Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D ␤-lactamase, OXA-1. The oxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosa were conferred by OXA-1 as well.The most frequent mechanisms of resistance to extendedspectrum cephalosporins in Pseudomonas aeruginosa are derepression of the chromosomal AmpC ␤-lactamase, impermeability of the outer membrane, and increased efflux (5). These various processes, independently or conjointly, confer resistance to ceftazidime. Extended-spectrum ␤-lactamases (ESBLs) belonging to each of the Ambler ␤-lactamase classes are described in P. aeruginosa (20). Among the clavulanic acid-inhibited Ambler class A enzymes, TEM-and SHV-related ESBLs, PER-1 and VEB-1 hydrolyze ceftazidime and cefepime significantly (20). The Ambler class B enzymes, IMP-1, VIM-1, and VIM-2, have the broadest hydrolysis profiles, which include hydrolysis of ceftazidime and cefepime (20,25). The last group of ESBLs includes OXA-18, the OXA-2 and OXA-10 derivatives (18,20), and OXA-24 and ARI-1, with the last two also hydrolyzing carbapenems (3, 9). Among the OXA-10 variants, predominantly compromise the activity of ceftazidime, while OXA-17 mainly degrades cefotaxime (8, 18). The OXA-10 variants confer resistance to cefepime usually at a lower level than they do to ceftazidime. A recent survey conducted with more than 2,000 isolates of P. aeruginosa showed that the rates of susceptibility to ceftazidime and cefepime are similar, being 78.8 to 81.9% and 80 to 83.4%, respectively (27).The aim of the present work was to characterize the mechanism(s) involved in a peculiar antibiotic resistance phenotype that combines resistance to cefepime and susceptibility to ceftazidime. MATERIALS AND METHODSBacterial strains and plasmids. P. aeruginosa SOF-1 was isolated from a rectal swab specimen from a 1-month-old child hospitalized at the Hôpital de Bicêtre (Le Kremlin-Bicêtre, France) in 1999. This isolation was the result of systematic screening for multidrug-resistant gram-negative isolates from patients admitted to the intensive care units of this hospital. It was identified with the API 20 NE system (bioMérieux, Marcy l'Etoile, France). Ciprofloxacin-or rifampin-resistant P. aeruginosa PU21 obtained in vitro and rifampin-resistant Escherichia coli K-12 C600 obtained in vitro were used as recipient strains for conjugation, and E. coli DH10B and P. aeruginosa 104116 (Institut Pasteur Collection, Paris, France) reference strains (23-25) were used for cloning experiments. E. coli NCTC 50192 carrying plasmids of 154, 66, 38, and 7 kb served as a control in a plasmid-sizing study (33). Plasmids pPCRScript-Cam (SKϩ; Stratagene, Amsterdam, The Netherlands), whic...

The genus Chryseobacterium, defined in 1994 by Vandamme et al., comprises six species, including Chryseobacterium meningosepticum (previously Flavobacterium meningosepticum) and Chryseobacterium indologenes (previously Flavobacterium indologenes), which are the most common clinical species of this genus (29). C. meningosepticum isolates are associated with meningitis in newborns or in immunocompromised patients (4). C. indologenes is responsible mostly for nosocomial infections linked to the use of intravascular devices (10, 28). In 1997, Hsueh et al. reported 36 cases of infections caused by C. indologenes over a 3-year period at the National Taiwan University Hospital (11). These infections were intra-abdominal infections, biliary tract infections, or wound sepsis (11). C. indologenes is also implicated in pneumonia (9). C. indologenes is resistant to nearly all penicillins, except piperacillin, to restricted-spectrum cephalosporins, to aztreonam, and to carbapenems (1). Among ␤-lactams, only extended-spectrum cephalosporins show in vitro activity against C. indologenes (8). Minocycline and ciprofloxacin seem to be the most effective antibiotics and have been successfully used to treat C. indologenes infections (10).Recently, two Ambler class B ␤-lactamases have been characterized for C. meningosepticum, which is also resistant to all ␤-lactams, including carbapenems (2, 26). The unrelated BlaB and GOB-1 exhibit a broad-spectrum profile and are chromosome encoded. These carbapenem-hydrolyzing ␤-lactamases (CH␤Ls) have molecular and biochemical heterogeneity among C. meningosepticum isolates. We have characterized a ␤-lactamase, IND-1, from C. indologenes strain 001 (1). Since plasmid-mediated CH␤Ls have been increasingly reported for gram-negative species worldwide (5,12,18,23,24,25), it was of interest to identify naturally occurring CH␤Ls of gramnegative species and to search for their degree of identity with the plasmid-mediated CH␤Ls. Thus, we performed an extended genetic and biochemical study of ␤-lactamase-mediated resistance to carbapenems in C. indologenes. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this work are listed in Table 1. C. indologenes 002, 007, 008, and 009 were isolated from bronchoalveolar brush border, blood culture, rectal swab, and biliary liquid drainage, respectively, at the Hôpital de Bicêtre (Le Kremlin-Bicêtre, France) from 1997 to 1999. C. indologenes 003 and 004 were isolated from blood cultures at the Hôpital Robert Debré (Paris, France). C. indologenes 005 was from the Assistance Publique-Hôpitaux de Marseille (Marseille, France). C. indologenes 006 was isolated at the Hôpital de la Pitié Salpêtrière (Paris, France), and the C. indologenes reference strain CIP101026 was from the Pasteur Institute (Paris, France) strain collection. Each C. indologenes isolate was identified according to standard biochemical techniques (19,29,31), and their identification was checked at the Center for Bacterial Identification at the Pasteur Institute. These C. indolog...