A clinical isolate ofPseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a 1-lactamase. This f-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pl of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pl of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km)* PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type 13-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum 13-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.More than 50 biochemically distinct P-lactamases responsible for resistance to ,-lactams have been reported in gram-negative bacteria. The resistance of broad-spectrum cephalosporins to these ,B-lactamases was a widely accepted concept in the beginning of the 1980s. However, overproduction of chromosomally mediated cephalosporinases has been described as responsible for failure of treatment of gram-negative bacterial infections with extended-spectrum cephalosporins (39). Since 1983, plasmid-mediated extended-spectrum 3-lactamases have been reported, primarily in Kiebsiella pneumoniae and then in numerous Enterobacteraceae species (16, 34). These enzymes hydrolyze extended-spectrum cephalosporins and aztreonam to various extents but usually neither cephamycins (cefoxitin and moxalactam) nor carbapenems (imipenem and meropenem). A common feature of these enzymes is inhibition of their activity by clavulanic acid. These enzymes are Ambler class A 1-lactamases, members of the TEM or SHV series that differ by a few point mutations in their structural genes (16,34 resistance to extended-spectrum cephalosporins. In this species, TEM-1 and TEM-2 1-lactamases confer additional resistance to ureidopenicillins (26). The OXA-type (oxacillin-hydrolyzing) enzymes possess high-level hydrolytic activity against cloxacillin, oxacillin, and methicillin (9, 10). Their activities are inhibited by clavulanic acid but to a lesser extent than TEM-or SHV-der...

Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing ␤-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, the bla VIM-2 cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes, aacA29a and aacA29b, were located at the 5 and 3 end of the bla VIM-2 gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5 end of the qacE cassette. The deduced amino acid sequence AAC(6)-29a protein shared 96% identity with AAC(6)-29b but only 34% identity with the aacA7-encoded AAC(6)-I1, the closest relative of the AAC(6)-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6)-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.

A study was performed to compare matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), linked to a recently engineered microbial identification database, and two rapid identification (ID) automated systems, BD Phoenix (Becton Dickinson Diagnostic Systems, France) and VITEK-2 (bioMérieux, Marcy L'Etoile, France), for the ID of coagulase-negative staphylococci (CoNS). Two hundred and thirty-four clinical isolates of CoNS representing 20 species were analyzed. All CoNS isolates were characterized by sodA gene sequencing, allowing interpretation of the ID results obtained using the respective database of each apparatus. Overall correct ID results were obtained in 93.2%, 75.6% and 75.2% of the cases with the MALDI-TOF-MS, Phoenix and VITEK-2 systems, respectively. Mis-ID and absence of results occurred in 1.7% and 5.1% of the cases with MALDI-TOF-MS, in 23.1% and 1.3% with the Phoenix, and in 13.7% and 0.9% with the VITEK-2 systems, respectively. In addition, with the latter automate, 10.3% of the IDs were proposed with remote possibility. When excluding the CoNS species not included in the databases of at least one of the three systems, the final percentage of correct results, Mis-ID and absence of ID were 97.4%, 1.3% and 1.3% with MALDI-TOF-MS, 79%, 21% and 0% with the Phoenix, and 78.6%, 10.3% and 0.9% with the VITEK-2 system, respectively. The present study demonstrates the robustness and high sensitivity of our microbial identification database used with MALDI-TOF-MS technology. This approach represents a powerful tool for the fast ID of clinical CoNS isolates.

Rhodococcus equi is an intracellular facultative, Gram-positive cocco-bacillary organism of increasing importance as a pulmonary pathogen in HIV-positive patients. This study was carried out to evaluate the optimal antibiotic combinations for treating such infections. Four human R. equi isolates and one reference strain were tested for their susceptibilities to 36 antibiotics. In-vitro the most active antibiotics were amikacin, gentamicin, netilmicin, erythromycin, clarithromycin, roxithromycin, ciprofloxacin, sparfloxacin, rifampicin, vancomycin, teicoplanin, doxycycline, minocycline, imipenem, meropenem and trimethoprim/sulphamethoxazole. The only bactericidal antibiotics were the aminoglycosides, ciprofloxacin, sparfloxacin and vancomycin. As determined by FIC indices, four combinations were synergistic: rifampicin-erythromycin, rifampicin-minocycline, erythromycin-minocycline and imipenem-amikacin. However, no antibiotic combinations were synergistic with the time-kill kinetic method at achievable serum concentrations or at ten-fold and half-fold the MICs. Frequencies of selection of antibiotic-resistant mutants determined at concentrations of five- and ten-fold the MICs ranged from less than 1 x 10(-8) for erythromycin and trimethoprim/sulphamethoxazole to 5 x 10(-4) for amikacin. These results may be of help in selecting the antibiotics for treating infected HIV-positive patients.

An AmpC-type ␤-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C ␤-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the ⍀-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.

Analysis of 15 European clinicalEnterobacteriaceae isolates showed that differences in the genetic context of bla CMY-2 -like genes reflected the replicon type, usually IncA/C or IncI1. These bla CMY-2 loci may originate from the same ISEcp1-mediated mobilization from the Citrobacter freundii chromosome as structures described in earlier studies.

A clinical isolate of Pseudomonas aeruginosa RP-1 produced the extended-spectrum β-lactamase (ESBL) SHV-2a. Its gene was expressed from a composite promoter made of the −35 region derived from the left inverted repeat of IS26 and the −10 region from the bla SHV-2a promoter itself. The DNA sequences immediately surrounding bla SHV-2awere homologous to plasmid pMPA2a from Klebsiella pneumoniae KpZU-3, while further away and 3′ to thebla SHV-2a gene, a sequence corresponding to the left end of Tn1721 was detected, thus indicating a likely enterobacterial origin of this ESBL gene.

In studying the interplay between mutation frequencies and antibiotic resistance among Escherichia coli natural isolates, we observed that modest modifications of mutation frequency may significantly influence the evolution of antibiotic resistance. The strains having intermediate mutation frequencies have significantly more antibiotic resistances than strains having low and high mutation frequencies.