Genetic and biochemical characterization of TRU-1, the endogenous class C beta-lactamase from Aeromonas enteropelogenes.

Aeromonas enteropelogenes (formerly A. tructi) was described to be an ampicillin-susceptible and cephalothin-resistant Aeromonas species, which suggests the production of a cephalosporinase. Strain ATCC 49803 was susceptible to amoxicillin, cefotaxime, and imipenem but resistant to cefazolin (MICs of 2, 0.032, 0.125, and >256 microg/ml, respectively) and produced an inducible beta-lactamase. Cefotaxime-resistant mutants (MIC, 32 microg/ml) that showed constitutive beta-lactamase production could be selected in vitro. The gene coding for the cephalosporinase of A. enteropelogenes ATCC 49803 was cloned, and its biochemical properties were investigated. Escherichia coli transformants showing resistance to various beta-lactams carried a 3.5-kb plasmid insert whose sequence revealed a 1,146-bp open reading frame (ORF) encoding a class C beta-lactamase, named TRU-1, showing the highest identity scores with A. punctata CAV-1 (75%), A. salmonicida AmpC (75%), and A. hydrophila CepH (71%). The bla(TRU-1) locus includes open reading frames (ORFs) showing significant homology with genes found in the genomes of other Aeromonas species, although it exhibits a different organization, as reflected by the presence of additional ORFs located downstream of the beta-lactamase gene in the A. hydrophila and A. salmonicida genomes. Specific PCR assays were negative for cphA-like and bla(OXA-12)-like genes in three A. enteropelogenes ATCC strains. Purified TRU-1 showed a broad substrate profile, efficiently hydrolyzing benzylpenicillin, cephalothin, cefoxitin, and, although with significantly lower turnover rates, oxyiminocephalosporins. Cephaloridine and cefepime were poorly recognized by the enzyme, as reflected by the high K(m) values observed with these substrates. Thus far, A. enteropelogenes represents the only known example of an Aeromonas species that produces only one beta-lactamase belonging to molecular class C.

Sequence analysis and biochemical characterisation of chromosomal CAV-1 (Aeromonas caviae), the parental cephalosporinase of plasmid-mediated AmpC 'FOX' cluster.

Aeromonas caviae CIP 74.32 was resistant to amoxicillin, ticarcillin and cephalothin, and susceptible to cefoxitin, cefotaxime, ceftazidime, aztreonam and imipenem. This strain produced a cephalosporinase (pI 7.2) and an oxacillinase (pI 8.5). The cephalosporinase gene cav-1 was cloned and sequenced. Unlike A. caviae donor, Escherichia coli pNCE50 transformant producing CAV-1 beta-lactamase was resistant to cefoxitin. The deduced protein sequence CAV-1 contained 382 amino acids, and shared >96% homology with FOX-1 to FOX-5 cephalosporinase. CAV-1 presented only two amino acid substitutions (Thr270Ser and Arg271Ala) with FOX-1. CAV-1 is the chromosomal putative ancestor of the FOX family, a cluster of class C/group 1 plasmidic cephalosporinases spreading in Klebsiella and E. coli clinical isolates via conjugative plasmids.

[The buccal-dental health of drug addicts treated in the University hospital centre in Nice]. / Etat de santé bucco-dentaire des toxicomanes suivis au CHU de Nice.

OBJECTIVE: Drug-addicts often suffer from rapidly progressive extensive dental decay. The objectives of this study were to determine the impact of illicit drugs on buccal and dental health and the use of illicit drugs for toothache. METHODS: Two groups of intravenous and non-intravenous drug-addicts were compared with two control groups of age matched non-addicted subjects. During a routine dental examination, medical and addictive history, periodontal and dental health and dental complaints were recorded. RESULTS: This study showed that intravenous heroin was responsible for rapidly progressive dental decay, even in four drug-addicts with satisfactory dental hygiene. Intravenous heroin users (14 women, 38 men, mean age 35) had a mean number of 10 missing and 10 decayed teeth, 6 of them to be extracted, and needed two dentures with 8 teeth each. Their masticatory function (45%) and smile did not permit normal alimentation or social life. Non-intravenous drug users (9 women, 29 men, mean age 26) had a mean number of one missing tooth and 4 decayed teeth to be treated. When compared to control groups, drug users of both categories exhibited more decayed teeth, reduced masticatory function and a lower periodontal health correlated with inadequate dental hygiene. Finally, 52% of heroin users and 21% of other illicit drug users admitted the use of illicit drugs as analgesics for toothache. CONCLUSION: The management of toothache should be proposed in the cessation protocols and dentures provided to intravenous drug-addicts, before any attempt at social reinsertion.

Chromosome- and plasmid-encoded beta-lactamases in Capnocytophaga spp.

Chromosome- and plasmid-encoded CfxA2 and CfxA3 beta-lactamases were detected in Capnocytophaga spp. from oral sources in France, Norway, and the United States. Unidentified chromosome-encoded beta-lactamases were present in Capnocytophaga sputigena. Nucleotide sequence analysis of the CfxA3-encoding plasmid from C. ochracea revealed an unreported insertion sequence (ISCoc1) upstream of the cfxA gene.

Sequence analysis of cfxA2-like beta-lactamases in Prevotella species.

Sixty-two strains of oral (32) and non-oral (30) Prevotella producing beta-lactamases were screened for cfxA by PCR, using an intragenic primer pair. All 62 were cfxA/cfxA2 positive. Fourteen of these strains, representing seven pigmented and seven non-pigmented Prevotella species were submitted to further PCR with specific primers that amplified the whole beta-lactamase structural gene (966 bp). After cloning and sequencing, the deduced amino acid sequences were compared with that of Bacteroides vulgatus CfxA beta-lactamase. All 14 sequences possessed the E272K substitution characteristic of CfxA2. CfxA sensu stricto was not observed in the present series. G83D, F/V189L, W193L and D239Y substitutions were observed more than once, without species specificity. This sequence analysis indicates that most oral and non-oral beta-lactamase-producing Prevotella isolates from French patients produce variants of the CfxA enzyme.