Evaluation of the in vitro activity of ertapenem and nine other comparator agents against 337 anaerobic bacteria

International Journal of Antimicrobial Agents 28 (2006) 25–35

Evaluation of the in vitro activity of ertapenem and nine other comparator agents against 337 anaerobic bacteria J. Behra-Miellet, L. Dubreuil ∗ , L. Calvet Facult´e de Pharmacie, 3 rue du Professeur Laguesse, BP83, 59006 Lille Cedex, France Received 24 November 2005; accepted 30 January 2006

Abstract Ertapenem activity in vitro was compared with that of nine reference antibiotics against 337 anaerobes by determining minimal inhibition concentrations (MICs). Amongst 246 Gram-negative anaerobes, 4, 8, 3, 4, 7, 2 and 52 strains showed resistance to ertapenem, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin/tazobactam, cefoxitin, imipenem and clindamycin, respectively, and all strains were inhibited by metronidazole. Ertapenem MIC50 values were 0.5, 0.25, 0.06 and ≤0.03 mg/L for the Bacteroides fragilis group, Prevotella spp., fusobacteria and Gram-positive cocci, respectively. Overall resistance rates were 2.1%, 51.3%, 2.4%, 1.2%, 1.5%, 7.1%, 0.6%, 22% and 1.5% for ertapenem, amoxicillin, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin/tazobactam, cefoxitin, imipenem, clindamycin and metronidazole, respectively. Ertapenem showed a broad spectrum and good activity against anaerobes. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Ertapenem; Carbapenems; Anaerobes; MIC determination

1. Introduction Ertapenem is a once-daily parenteral 1-␤-methyl carbapenem administered as a single agent via either the intravenous or intramuscular route [1–4]. It exhibits a long plasma half-life (t1/2 ) of ca. 4 h owing to its high plasma protein binding [5] and stability against human renal dehydropeptidase. This antimicrobial agent appears to respond to a medical need owing to its pharmacokinetics, which is superior to that of imipenem and meropenem, its effectiveness against a wide range of Gram-negative and Gram-positive aerobic and anaerobic bacteria [6–15] and its potential as an outpatient parenteral antimicrobial therapy [16]. Although ertapenem shows limited activity against Enterococcus spp., Pseudomonas aeruginosa and other non-fermentative Gramnegative bacteria associated with nosocomial infections, this new carbapenem is, however, promising for the treatment of a variety of community-acquired infections [17–26]

∗

Corresponding author. Tel.: +33 3 2096 4008; fax: +33 3 2096 4008. E-mail address: [email protected] (L. Dubreuil).

owing to its good tissue penetration [27], excellent tolerability and safety profile [28]. Thus, ertapenem can be used for the treatment of intra-abdominal and gynaecological infections, community-acquired pneumonia in Europe, and worldwide for skin infections and complicated urinary tract infections. However, the severity of bacterial infections is often due to anaerobes, especially when aerobic and anaerobic microorganisms are both involved [8,29]. These anaerobes are difficult to isolate and are rarely tested for their susceptibility to antibiotics. Inadequate empirical antibiotic therapy results in increased treatment failure and mortality. Thus, the aim of this study was to assess the anti-anaerobic activity of ertapenem. Its antimicrobial activity was evaluated against 337 clinical anaerobic isolates together with appropriate reference and control strains. Nine comparator agents were also tested to determine minimal inhibitory concentrations (MICs) using an agar dilution method according to approved standards of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards (NCCLS)) [30–35]. Resistance rates were calculated using NCCLS breakpoints.

0924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2006.01.011

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J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

MIC90 4 >64 4 >256 8 16 32 1 >256 4

2. Materials and methods

MIC (mg/L) Range ≤0.03–>128 ≤0.06–>64 ≤0.06–>64 ≤0.125–>256 ≤0.125–>256 ≤0.125–>256 ≤0.25–256 ≤0.03–64 ≤0.06–>256 ≤0.06–16 4

37 4 42 1 2 15

1 6 2 2

2 1 10

3

31c 2c 26 2 2

1

2

23

18 1 2

>256 256 2c 128 64 32

7 3

7 16 3 10 5 12 45

3

54 1 32 9 19 42

16 8

13

4 17 1 5 2 9 15 28

15 27 29

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole Bacteroides spp. (all strains) (n = 150)a

34b

24 35b 6b

35 5

29 2 30 1 11b 21 20 6

28 4

17 13 76

2 18 1 4 2 12 7 2 2 11 14 1 19 3 4 1 10 16 0.5 36 1 22 0.25 30

MIC distribution (mg/L) 0.03 0.06 0.125 17b 3 1 4b 48b 27 3b 43b 69b Antibiotic

Standard laboratory powders were supplied as follows: ertapenem and imipenem (Merck Sharp Dohme, Paris, France), cefoxitin (Merck, Rahway, NJ), clindamycin (Pfizer, Paris, France), metronidazole (SanofiAventis, Paris, France), amoxicillin, ticarcillin and clavulanic acid (GlaxoSmithKline, Nanterre, France) and piperacillin and tazobactam (Wyeth-Ayerst, West Chester, PA). Antimicrobials were reconstituted according to each manufacturer’s instructions. Serial two-fold dilutions of antimicrobial agents were prepared on the day of the test and were added to the media according to the recommendations of Ericsson and Sherris [38] in various concentrations in distilled water: amoxicillin (0.06–64 mg/L), ticarcillin and piperacillin (0.125–256 mg/L), cefoxitin (0.25–128 mg/L), ertapenem, imipenem and metronidazole (0.03–128 mg/L) and clindamycin (0.06–256 mg/L). Metronidazole was first dissolved in 2 mL of methanol.

Microorganism (no. of strains)

2.2. Antimicrobial agents

Table 1 Activities of 10 antibiotics against Bacteroides fragilis group strains and other Gram-negative anaerobes

The 337 obligate anaerobic bacterial strains were isolated from human clinical specimens from October 2002 to March 2003 and were identified according to classical methods [36]. Bacteroides fragilis group strains and Clostridia spp. were isolated mainly from abdominal infections and blood cultures, whereas Prevotella, Porphyromonas and Fusobacterium spp. were isolated from sputum and pleural aspirates and infected ear, nose and throat. The anaerobe strains isolated were studied together with appropriate reference and control strains of the American Type Culture Collection (ATCC): (B. fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, Eggerthella lenta ATCC 43055 and Clostridium perfringens ATCC 13124). The major groups tested included B. fragilis group spp. (147), Prevotella spp. (52), Fusobacterium spp. (30), Clostridium spp. (45) and Peptostreptococcus spp. (28). The number and species of isolates tested are given in Tables 1 and 2. Anaerobes were subcultured in Rosenow medium (BioRad, Marnes-la-Coquette, France) and stored at −20 ◦ C in a freezer if not used immediately. Before each test, bacterial purity was checked by subculturing in Columbia agar medium (bioM´erieux, Marcy l’Etoile, France) with 5% lysed defibrinated horse blood (Eurobio, Les Ulis, France) or on laked blood kanamycin–vancomycin plates (Serlabo, Paris, France) for Bacteroides and Prevotella spp., or on josamycin–norfloxacin plates for fusobacteria [37]. Gram staining was also performed. ␤-Lactamase production was determined using the nitrocefin disk method (C´efinase® ; bioM´erieux).

MIC50 0.5 16 0.125 32 0.5 1 8 0.125 0.5 1

2.1. Bacterial strains

Prevotella spp. (n = 52)d

Porphyromonas spp. (n = 8)e

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole

16b

22b

2 18b 27b

3 4 4 17b 31b 46b

10 25b 19b

4 8

5 6b 6b

2 1 1 7b 7b 7b

6 20b 9 2

5 4 16

2 1 1

1 2

4 4 9 3 4 1 3 6

3 1 2 2 2 13

3 6 2 1

2 6 2 5 1

6

6

1 1

1 4

14c

7

3 7

1

1 1 4

1 2

1 1

2 2 2

1 1 1 1 1

1

1

1 2

3 1 1

1 1 1

1 1 1

2 2

1

1 1 1

1

6

3

3

1

1

7 27b 26b

2 25b 24b

3

4

1 1

26b 24b

10 1 6 2 1 1 2 1

1

4b 1b

1 26b 26b 27b

2 1 1 2 1

1 1

7b 1 1 1

7b

14b

7 1 7

1

≤0.03–16 ≤0.06–>64 ≤0.06–16 ≤0.125–>256 ≤0.125–16 ≤0.125–4 ≤0.25–32 ≤0.03–2 ≤0.06–>256 ≤0.06–16

0.25 8 ≤0.06 4 ≤0.125 ≤0.125 2 0.06 0.125 0.5

4 >64 2 >256 2 1 16 0.5 256 16

0.06–0.125 ≤0.06–0.25 ≤0.06–0.25 ≤0.125–16 ≤0.125–16 ≤0.125–16 ≤0.25–16 ≤0.03–0.25 ≤0.06–128 ≤0.06–4 ≤0.03–4 ≤0.06–64 ≤0.06–32 ≤0.125–128 ≤0.125–64 ≤0.125–16 ≤0.25–32 ≤0.03–4 ≤0.06–64 ≤0.06–8

0.06 0.06 ≤0.06 ≤0.125 ≤0.125 ≤0.125 ≤0.25 ≤0.03 ≤0.06 ≤0.06

2 ≤0.06 0.125 0.5 0.25 1 1 0.5 0.5 0.25

Ertapenem 47b 22 6 39 38 31 24 25 10 2 2c ≤0.03–>128 0.5 4 6 3 2 5 1 1 22 60 38 4 45c ≤0.06–>64 16 >64 Amoxicillin 59b b c Amoxicillin/clavulanic acid 112 33 38 23 11 8 7 3 3 5 1 2 ≤0.06–>64 0.125 2 2 4 3 7 5 12 38 47 17 27 3 24 ≤0.125–>256 16 256 Ticarcillin 57b b 37 30 12 21 12 6 12 1 1 2 1 ≤0.125–>256 0.25 4 Ticarcillin/clavulanic acid 111 2 4 19 10 21 12 22 2 1 2 2 ≤0.125–>256 ≤0.125 16 Piperacillin/tazobactam 149b b 3 6 41 53 51 17 6 1 ≤0.25–256 8 16 Cefoxitin 68 36 40 32 22 20 3 1 2 ≤0.03–64 0.06 1 Imipenem 90b b Clindamycin 93 16 23 33 13 15 1 8 1 3 7 7 26 ≤0.06–>256 0.25 >256 b 2 9 50 85 20 15 4 10 ≤0.06–16 1 4 Metronidazole 51 MIC, minimal inhibitory concentration; MIC50 and MIC90 , MICs for 50% and 90% of the organisms, respectively. a Including strains of the B. fragilis group: B. fragilis (86), B. thetaiotaomicron (21), B. ovatus (9), B. vulgatus (9), B. distasonis (7), B. uniformis (5), B. caccae (3), B. merdae (2), B. stercoris (2) and Bacteroides spp. (3); and strains of other Bacteroides spp.: B. capillosus (1), and B. ureolyticus (2). b ≤MIC value indicated. c >1/2 MIC value indicated. d Including P. loescheii (23), P. oralis (7), P. buccae (4), P. intermedia (3), P. bivia (3). P. oris (1), P. corporis (1), P. melaninogenica (2) and Prevotella spp. (8). e Including P. asaccharolytica (6) and Porphyromonas spp. (2). Gram-negative anaerobes (all strains) (n = 246)

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

Fusobacterium spp. (n = 30)f

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole

f Including F. nucleatum (22), F. necrophorum (4), F. mortiferum (1), F. varium (1) and Fusobacterium spp. (2).

27

28

Table 2 Activities of 10 antibiotics against Gram-positive anaerobes Microorganism (no. of strains)

Antibiotic

MIC distribution (mg/L) 0.03

Clostridium difficile (n = 10)

Other Clostridium spp. (n = 20)b

Other non-sporulated Gram-positive bacilli (n = 18)c

Gram-positive cocci (n = 28)e

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole

0.125

MIC (mg/L) 0.25

0.5

1

6 6

1 3 3

2

4 3

8 5

6 5 1a

2 12a 14a

2 4 1

4a 12a 1a

5 5a

1

1 1a 2a

1 1 4 1a 1a 3a

2 6 1 2a 6 1 1 5 7 9

3a 4

2a 3a 6a 9a

6 1

2 13a 13a

14a

1 4 3 3 3a 5a 2a 1

15a 1a 23a 18a 23a

23a 8a 2a

2 2 6 2 21a 23a 22a 2 2 2

5

1 1 1 1

1 1

2 3

3 10

1 1

1 1 1

2

3 4 6

6 6 1

1 1 1 10

5

2 2 2 1 17 1 3 7

256

>256

1

1

3 5 1 1

1

2

2 2 5 2 4 1 2

1 1

1

6

3 1

1 1 2 2 1 1

1

9 2 2 1

10 2 2

2 2 3

2

1 6

1

1 1 1 1

1

4 1 1 2

1 2 1 1

1 1 2 1

1

6

2

1 1 3 3

3

1

2

1 1 1

1 1 1

1

1

3d

1 1 1

4 2 3 1 6

1 1 2

1 2

1 1 1

1

2

1

6

1

1

Range 4–16 0.5–2 0.25–1 16–64 8–64 8–64 64–64 4–8 4–>256 ≤0.06–2

MIC50

MIC90

0.5 0.5 0.25 16 8 2 8 0.5 4 0.25

16 2 0.5 32 32 16 64 2 >256 2

≤0.03–16 ≤0.06–0.5 ≤0.06–0.5 0.25–4 ≤0.125–2 ≤0.125–1 ≤0.25–2 ≤0.03–8 ≤0.06–256 0.25–2

1

1 1

1

1 1 3 3a 2 9 11

3

128

4 3

1

2 1 1 2 3 4 4 3 2 2

64

7

1 1 10 3 1 10

4 7 4 1 2 1 1 5 4 4

32

1

3 3

1a

16 2

0.06–8 ≤0.06–8 ≤0.06–1 ≤0.125–256 ≤0.125–128 ≤0.125–128 ≤0.25–128 ≤0.03–2 ≤0.06–>256 ≤0.06–4 ≤0.03–1 ≤0.06–4 ≤0.06–4 ≤0.125–64 ≤0.125–32 ≤0.125–32 0.25–16 ≤0.03–0.25 ≤0.06–2 ≤0.06–>64 ≤0.03–2 ≤0.06–8 ≤0.06–2 ≤0.125–1 ≤0.125–16 ≤0.125–8 ≤0.25–16 ≤0.03–0.5 ≤0.06–128 ≤0.06–1

≤0.03 ≤0.06 ≤0.06 ≤0.125 ≤0.125 ≤0.125 0.5 ≤0.03 0.25 0.25

0.25 0.5 0.125 1 0.5 0.25 2 0.125 64 1

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

Clostridium perfringens (n = 15)

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole

0.06

Gram-positive anaerobes (all strains) (n = 91)

1 3d 4 1

2

1 1 1 1

Ertapenem 33a 5 11 12 6 2 1 10 8 3 Amoxicillin 44a 11 9 16 5 3 1 2 a Amoxicillin/clavulanic acid 52 9 11 13 4 1 1 Ticarcillin 25a 5 16 10 4 4 1 14 8 a Ticarcillin/clavulanic acid 33 10 10 4 5 2 12 4 9 a Piperacillin/tazobactam 39 8 8 5 7 3 6 10 2 Cefoxitin 12a 28 6 7 10 4 4 3 a Imipenem 40 5 4 15 6 5 4 7 5 Clindamycin 31a 2 12 9 5 3 7 5 3 a 5 19 17 18 10 4 1 2 1 Metronidazole 10 MIC, minimal inhibitory concentration; MIC50 and MIC90 , MICs for 50% and 90% of the organisms, respectively. a ≤MIC value indicated. b Including C. innocuum (9), C. clostridioforme (7), C. sporogenes (2), C. bifermentans (1) and C. subterminale (1). c Including Bifidobacterium spp. (7), Bifidobacterium lactis (1), Eubacterium spp. (3), Eggerthella lenta (1), Collinsella aerofaciens (1), Pseudoramibacter sp. (1) and Propionibacterium acnes (4). d >1/2 MIC value indicated. e Including Anaerococcus prevotii (6), Finegoldia magna (9), Peptostreptococcus spp. (7), Micromonas micros (3), Peptostreptococcus anaerobius (2) and Ruminococcus sp. (1).

2 1 2 16

1

7

≤0.03–16 ≤0.06–8 ≤0.06–4 ≤0.125–256 ≤0.125–128 ≤0.125–128 ≤0.25–128 ≤0.03–8 ≤0.06–>256 ≤0.06–>64

0.125 0.125 ≤0.06 0.5 0.5 0.25 1 0.125 0.5 0.25

8 1 0.5 32 32 16 64 4 128 4

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

29

2.3. Determination of MICs Susceptibility testing was performed by the reference agar dilution method according to the methods of the NCCLS (M11-A5 and M11-A6) [30,31]. Brucella agar (Difco, Le Pont de Claix, France) with 5% lysed horse blood (Eurobio) was the basal medium. Amoxicillin and ticarcillin were diluted alone or with clavulanic acid at a constant concentration of 2 mg/L, as is usual in most European countries, and piperacillin was used with tazobactam at a constant concentration of 4 mg/L. To conform to the interpretative categories of the NCCLS, a plate was added containing 8/4 mg/L amoxicillin/clavulanic acid (for resistance rate estimation). An actively growing subculture was diluted in Brucella broth (Difco) to a 0.5 McFarland standard. The inocula were ca. 7.5 × 107 or 108 colony-forming units (CFU)/mL. For fastidious strains, 0.1 mL of lysed blood (Eurobio) per 10 mL culture tube could be added to the medium as well as 5 ␮g/L haemin (Sigma, Saint-Quentin-Fallavier, France), 0.1 ␮g/L menadione (Merck-Eurolab, Fontenay-sous-bois, France) and 1 g/L sodium bicarbonate. A Steers replicator (Mast Systems, London, UK) was used to deliver inocula of ca. 105 CFU per spot (2–3 ␮L). Plates were incubated in an anaerobic chamber (Concept +/400; Jouan, Saint-Herblain, France). Two plates of Brucella blood agar were inoculated without antimicrobial agent and were incubated anaerobically as a control for growth comparison and viability determination of anaerobes, and aerobically to detect any aerobic contamination. MICs were read after 48 h of incubation at 35–36 ◦ C or after 72 h for fastidious strains. For ␤-lactam/␤lactamase inhibitor combinations, MICs correspond to the ␤-lactam concentration (clavulanic acid or tazobactam were used at a constant concentration in plates, as described above). Susceptibility and resistance rates were then calculated. Differences were observed between French breakpoint values in the 2005 report of the Comit´e de l’Antibiogramme de la Soci´et´e Franc¸aise de Microbiologie (CA-SFM) [39] and those of the NCCLS, even if the specific values for anaerobes were almost the same. High breakpoints used for this study are those of the NCCLS recommendations (as indicated in the NCCLS M11-A6 norma [31]). If breakpoints were not available (i.e. for amoxicillin), the French CA-SFM values were used. Low breakpoints were those of the NCCLS recommendations except for amoxicillin where the French CA-SFM breakpoints were used (≤4 mg/L for Gram-positive anaerobes and ≤0.5 mg/L for Gram-negative anaerobes) or for metronidazole. For metronidazole, the high breakpoints were the same but the low CA-SFM breakpoint enabled the detection of strains with reduced susceptibility to metronidazole (MICs of 8 mg/L and 16 mg/L). For amoxicillin/clavulanic acid, the low breakpoint of 4/2 mg/L is the same according to CA-SFM and NCCLS breakpoints. Resistance rates estimated at high breakpoints for MICs >16/2 mg/L for the CA-SFM or 16/8 mg/L (>8/4 mg/L) for the NCCLS were

0.25 8 0.125 16 0.25 ≤0.125 4 0.06 0.25 1

Range

≤0.03–>128 ≤0.06–>64 ≤0.06–>64 ≤0.125–>256 ≤0.125–>256 ≤0.125–>256 ≤0.25–256 ≤0.03–64 ≤0.06–>256 ≤0.06–>64

0.06

27 103a 164a

41 124a 61a

0.03

80a

130a

9

24 1 2

8 3b

4

45b 2b 28 3 3 1

4 1 19 2 3 22 2 7 1

64

1

38 5 55 10 4 20

32

4 12

5 60 3 52 16 32 55

Ertapenem Amoxicillin Amoxicillin/clavulanic acid Ticarcillin Ticarcillin/clavulanic acid Piperacillin/tazobactam Cefoxitin Imipenem Clindamycin Metronidazole All anaerobes (n = 337)

MIC, minimal inhibitory concentration; MIC50 and MIC90 , MICs for 50% and 90% of the organisms, respectively. a ≤MIC value indicated. b >1/2 MIC value indicated.

16 8

18 24 3 13 18 18 57 5 13 5 35 2 8 9 14 24 51 8 8 19

4 2

25 4 9 11 26 17 13 7 18 30 33 10 15 13 16 24 9 25 18 103

1 0.5

44 18 36 20 40 12 28 28 42 67 51 12 49 7 47 10 80a 47 35 28

0.25 Antibiotic Microorganism (no. of strains)

Table 3 Activities of 10 antibiotics against 337 anaerobic bacteria

17 17 42 82a 144a 188a

3.2. Gram-negative anaerobes 3.2.1. Bacteroides fragilis group and other Bacteroides spp. The B. fragilis group represented 147 strains of the 150 Bacteroides spp. tested. No resistance was found for the three other Bacteroides spp. isolates for the nine antibiotics, with MICs ≤1 mg/L. The MIC values and the percentage of resistant strains to ten antibiotics are shown in Tables 1 and 4, respectively. Ertapenem MICs ranged from ≤0.03 mg/L to >128 mg/L (Table 1). Of the B. fragilis group, 138/147 strains had MICs ≤4 mg/L, showing 93.9% of the anaerobes as susceptible to ertapenem. Two B. fragilis strains (1.4% of the B. fragilis group) were resistant to ertapenem, with a MIC >128 mg/L (Table 4). Imipenem showed lower MIC50 and MIC90 values compared with ertapenem (Table 1), but the two strains of B. fragilis described above were also resistant to this carbapenem (as well as to all ␤-lactams) and one of the two strains was resistant to clindamycin. Amongst the 147 strains of the B. fragilis group, 98.6% were inhibited by ertapenem. For amoxicillin/clavulanic acid, seven strains of the B. fragilis group were detected as resistant. No resistance to metronidazole was seen. The percentages of susceptible isolates of the B. fragilis group to metronidazole calculated using the NCCLS and CA-SFM low breakpoints were 98% and 95.9%, respectively (Table 5). Resistance rates of these Bacteroides spp. to ticarcillin/clavulanic acid, piperacillin/tazobactam, cefoxitin and clindamycin were 2%, 2.7%, 4.8% and 26.5%, respectively.

2b

128

256

>256

MICs for the control strains (for each batch and series) were in the ranges recommended by the NCCLS M11-A6 standard [31]. Therefore, the MIC values could be interpreted for each strain and antimicrobial agent. Distributions of the MIC values, as well as MIC50 and MIC90 values (MICs for 50% and 90% of the organisms, respectively), of each antibiotic for each group of bacteria are listed in Table 1 for Bacteroides spp. and Gramnegative bacteria other than Bacteroides spp., in Table 2 for the Gram-positive anaerobes and in Table 3 for all anaerobes studied. Resistance rates of the strains, calculated using the NCCLS breakpoints (if available) are presented in Table 4. For amoxicillin, the French values of the CA-SFM are used corresponding to Gram-negative and Gram-positive anaerobes. Susceptibility rates are represented in Table 5. For metronidazole, NCCLS and CA-SFM low breakpoints are used.

1

3.1. MIC determination

44 18 7

3. Results

33

MIC50 MIC (mg/L) MIC distribution (mg/L)

identical in this study as well as in all studies that we have carried out until now.

4 >64 1 128 8 16 32 1 256 4

MIC90

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

0.125

30

amoxicillin (the French value of the CA-SFM is given) and for amoxicillin/clavulanic acid (the CA-SFM value >16/2 mg/L gives the same results as

0 0 0 0 0 0 0 0 0 0 0 27.8 0 5.5 1.5 2.3 0 0 1.4 0 0 0 0.8 0 0 0 0 0 0 0.6 5.8 4.8 2.5 4.8 0 0 0 2.8 100 0 35 0 0 18.7 7.1

19.8 42.9 32.5 26.5 17.3 25 6.7 21.1 50 13.3 50 0 17.9 24.2 22

Imipenem (≥16 mg/L)

27.9 33.3 37.5 31.3 13.5 0 3.3 22 0 0 10 0 0 2.2 16.6

3.2.3. All Gram-negative anaerobes MIC results and resistance rates to antibiotics are shown in Tables 1 and 4, respectively. Four strains (1.6%) and two strains (0.8%) of 246 Gram-negative anaerobes were resistant to ertapenem (as well as to piperacillin/tazobactam) and to imipenem, respectively, whereas no resistance was found for metronidazole among Gram-negative anaerobes. Amongst these strains, 94.3% and 99.2% were susceptible to ertapenem (as well as to amoxicillin/clavulanic acid) and to imipenem, respectively. For metronidazole, 94.3% or 95.9% of the strains were susceptible using CA-SFM (≤4 mg/L) or NCCLS (≤8 mg/L) low breakpoints, respectively (Table 5). For piperacillin/tazobactam, 88.2% of the strains were found to be susceptible. d Value taking into account strains with a positive nitrocefin test.

Amoxicillin (>16 mg/Lb ; >1 mg/Lc ) 96.5 95.2 97.5 96.6 55.8d 0 6.7d 70.3 0 0 0 0 0 0 51.3 Ertapenem (≥16 mg/L)

31

3.2.2. Other Gram-negative anaerobes The activity of the ten antibiotics against each type of Gram-negative anaerobe is shown in Table 1 and the resistance rates of the strains tested are given in Table 4 (except for the six Veillonella strains). Resistance to ertapenem was reported for only two Prevotella strains (3.8%), with MICs not exceeding 16 mg/L. A rate of 100% of susceptible strains to ertapenem was noted amongst the Porphyromonas, Fusobacterium (Table 5) and Veillonella spp., whereas MICs were ≤4 mg/L for 90.4% of the Prevotella strains. No resistant strains of Prevotella, Fusobacteria and Veillonella spp. to imipenem, metronidazole, ticarcillin/clavulanic acid, piperacillin/tazobactam or cefoxitin were found. For metronidazole, the susceptible strain rate of Prevotella spp. reached 86.5% at the NCCLS and CA-SFM low breakpoints. Resistance to amoxicillin and ticarcillin was detected for 51.9% and 13.5% of the Prevotella strains and for 6.7% and 3.3% of the Fusobacterium strains by calculating MICs. In fact, ␤lactamase production was detected amongst 29/52 (55.8%) Prevotella spp. and two Fusobacterium strains. Thus, these strains were reported as resistant to amoxicillin. No strains of Porphyromonas and Veillonella were resistant to the ␤lactams tested or to metronidazole. Nine strains of Prevotella (17.3%), two Porphyromonas strains (25%) and two Fusobacterium strains (6.7%) were shown to be resistant to clindamycin.

Amoxicillin/ clavulanic acid (≥32/2 mg/L) 5.8 9.5 0 4.8 0 0 3.3 3.3 0 0 0 0 0 0 2.4

Bacteroides fragilis (n = 86) 2.3 Bacteroides thetaiotaomicron (n = 21) 0 Other B. fragilis group (n = 40) 0 B. fragilis group (all strains) (n = 147) 1.4 Prevotella spp. (n = 52) 3.8 Porphyromonas spp. (n = 8) 0 Fusobacterium spp. (n = 30) 0 Gram-negative anaerobes (all strains) (n = 246) 1.6 Clostridium difficile (n = 10) 20 Clostridium perfringens (n = 15) 6.7 Other Clostridium spp. (n = 20) 0 Other non-sporulated Gram-positive bacilli (n = 18) 0 Gram-positive cocci (n = 28) 0 Gram-positive anaerobes (all strains) (n = 91) 3.3 All anaerobes (n = 337) 2.1 CA-SFM, Comit´e de l’Antibiogramme de la Soci´et´e Franc¸aise de Microbiologie. a Breakpoints corresponding to the National Committee for Clinical Laboratory Standards recommendations, except for ≥16/8 mg/L). b Value corresponding to Gram-positive anaerobes (CA-SFM recommendations). c Value corresponding to Gram-negative anaerobes (CA-SFM recommendations).

Microorganism (no. of strains)

Table 4 Resistance rates to the antibiotics tested

% of resistant strains (breakpointsa given in parentheses)

Ticarcillin (≥128 mg/L)

Ticarcillin/ clavulanic acid (≥128/2 mg/L) 3.5 0 0 2 0 0 0 1.2 0 0 5 0 0 1.1 1.2

Piperacillin/ tazobactam (≥128/4 mg/L) 4.7 0 0 2.7 0 0 0 1.6 0 0 5 0 0 1.1 1.5

Cefoxitin (≥64 mg/L)

Clindamycin (≥8 mg/L)

Metronidazole (≥32 mg/L)

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

3.3. Gram-positive anaerobes 3.3.1. Clostridia As shown in Tables 2 and 4, 3/45 strains of clostridia (2/10 Clostridium difficile and 1/15 C. perfringens) were resistant to ertapenem and 17/45 Clostridium spp. were resistant to clindamycin and cefoxitin. One strain of Clostridium showed resistance to ticarcillin/clavulanic acid or piperacillin/tazobactam. In contrast, no resistance of clostridia was found to amoxicillin, amoxicillin/clavulanic acid, imipenem or metronidazole. Amongst the C. difficile strains, 30% were susceptible to ertapenem, ticarcillin, ticarcillin/clavulanic acid and piperacillin/tazobactam, and 60% were susceptible to imipenem and 100% to metronidazole (Table 5).

32

Microorganism (no. of strains)

% of susceptible strains (breakpointsa given in parentheses) Ertapenem (≤4 mg/L)

Amoxicillin (≤4 mg/Lb ; ≤0.5 mg/Lc )

Amoxicillin/clavulanic acid (≤4/2 mg/L)

Ticarcillin (≤16 mg/L)

Ticarcillin/clavulanic acid (≤16/2 mg/L)

No. % No. % No. % No. % No. Bacteroides fragilis (n = 86) 79 91.9 1 1.2 79 91.9 32 37.2 82 Bacteroides thetaiotaomicron 20 95.2 1 4.8 19 90.5 6 28.6 21 (n = 21) Other B. fragilis group (n = 40) 39 97.5 0 0 38 95 11 27.5 40 B. fragilis group (all strains) 138 93.9 2 1.4 136 92.5 49 33.3 143 (n = 147) Prevotella spp. (n = 52) 47 90.4 24 46.2 50 96.2 34 65.4 52 Porphyromonas spp. (n = 8) 8 100 8 100 8 100 8 100 8 Fusobacterium spp. (n = 30) 30 100 28 93.3 29 96.7 29 96.7 29 Gram-negative anaerobes (all 232 94.3 70 28.5 232 94.3 128 52 241 strains) (n = 246) Clostridium difficile (n = 10) 3 30 10 100 10 100 3 30 3 Clostridium perfringens (n = 15) 14 93.3 15 100 15 100 15 100 15 Other Clostridium spp. (n = 20) 17 85 19 95 20 100 16 80 17 Other non-sporulated 18 100 18 100 18 100 17 94.4 17 Gram-positive bacilli (n = 18) Gram-positive cocci (n = 28) 28 100 27 96.4 28 100 28 100 28 Gram-positive anaerobes (all 80 87.9 89 97.8 91 100 79 86.8 80 strains) (n = 91) All anaerobes (n = 337) 312 92.6 159 47.2 323 95.8 207 61.4 321 CA-SFM, Comit´e de l’Antibiogramme de la Soci´et´e Franc¸aise de Microbiologie; NCCLS, National Committee for Clinical Laboratory Standards. a Breakpoints corresponding to the CA-SFM and NCCLS recommendations. b Breakpoint corresponding to Gram-positive anaerobes (CA-SFM recommendations). c Breakpoint corresponding to Gram-negative anaerobes (CA-SFM recommendations). d Breakpoint corresponding to CA-SFM recommendations.

Piperacillin/tazobactam (≤8/4 g/L)

Cefoxitin (≤16 mg/L)

Imipenem (≤4 mg/L)

Clindamycin (≤2 mg/L)

Metronidazole (≤4 mg/Ld ; ≤8 mg/L)

% 95.3 100

No. 79 14

% 91.9 66.7

No. 75 15

% 87.2 71.4

No. 84 21

% 97.7 100

No. 69 12

% 80.2 57.1

No. 81; 83 21; 21

% 94.2; 96.5 100; 100

100 97.3

28 121

70 82.3

35 125

87.5 85

40 145

100 98.6

27 108

67.5 73.5

39; 40 141; 144

97.5; 100 95.9; 98

100 100 96.7 98

52 8 28 217

100 100 93.3 88.2

51 8 29 222

98.1 100 96.7 90.2

52 8 30 244

100 100 100 99.2

42 6 28 193

80.8 75 93.3 78.5

45; 45 8; 8 29; 30 232; 236

86.5; 86.5 100; 100 96.7; 100 94.3; 95.9

30 100 85 94.4

3 15 16 14

30 100 80 77.8

0 15 10 18

0 100 50 100

6 14 20 18

60 93.3 100 100

0 12 9 18

10; 10 15; 15 20; 20 10; 11

100; 100 100; 100 100; 100 55.6; 61.1

100 87.9

28 76

100 83.5

28 71

100 78

28 86

100 94.5

23 62

82.1 68.1

28; 28 83; 84

100; 100 91.2; 92.3

95.3

293

86.9

293

330

97.9

255

75.7

315; 320

93.5; 95

86.9

0 80 45 100

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

Table 5 Anaerobes susceptible to the antibiotics tested (compared %)

J. Behra-Miellet et al. / International Journal of Antimicrobial Agents 28 (2006) 25–35

3.3.2. Other non-sporulated Gram-positive bacilli According to Tables 2 and 5, the 18 other nonsporulated Gram-positive bacilli (including 4 strains of Propionibacterium acnes) were susceptible to ertapenem, imipenem, amoxicillin, amoxicillin/clavulanic acid, cefoxitin and clindamycin, but 94.4% were susceptible to ticarcillin and ticarcillin/clavulanic acid, 77.8% were susceptible to piperacillin/tazobactam and 55.6% (for CA-SFM breakpoint ≤4 mg/L) or 61.1% (for NCCLS breakpoint ≤8 mg/L) were susceptible to metronidazole (Table 5). No resistance of propionibacteria was detected to antibiotics, except for metronidazole (4/4 strains with MICs ≥32 mg/L), and all were susceptible to antibiotics except metronidazole. One strain of Bifidobacterium only showed resistance to metronidazole, whereas the non-sporulated Gram-positive bacilli were all non-resistant to the other antibiotics tested. 3.3.3. Gram-positive cocci MICs are given in Table 2 and percentages of resistance in Table 4. All strains (n = 28) were susceptible to metronidazole and to the ␤-lactams tested (with NCCLS breakpoints, or with CA-SFM low breakpoints if not available), except for amoxicillin (96.4% of susceptible strains) (Table 5). Amongst the 28 strains, 82.1% were susceptible to clindamycin and 17.9% were resistant to this antimicrobial agent (Table 4). 3.3.4. All Gram-positive anaerobes No resistance of Gram-positive anaerobes was shown to amoxicillin, amoxicillin/clavulanic acid and imipenem. Of the 91 strains, 97.8%, 100% and 94.5% were susceptible to these antibiotics, respectively (Table 5). Only 3.3% of Gram-positive anaerobes showed resistance to ertapenem (with 87.9% of susceptible strains amongst these bacteria). These results are similar to those obtained for ticarcillin, ticarcillin/clavulanic acid and piperacillin/tazobactam. For cefoxitin and clindamycin, resistance was observed for 18.7% and 24.2% of the strains, respectively (with 78% and 68.1% of susceptible strains). Resistance to metronidazole (5.5%) is largely due to P. acnes. 3.4. All anaerobes The activity of ten antibiotics against all of the anaerobes tested are reported in Table 3 (calculated according to the NCCLS breakpoints, or CA-SFM breakpoints if not available). As shown in Table 4, resistance rates for ertapenem and imipenem were 2.1% and 0.6%, respectively. Amoxicillin/clavulanic acid, ticarcillin, cefoxitin and clindamycin were less active than ertapenem, with 2.4%, 16.6%, 7.1% and 22% of resistant strains, respectively, whereas ticarcillin/clavulanic acid, piperacillin/tazobactam and metronidazole showed resistance rates near 1.5%. Amongst the 337 anaerobes tested, susceptibility rates (at low breakpoints) were 92.6%, 97.9%, 47.2%, 95.8%, 61.4%, 95.3%, 86.9%, 86.9% and 75.7% for ertapenem, imipenem, amoxicillin,

33

amoxicillin/clavulanic acid, ticarcillin, ticarcillin/clavulanic acid, piperacillin/tazobactam, cefoxitin and clindamycin, respectively (Table 5). For metronidazole, 95% or 93.5% of the anaerobes tested were found to be susceptible using the low NCCLS breakpoint or the CA-SFM breakpoint, respectively.

4. Discussion Although MICs were >128 mg/L for two strains of B. fragilis, ertapenem concentrations of 0.5 mg/L and 4 mg/L represent the MIC50 and MIC90 for the B. fragilis group, which is known to be particularly resistant to antibiotics amongst the anaerobes. Only imipenem had concentrations of 0.125 mg/L and 1 mg/L for MIC50 and MIC90 , respectively. If we compare our results with those of previous studies, similar values were found by Hoellman et al. [9] (MIC50 and MIC90 of 0.5 mg/L and 2 mg/L), Pelak et al. [10] (MIC50 and MIC90 of 1 mg/L and 4 mg/L), Wexler in a recent review [13] (MIC90 1–4 mg/L) and Zhanel et al. [26] (MIC50 and MIC90 of 0.5 mg/L and 2 mg/L). In our study, strains of the B. fragilis group with amoxicillin MICs >1 mg/L were ␤-lactamase-positive (tested by nitrocefin). This chromosomal enzyme is able to inactivate cefalothin, cefuroxime and third-generation cephalosporins. As shown in Table 4 and largely demonstrated previously, cefoxitin, a cephamycin antibiotic, still proves to have good activity (resistance rate of 4.8%) whereas resistance to cefotetan and clindamycin (26.5% in our study) is high in France. Resistance to metronidazole was not observed amongst the B. fragilis group strains, but decreased susceptibility to metronidazole was observed according the CA-SFM or NCCLS low breakpoints (4.1% or 2%, respectively), similar to previous studies (2–3%) [40,41]. The resistance rate to imipenem of B. fragilis is still low (2.3%) and is generally due to carbapenemase production that confers cross-resistance to all ␤-lactams. It is alarming to notice that one of the two strains resistant to ertapenem and imipenem (MIC >128 mg/L and MIC = 64 mg/L, respectively) was classified as intermediate for metronidazole according to the CA-SFM low breakpoint (MIC = 8 mg/L) and was also resistant to all the ␤-lactams tested, but not to clindamycin. These strains were isolated from the intra-abdominal sputum and vaginal infection of patients previously untreated by antimicrobial agents. Another B. fragilis strain showed MICs of 2, 8 and 16 mg/L for imipenem, ertapenem and metronidazole, respectively. The other strains intermediate for metronidazole were susceptible to the carbapenems tested. The five strains susceptible to imipenem and resistant to amoxicillin/clavulanic acid could correspond to strains overproducing ␤-lactamase and/or with a lack of porins [42,43]. In summary, ertapenem, imipenem and metronidazole demonstrated the lowest in vitro resistance rates against the B. fragilis group frequently involved in intra-abdominal infections. A good in vitro susceptibility rate of 93.9% was found at the low NCCLS

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breakpoint for ertapenem, comparable with that of amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, imipenem and metronidazole, and better than the susceptibility rate of piperacillin/tazobactam (82.3%), cefoxitin (85%) and clindamycin (73.5%). The high resistance rates noted for the other Gramnegative anaerobes Prevotella and Fusobacterium spp. to amoxicillin were due to ␤-lactamase production detected amongst 29/52 Prevotella strains and 2/30 Fusobacterium strains by the nitrocefin test. This ␤-lactamase production is also the cause of the poor antimicrobial activities commonly described for first- and second-generation cephalosporins and to a lesser extent for cefotaxime against Prevotella spp. Ertapenem demonstrated good activity against Prevotella spp., with only two strains resistant to ertapenem (MICs of 16 mg/L) from intra-abdominal or cutaneous sputum after digestive surgery and in the latter case after treatment by amoxicillin/clavulanic acid, and inhibited all Fusobacteria; no resistance was observed against imipenem, the ␤lactam/␤-lactamase inhibitor combinations and metronidazole, which showed excellent activity. In their recent review on ertapenem, Zhanel et al. [26] showed that for Fusobacterium and Prevotella spp., ertapenem MICs could reach 8 mg/L and 2 mg/L, respectively. As far as ertapenem MIC90 values are concerned, the value found in our study (Table 1) was a little higher than the MIC90 reported by Wexler [13] for Prevotella (4 mg/L and 0.125–0.5 mg/L in our study and in the review, respectively); for Fusobacterium, values were similar. For Gram-positive anaerobes, propionibacteria resistance to metronidazole is intrinsic. Ertapenem showed good activity against these anaerobes, with only 3 strains (amongst 91) resistant to this antibiotic (2 C. difficile and 1 C. perfringens). No previous treatment by antimicrobial agent(s) was specified for the corresponding patients. Wexler [13] also described a few strains of C. difficile and Clostridium innocuum with MICs of 8 mg/L and 4 mg/L, respectively. All the antibiotics tested showed good activity against the 28 Gram-positive cocci, except clindamycin (17.9% of strains were resistant to this antimicrobial agent). In our study, the 87.9% rate of susceptible Gram-positive strains to ertapenem was the same for ticarcillin/clavulanic acid and even better than the 83.5% value calculated for piperacillin/tazobactam. As the latter is often used as an empirical treatment for skin and skin-structure infections or to treat polymicrobial complicated intra-abdominal infections as well as acute pelvic infections [29,44,45], these results require consideration. Ertapenem could constitute a promising alternative treatment. In our study, the rates of susceptible strains for all anaerobes were 92.6% for ertapenem and 86.9% for piperacillin/tazobactam. These values are a little lower than those found by Goldstein et al. [44] who studied the in vitro activity of ertapenem compared with piperacillin/tazobactam against anaerobes recovered from patients with skin and skinstructure infections: 137 (97.2%) of 141 anaerobes investi-

gated (including 53% of Gram-negative species) were susceptible to ertapenem and 138 (97.9%) were susceptible to piperacillin/tazobactam. In conclusion, 330 of the 337 anaerobes in this study were inhibited by ertapenem at concentrations of ≤8 mg/L (corresponding to 2.1% of resistant strains). For the other antimicrobial agents, resistant rates <5% were observed only for amoxicillin/clavulanic acid (2.4%), ticarcillin/clavulanic acid (1.2%), piperacillin/tazobactam (1.5%), imipenem (0.6%) and metronidazole (1.5%). Clindamycin resistance was noted for many species, with a resistance rate of 22% of all the strains investigated. Further clinical trials will be needed to assess the efficacy of ertapenem, particularly as a treatment for various community-acquired infections.

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