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Doi:10.1016/s0924-8579(02)00352-7International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 b-lactamase production in Provotella and in vitro susceptibilities to L. Dubreuil a,, J. Behra-Miellet a, C. Vouillot a, S. Bland b, A. Sedallian b, F. Mory c a Faculte´ de Pharmacie, 3, rue du Professeur Laguesse, BP83, Lille Cedex 59006, France Received 21 May 2002; accepted 1 July 2002 This study looked for b-lactamase production in 100 Provotella isolates. MICs were determined for amoxycillin, ticarcillin, amoxycillin'/clavulanate, cephalothin, cefuroxime, cefixime, cefpodoxime and cefotaxime using the reference agar dilution method(standard M11 A4, NCCLS). Beta-lactamase activity was detected in 58 of the 100 isolates, 24 of 46 black-pigmented Provotella and34 of 54 non-pigmented Provotella. All b-lactamase-negative strains were susceptible to all b-lactam antibiotics with the exception ofcefuroxime and cefixime. Overall, resistance rates of Provotella strains were lower for ticarcillin (8%) and celefotaxime (12%) thanfor the other cephalosporins. All Provotella isolates were susceptible to amoxyillin and were all inhibited by 2 mg/l or less amoxcillin.
# 2002 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.
Keywords: Provotella ; b-lactamase production; Antibiotic resistance Beta-lactamase production steadily increased duringthe 1980s. Clinical failures of penicillin treatment for Anaerobic Gram-negative bacilli other than the orofacial infections have been documented to- Bacteroides fragilis group have been shown to be gether with reports suggesting that previous penicillin involved in clinical infections, either alone or mixed therapy increases the incidence of penicillin-resistant with other species. Although Fusobacterium and Pro- Provotella . Resistance to metronidazole, combina- votella are the main anaerobic bacilli isolated from tions of penicillins and b-lactamase inhibitors or imipe- human pathological samples, b-lactamase production nem is rare and many laboratories now consider identification and susceptibility testing of Provotella to be unnecessary. Oral cephalosporins have a limited anti- in pyogenic orofacial and upper respiratory tract infec- anaerobic activity, especially against the B. fragilis tions (chronic sinusitis and otitis). In elderly people, group, but this activity may vary for other species.
micro-aspiration of saliva may inoculate the lungs and Some oral cephalosporins are used to treat community- cause pulmonary infections. There have been scattered acquired anaerobic infections, although their anti-anae- reports on the antibiotic susceptibility of Provotella robic activities have not been recently reviewed. The species, but only rare studies have been based on a large activity of b-lactam antibiotics is decreasing due to the number of Provotella strains apart from strains increasing incidence of b-lactamase-producing isolates.
derived from periodontal isolates. Until the latter half of It is, therefore, important to monitor resistance both to the 1970s, penicillins and cephalosporins were generally new drugs but also to widely prescribed antibiotics in still effective against oral Gram-negative anaerobes.
community-acquired infections in order to guide theirempirical use.
In this study, we collected strains of Provotella and tested the susceptibility of a sufficient number of isolates * Corresponding author. Tel./fax: '/33-3-2096-4008E-mail address: (L. Dubreuil).
to antimicrobial agents marketed in the community. As 0924-8579/02/$30 # 2002 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved.
PII: S 0 9 2 4 - 8 5 7 9 ( 0 2 ) 0 0 3 5 2 - 7 L. Dubreuil et al. / International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 Table 1b-lactamase production in Provotella isolates the b-lactamase of Provotella has been reported to be a fold dilutions of antimicrobial agents were prepared on cephalosporinase ticarcillin was added to this the day of the test and added to the media in various study, although this drug is available only in hospitals.
Beta-lactamase production was tested by using the (Cefinase†, Biome´rieux, France). According to Appel- A total of 100 Provotella strains were isolated from baum’s recommendations disks which did not turn human clinical sources (blood culture, pleural fluid, from yellow to red within 15 min at room temperature chronic sinusitis and otitis, lung abscess, etc.). and from vaginal samples for P. bivia and P. disiens . Strainsisolated from stools were excluded. The 100 isolates were studied together with appropriate reference andcontrol strains (B. fragilis ATCC 25285, B. thethaiotao- Susceptibility testing was performed by the reference micron ATCC 29741, Eggerthella lenta ATCC 43055 agar dilution method according to the standards of and C. perfringens ATCC 13124). All isolates were the National Committee for Clinical and Laboratory identified by standard criteria The P. intermedia Standards (M11-A4). Brucella blood agar (Difco, group includes three phenotypically indistinguishable France) with 5% lysed horse blood (Eurobio, Les Ulis, species: P. intermedia , P. nigrescens and P. pallens France) was the basic medium. Amoxycillin and ticar- this group is referred to as P. intermedia in this cillin were diluted with clavulanate tested at a constant paper. The numbers and species of isolates tested are concentration of 2 mg/ml, as is usual in most European countries. To comply with the interpretative categoriesof the NCCLS, we added two plates containing 8/4 and 16/8 mg/ml of amoxycillin and clavulanate combinations,respectively. A Mast multipoint inoculator was used to Standard laboratory powders were obtained from the deliver inocula of approximately 105 CFU per spot.
following sources: amoxycillin, ticarcillin, clavulanic Plates were incubated in an anaerobic chamber (Don Whitley†, AES, Combourg, France) and MICs were Marly-le-Roi, France), cefotaxime, cefixime, cefpodox- determined after 48 h of incubation at 35 8C and then ime (Aventis, Paris). Antimicrobials were reconstituted examined. Resistance rates were calculated at the according to each manufacturer’s instructions. Serial 2- NCCLS breakpoints. French (CA-SFM) breakpoints L. Dubreuil et al. / International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 were used for some oral cephalosporins, due to the lack Beta-lactamase production was detected in 58 of the 100 isolates, including 24 of 46 black-pigmented Provo- tella (52%) and 34 of 54 non-pigmented Provotella(63%). Beta-lactamase production varied according to the species (Beta-lactamase production wasmore frequent for P. melaninogenica (14/21) , P. bivia(15/20), and P. intermedia (7/11). The amoxycillin MIC breakpoint separating b-lactamase-positive and b-lacta-mase-negative isolates was ]/0.5 mg/l ( 3.2. In vitro susceptibility to b-lactam antibiotics Most antimicrobial agents were more active against b- lactamase-negative isolates than against b-lactamase- positive isolates (). All b-lactamase-negative strains were susceptible to all b-lactams, with theexception of one strain of P. oris and one strain of P.
bivia that were resistant to both cefuroxime and cefixime. Beta-lactamase-negative strains were all in-hibited by either 0.25 mg/l of amoxycillin'/clavulanate,1 mg/l of ticarcillin or cefpodoxime, or 2 mg/l of Amoxycillin-resistant isolates were detected in all species of Provotella with the exception of P. denticola ;the highest resistance rate was observed for Provotella bivia . Among b-lactamase-positive strains, amoxycillin MICs were higher for non-pigmented Provotella than for black-pigmented isolates (Amoxycillin MIC90 for b-lactamase-positive isolates was 9 dilutionshigher than that for b-lactamase-negative isolates.
All isolates in this study were susceptible to amoxycillin'/clavulanate. Most isolates were suscepti- ble to 64 mg/l of ticarcillin. Although the MICs ofcefpodoxime and cefotaxime were generally fairly simi- lar, cefpodoxime, which has lower antibiotic break- points, had poor activity against b-lactamase-positive for cefixime. Among the black-pigmented isolates, only one strain of P. intermedia was resistant to cefotaxime; most cefotaxime-resistant strains were, therefore, notpigmented (P. oralis , P. buccae and P. bivia ).
On the whole, resistance rates of Provotella strains were lower for ticarcillin (8%) and cefotaxime (12%) than for the other cephalosporins. All Provotella isolates inhibited by 2 mg/l or less of amoxycillin'/clavulanate were susceptible to this combination.
L. Dubreuil et al. / International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 Table 3Activity of three penicillins against 100 strains of Provotella : distribution of MICs Amoxycillinb-lactamase negative Provotella Amoxycillin-clavulanateb-lactamase negative Provotella Ticarcillinb-lactamase negative Provotella Table 4Activity of five cephalosporins against 100 strains of Provotella : distribution of MICs Cephalothinb-lactamase negative Provotella Cefuroximeb-lactamase negative Provotella Cefpodoximeb-lactamase negative Provotella Cefotaximeb-lactamase negative Provotella However, several studies have also reported lowerprevalences The various frequencies observedin other reports could be explained by geographical differences and isolate sampling differences .
Most studies of antimicrobial susceptibility of anae- robic bacteria are based on serial isolates from clinical 4.2. In vitro susceptibility to b-lactam antibiotics material and, therefore, focus on the more commonspecies and combine the results for less common The amoxycillin MIC distribution clearly distin- isolates. Consequently, little information is available guished Provotella isolates into two groups according about the susceptibility of less commonly isolated to b-lactamase production (breakpoint ]/0.5 mg/l). This Provotella species. Beta-lactamase production was low value was identical to the penicillin G breakpoint (19 Á/40%) in the 1980s and subsequently in- previously proposed by Matto et al. . Beta-lactamase creased to reach 70% of strains in 1994 . Since then, detection is relatively difficult and could be replaced in the frequency of b-lactamase-producing strains has routine by determination of amoxycillin MICs using the remained in the 60 Á/70% range . The prevalence E -test and the 0.38 mg/l breakpoint. As recommended of b-lactamase production of Provotella species is in by the NCCLS, all Gram-negative anaerobes should be agreement with several previous French studies .
screened for b-lactamase production with nitrocephin L. Dubreuil et al. / International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 Table 5Comparative in vitro activity of b-lactam antibiotics against Provotella species according to b-lactamase production Organism (number tested) and antimicrobial agent L. Dubreuil et al. / International Journal of Antimicrobial Agents 21 (2003) 267 Á/273 Organism (number tested) and antimicrobial agent and, if positive, should be reported as resistant to  King A, Downes J, Nord CE, Phillips I. Antimicrobial suscept- penicillin and ampicillin. No guidelines have been ibility of non-Bacteroides fragilis group anaerobic Gram-negativebacilli in Europe. Clin Microbiol Infect 1999;5:404 Á  Brook I, Calhoum L, Yocum P. b-lactamase-producing isolates of In this study, no clear cut-off point was defined for Bacteroides species from children. Antimicrob Agents Chemother oral cephalosporins between the two populations. How- ever, b-lactamase-negative Provotella were inhibited by  Heimdahl A, von Konow L, Nord CE. b-lactamase-producing either 1 mg/l of cefuroxime and cefpodoxime or 2 mg/l Bacteroides species in the oral cavity in relation to penicillin of cephalothin and cefixime. At the 1 mg/l French therapy. J Antimicrob Chemother 1981;8:225 Á/9.
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