32.0 128.0 128.0 0.5 \128.0 2.0
55.0 3.8 22.2 0.0 22.5 7.1 28.6 32.0 0.0 29.2 1.9
a
0.5–\128 0.03–128 2– \128 0.06–64 1– \128 2–64 1–\128 0.5– \128 0.03–1 0.03– \128 0.03–32
Resistance rates calculated using French breakpoints [14] described in the section material and methods.
F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
231
Table 2 Antimicrobial activities of 11 antibiotics against gram negative bacilli other than the B. fragilis group Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Porphyromonas (19) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–0.50 0.03–0.25 0.25–0.50 0.25–0.50 0.12 0.25–0.50 0.06–1.00 0.06–1.00 0.015–0.06 0.03–0.25 0.03–0.25
5]Pre6otella spp (56) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.06–\128.00 0.03–16.00 0.12–64.00 0.06–16.00 0.12–\128.00 0.06–4.00 0.03–16.00 0.06–64.00 0.015–0.12 0.03–0.06 0.015–2.00
Fusobacterium spp (27) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–8.00 0.03–1.00 0.06–4.00 0.06–2.00 0.06–2.00 0.06–2.00 0.03–32.00 0.06–4.00 0.015–0.25 0.03–8.00 0.03–0.25
Resistance rates(%) MIC 50%
MIC 90%
0.50 0.25 0.25 0.25 0.12 0.25 0.12 1.00 0.03 0.06 0.12
0.50 0.25 0.50 0.50 0.12 0.50 0.25 1.00 0.03 0.12 0.25
0 0 0 0 0 0 0 0 0 0 0
8.00 0.06 1.00 0.12 16.00 1.00 4.00 2.00 0.03 0.03 0.50
64.00 1.00 16.00 2.00 32.00 4.00 16.00 8.00 0.06 0.06 1.00
25 0 0 0 2 0 0 2 0 0 0
0.06 0.03 0.06 0.03 0.06 0.06 0.06 0.12 0.03 0.03 0.03
1.00 0.06 0.50 0.25 0.25 0.25 0.25 0.50 0.06 0.12 0.25
0 0 0 0 0 0 0 0 0 4 0
kanamycin-vancomycin plates (Serlabo,Bonneuil/ Marne,France) for Bacteroides spp. or josamycinnorfloxacin plates for Fusobacteria [9]. Purity was also checked by Gram staining. The Bacteroides fragilis group (209) included Bacteroides fragilis (109) B. thetaiotaomicron (28) B. o6atus (9) B. distasonis (16), B. caccae (3), B. merdae (2) B. uniformis (4) B. stercoris (3) B. eggerthii (2) B. 6ulgatus (33). Other strains were as follows: B. capillosus (1) B. spanchnicus (1) Anaerorhabdus furcosus (1) Dialister pneumosintes (1) Pre6otella intermedia /nigrescens (15) P. loescheii (1) P. melaninogenica (12) P. buccalis (1) P. buccae (7) P.oris (6), P. oralis (1) P. 6eroralis (1) P. bi6ia (10) P. disiens (2) Porphyromonas asaccharolytica (17) P. gingi6alis (2) Fusobacterium nucleatum (20) F. necrophorum (7) Bilophila wadsworthia (3) Veillonella par6ula (6) Clostridium difficile (37) C. perfringens (16) C. bifermentans (1) C. butyricum (1) C. clostridioforme (2), C. fallax (1), C. innocuum (4), C.
paraputrificum (1) C. ramosum (6) C. sphenoides (2), C. tertium (3), C. sp (4) Eubacterium lentum (17) E. biforme (1) E. 6entriosum (1) Pseudoramibacter alactolyticus (2) Propionibacterium acnes (8) P. granulosum (1), Bifidobacterium bifidum (5) Peptostreptococcus anaerobius (4) P. asaccharolyticus (4) P. indolyticus (1) P. magnus (10) P.micros (1) P. pre6otii (4) Ruminococcus gna6us (2). For quality control and assessment of reproducibility, four reference ATCC control strains were added to each batch of tests. The ATCC control strains, advocated by the M11 A3 Norma [10] of the National Committee for Clinical Laboratory Standards (NCCLS), were Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, Clostridium perfringens ATCC 13124 and Eubacterium lentum ATCC 43055. b-Lactamase production was assessed using a nitrocefin disk (cefinase® bioMe´rieux). As all strains of the B. fragilis group have a chromosomal cephalosporinase,
232
F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
Table 3 Antimicrobial activities of 11 antibiotics against gram positive bacilli Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Resistance rates (%) MIC 50%
MIC 90%
2 1 32 16 8 128 32 64 4 \128 1
0 0 0 0 0 89 3 95 0 54 0
C. difficile (37) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–2 0.06–2 0.5–32 0.5–32 0.12–8 32–128 8–\128 32–128 1–8 0.12–\128 0.03–4
0.50 0.50 8.00 8.00 4.00 64.00 16.00 64.00 4.00 4.00 0.25
Other clostridia (41) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
0.03–1 0.03–2 0.12–32 0.06–32 0.03–16 0.06– \128 0.03–\128 0.12–32 0.015–4 0.03–\128 0.03–2
0.06 0.06 4.00 0.50 0.06 1.00 4.00 2.00 0.06 0.25 0.25
1.0 0.5 16.0 16.0 8.0 32.0 \128.0 32.0 1.0 16.0 1.0
0 0 0 0 0 20 32 0 0 32 0
Non sporulated gram+ bacilli (35) Amoxicillin 0.03–2 +Clavulanic acid 0.03–2 Ticarcillin 0.25–64 +Clavulanic acid 0.06–64 Piperacillin 0.06–32 Cefoxitin 0.06–32 Cefotetan 0.03–64 Cefotaxime 0.06– \128 Imipenem 0.03–1 Clindamycin 0.03–16 Metronidazole 0.25–\128
0.50 0.50 4.00 1.00 1.00 8.00 8.00 1.00 0.12 0.03 0.50
2 1 32 2 16 16 64 \128 0.5 4 1
0 0 0 0 0 0 33 40 0 11 50
the test was applied only to gram negative bacilli other than B. fragilis and clostridia.
2.2. Antimicrobial agents Powders of known potency were supplied by the manufacturers or their French subsidiaries: metronidazole (Specia), amoxicillin, amoxicillin, ticarcillin, clavulanic acid (Smith Kline Beecham), piperacillin (Wyeth), cefoxitin, imipenem (Merck Sharp Dohme), cefotetan (Zeneca-Pharma),cefotaxime (Hoechst Marion Roussel), clindamycin (Upjohn) and metronidazole (Rhoˆne Poulenc-Rorer).
2.3. MIC determinations MICs were determined using a reference agar dilution method according to the Norma M11T [11] of the
NCCLS with further recommendations from the Norma M11 A3 [10]. Stock solutions of 512 mg/l of each antibiotic were prepared and two-fold dilutions were carried out in distilled water [12]. Metronidazole was first dissolved in 2 ml of methanol and then distilled water was added to the solution.Clavulanic acid was used in a fixed concentration of 2 mg/l when combined with amoxicillin or ticarcillin. Each antibiotic was incorporated into Wilkins Chalgren [13] agar (Oxoid-Unipath, Dardilly, France) with 5% added sterile defibrinated blood to provide adequate support for the growth of Fusobacteria, Peptostreptococcus spp, and Eubacterium spp. Plates contained serial doubling dilutions of antimicrobial agents (from 128 to 0.003 mg/l). All plates were used within 24 h of preparation. An actively growing culture in Rosenow medium was diluted in a Schaedler broth (BioMe´rieux) to reach and match the 0.5 point of a Mac Farland standard. The
F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
233
Table 4 Antimicrobial activities (MIC, in mg/l) of 11 antibiotics against anaerobic cocci Microorganism (N°)
MIC (mg/l)
Antibiotic
Range
Peptostreptococus spp (24) and Ruminococcus gna6us (2) Amoxicillin 0.03–16.0 +Clavulanic acid 0.03–0.5 Ticarcillin 0.06–8.0 +Clavulanic acid 0.03–8.0 Piperacillin 0.03–1.0 Cefoxitin 0.06–4.0 Cefotetan 0.06–32.0 Cefotaxime 0.12–4.0 Imipenem 0.015–4.0 Clindamycin 0.03–64.0 Metronidazole 0.03–2.0 Veillonella spp (6) Amoxicillin +Clavulanic acid Ticarcillin +Clavulanic acid Piperacillin Cefoxitin Cefotetan Cefotaxime Imipenem Clindamycin Metronidazole
Resistance rates (%) MIC 50%
MIC 90%
0.12 0.03 1.00 1.00 0.12 0.50 0.50 1.00 0.03 0.12 0.50
0.50 0.25 4.00 2.00 0.50 2.00 8.00 4.00 0.25 32.00 1.00
0.03–2.00 0.03–4.00 0.5–32.00 1–32.00 1–64.00 0.06–4.00 0.12–8.00 0.12–2.00 0.015–1.00 0.03–0.12 0.12–1.00
inocula were approximately 7.5×107 to 1×108 UFC/ ml. For fastidious strains, the following were added to the Schaedler broth: Hemin (5 mcg/l), menadione (0.1 mcg/l), sodium bicarbonate (1 g/l) and 1 ml of laked blood per tube containing 100 ml. The previous inocula (2–3 ml) were delivered by a Steers replicator (Mast Systems, London) and resulted in a final inocula of 105 CFU per spot of inoculation on the agar plates. At the end of each series of tests, two plates of Wilkins Chalgren agar were inoculated without antimicrobial agent. One plate was incubated anaerobically to determine the viability of the organisms and to serve as a control for growth comparison and the other aerobically to indicate possible aerobic contamination. Incubation of the tested plates containing the antibiotics was performed in an anaerobic chamber (Forma Scientific) at 35° – 36° C. Reading of the MICs was made after 48 h of incubation. The MIC for an organism was noted as the lowest concentration of an antimicrobial agent yielding no growth. Resistance rates were calculated in relation with French breakpoints (mg/l), which are identical for most antibiotics to those of the NCCLS with the exception of amoxicillin (\16), amoxicillin-clavulanate ( \ 16/2), metronidazole (\4),clindamycin (\2) [14].
0 0 0 0 0 0 0 0 0 28 0 0 0 0 0 0 0 0 0 0 0 0
3. Results Over all 463 anaerobes, no resistance could be detected for either imipenem or the ticarcillin clavulanic acid combination. MIC50 and MIC90 values for each antibiotic together with resistance rates and for each group of anaerobic bacteria are listed in the tables.
3.1. Bacteroides fragilis group (Table 1) Seven strains were resistant to amoxicillin-clavulanate but susceptible to ticarcillin- clavulanic acid and cefoxitin: including two B. fragilis, three B. thetaiotaomicron and two B. 6ulgatus strains. Among these seven strains, only the B. fragilis strains remained susceptible to cefotetan. Resistance to cefotetan was rare for B. fragilis, but frequent for other species such as B. thetaiotaomicron and B. distasonis. Half of the clindamycin resistant strains were susceptible to all other antibiotics with the exception of amoxicillin. Metronidazole resistance (CMI\4 mg/l) was observed for four strains but cross-resistance to clindamycin and metronidazole did not occur. Crossresistance to ticarcillin, piperacillin, cefoxitin, cefotetan, cefotaxime and clindamycin was observed for one of each strain of B. fragilis and B. uniformis.
AMC (\16/2) 3.8 0.0
0.0 1.8
AMX (\16)
55.0
7.4
0.0 28.7
Antibiotic (breakpoint in mg/l)
0.0 10.9
0.0
22.2
TIC (\64)
0 0
0
0
TCC (\64/2)
0.0 11.8
4.3
22.5
PIP (\64)
25.6 11.7
0.0
7.1
CFX (\32)
11.0 16.9
0.0
28.6
CTT (\32)
37.3 24.0
1.1
32.0
CTX (\32)
0 0
0
0
IMI (\8)
27.9 25.0
4.8
29.2
CLN (\2)
15.0 5.6
0.0
1.9
MOL (\4)
AMX, amoxicillin; AMC, amoxicillin-clavulanate; TIC, ticarcillin; TCC, ticarcillin+clavulanic acid; PIP, piperacillin; CFX, cefoxitin; CTT, cefotetan; CTI, cefotaximes; IMI, imipenem; CLN, clindamycin and MOL, metronidazole.
B. fragilis group Other gram negative bacilli Gram positive All
Anaerobes
Table 5 Antibiotic resistance rates (%) in anaerobes
234 F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
F. Mory et al. / International Journal of Antimicrobial Agents 10 (1998) 229–236
3.2. Gram negati6e bacilli other than the B. fragilis group (Table 2) Porphyromonas strains were very susceptible (CMIB 1 mg/l) to all the antibiotics investigated. b-Lactamase production was never detected by the nitrocefin disk. In contrast b-lactamase production was frequent (84%) among Pre6otella species. Resistant strains were observed for amoxicillin, piperacillin (one strain of P. bi6ia) and cefotaxime (one strain of P. oris). Black pigmented Pre6otella were all inhibited by coamoxi-clav and ticarcillin at a concentration of 1mg/l whereas higher concentrations are needed to inhibit all the non-pigmented Prevotella. Most Fusobacterium species demonstrated low antibiotic MIC values and only one strain of F. necrophorum was resistant to clindamycin. 7.4% of the Fusobacterium strains produced a penicillinase. The three strains of Bilophila wadsworthia were resistant to amoxicillin and piperacillin (b-lactamase+) but suceptible to amoxicillin-clavulanate, ticarcillin, cefoxitin, cefotetan, imipenem, clindamycin and metronidazole. Considering all the 115 gram negative rods other than the B. fragilis group, no resistance could be detected for amoxicillin-clavulanate, ticarcillin either alone or combined with clavulanic acid, cefoxitin, cefotetan, imipenem and metronidazole.
3.3. Gram positi6e bacilli (Table 3) C. difficile strains were susceptible to all penicillins and to metronidazole. We observed intrinsic resistance to imipenem (low level) and to all cephalosporins. Resistance to clindamycin occurred for 54% of strains investigated. In contrast C. perfringens was susceptible to all antibiotics studied herein. Other clostridia were susceptible to penicillins, imipenem and metronidazole whereas resistance occurred with cefoxitin (20%), cefotetan and clindamycin (both 32%).Non sporulated gram positive bacilli were all inhibited by penicillins, cefoxitin and imipenem. Resistance to cefotaxime was common among strains of Eubacterium. Most strains of Bifidobacterium and all propionibacteria were resistant to metronidazole. Resistance to clindamycin was also common (22%) among P. acnes. The 113 gram positive bacilli were all inhibited by amoxicillin, ticarcillin, piperacillin and imipenem.
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4. Discussion Assessment of any evolution in antibiotic resistance must be treated with great caution because the numerous methodological problems may result in invalid comparisons[15]. Change in antibiotic testing methodology may reflect spurious evolution in antibiotic resistance rates but we have used the same constant NCCLS methodology for the last twenty years in our outwork on possible antibiotic resistance. Previous French reports [1,2,16–20] demonstrated that resistance to imipenem and to metronidazole remained rare in the B. fragilis group. Decreased susceptibility [3,4] to metronidazole (MIC from 4 to 16 mg/l) was observed for less than 5% of the strains [17,19]. Resistance to most antibiotics is stable whereas resistance to clindamycin is still increasing in France. Clindamycin resistance rates increased from 14% in 1992 [1], to 20.6%, 1995 [20], reaching 29.2% in this study (Table 5). b-Lactamase production is common among Pre6otella and is becoming more frequent; in contrast it remains rare among strains of Fusobacterium.. In relation probably to the cephalosporinase activity of Pre6otella enzymes [6], ticarcillin had greater potency than piperacillin. Although we isolated few B. wadswortia our results are in agreement with those of other articles [21]. The penicillins were very effective against non sporulated gram positive anaerobes. Propionibacterium spp is naturally resistant to metronidazole (MIC= 32 mg/l). Higher clindamycin rates are observed among P. acnes and Peptostreptococcus strains. Gram positive cocci are generally known to be susceptible to metronidazole, the resistance rate being below 10% in France [16,17]. Resistance to clindamycin has increased among gram positive cocci from 9.8% in 1995 [20] to 28% at present. Over the whole range of anaerobes, higher resistance rates were observed in this study for clindamycin compared to previous results. Multidrug resistance to all b-lactams, clindamycin and metronidazole has not been observed at present among B. fragilis. Although resistance to some antibiotics is increasing, the lack of cross resistance to clindamycin and metronidazole is of interest. The good activity of imipenem and of the two combinations of clavulanic acid with either amoxicillin or ticarcillin offers some optimism for the future.
3.4. Anaerobic cocci (Table 4) References Peptostreptococci were inhibited by low concentrations of amoxicillin with the exception of one strain of P. micros (CMI=8 mg/l). No b-lactamase could be detected and MICs for ticarcillin and piperacillin were 2 and 1 mg/l, respectively. A high prevalence of resistance to clindamycin (28%) was observed. All Veillonella strains were inhibited by all the antibiotics investigated.
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