Based on the DNA sequences of bla EM and blaTEMZ, which encode parental penicillinases TEM-1 and TEM-2, respectively, and of blaTEM3, blaTEM-4 blaTEMS, blaTEM46, and blaTEM-79 which encode extendedspectrum 3-lactamases, we designed heptadecanucleotides to discriminate point mutations in five loci. Determination of the hybridization profiles by colony hybridization with this selection of probes, termed "oligotyping," allowed characterization of the TEM variants present in 265 clinical isolates of the family Enterobacteriaceae that exhibit synergism between a peniciflinase inhibitor and broad-spectrum cephalosporins. Among the 222 strains harboring TEM enzymes, Klebsiella pneumoniae (48%) and Escherichia coli (21%) were predominant, and TEM-3 was the most common enzyme (60%). Penicillinases TEM-1 and TEM-2 were detected alone (15 and 1%, respectively), combined (1%), or associated with another TEM P-lactamase (17 and 6%, respectively). Fourteen variants, including seven new enzymes, were detected. One, TEM-13, was a new penicillinase with the same isoelectric point and substrate range as TEM-2 but differed by a single amino acid substitution, whereas the others, TEM-14 to TEM-19, were extended-spectrum ,I-lactamases that consisted of novel combinations of known amino acid substitutions. Different TEM variants were found to coexist within the same cells. A patient could harbor two or three different strains that encoded the same enzyme or two indistinguishable isolates that produced distinct TEM 13-lactamases.3-Lactams constitute one of the most important families of antibiotics, but resistance to these drugs has emerged following their wide use in therapy. Gram-negative bacteria are most often resistant as a result of their production of ,B-lactamases (17), and numerous enzymes that differ in their substrate ranges have been described (34). The utilization of 3-lactams resistant to hydrolysis by penicillinases, such as broad-spectrum cephalosporins, led to the selection of new enzymes. In 1983, 5 years after the introduction of this class of drugs in Europe, Knothe et al. (13) reported transferable resistance to cefotaxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Biochemical and DNA-DNA hybridization studies (12) indicated that the enzyme responsible for this new resistance phenotype was closely related to SHV-1 penicillinase and was designated SHV-2. Starting in 1984, nosocomial outbreaks of multiresistant members of the family Enterobacteriaceae highly resistant to cefotaxime and ceftazidime occurred in French hospitals (4,11,27,28). Resistance was due to a new, plasmid-mediated, extended-spectrum ,B-lactamase named CTX-1 (27). Molecular analysis (30) and nucleotide sequence determination (29) of blaTEM-3, the structural gene for the enzyme, indicated that the P-lactamase was a double point mutation of TEM-2 penicillinase and was therefore redesignated TEM-3 (30). Since then, similar enzymes of either the SHV type (5, 9) or the TEM type (3,8,22,24)
The epidemiology of integron-mediated antibiotic-resistant genes in clinical enterobacteria from a single location was investigated. Forty-nine isolates (kindly provided by Dr. D. Sirot, Clermont-Ferrand, France) were selected for transferable resistance to aminoglycosides or to other antibiotics. Total DNA prepared from these strains was screened for the presence of conserved segments of integrons by PCR. The nature and frequency of inserted resistance gene cassettes were determined by direct nucleotide sequencing and were related to the resistances expressed by the strain. Integron hot-spots were present in 59% of the strains from 6 species, in either one or two copies. For amplicons sequenced, one or two antibiotic-resistant genes were found in various combinations, and were always expressed at the phenotypic level. They included the aminoglycoside resistance genes ant(3")-Ia and aac(6')-Ib (75%), as well as dhfr-I,-VII (21.4%) and blaOXA-1 (3.6%). Almost half of the transferable resistance to aminoglycosides (53%) was mediated by integron hot-spots in strains characterized at the nucleotide level. The proportion rose to 100% for the AAC(6')-I resistance profile. This study emphasizes the important contribution of integrons to aminoglycoside resistance within enterobacteria from a clinical setting.
Species identification within the genus Mycobacteriumand subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detectingMycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.
In order to establish the taxonomic value of 16s rRNA and 23s rRNA for distinguishing Listeria species, the complete 23s rRNA sequences for all Listeria species were determined by using the type strains. We designed and experimentally validated a universal 23s rRNA sequencing method, which included PCR amplification of the rDNA gene and direct cycle sequencing of the amplicon with eubacterial primers. The results of our sequence comparison indicated that the genus Listeria can be divided into two subgroups; one subgroup is composed of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, whereas the other subgroup includes Listeria grayi subsp. grayi and Listeria grayi subsp. murrayi. A phylogenetic analysis revealed that these species diverged recently. These results are consistent with 16s rRNA sequence analysis data. For application purposes, one 16s rRNA region that can be used to distinguish each Listeria species except L. monocytogenes and L. innocua has been described. In this study we found four 23s rRNA signature regions which, when used in combination, can be used to distinguish the species.Traditionally, bacterial classifications are based on phenotypic characteristics and principally involve analyses of differential metabolic properties. Recently, however, molecular analyses of phylogenetic markers have resulted in a second type of classification based on data from nucleic acid sequence comparisons. The genes that encode these markers possess the features of molecular clocks, and the numbers of mutations that occur in their nucleotide sequences are proportional to elapsed time (7, 17).Among such markers, rRNAs are particularly useful because these molecules are present in every living cell and their function is highly conserved. Moreover, rRNAs share a mosaic structure composed of a succession of several domains that are more or less conserved as a result of variable evolution rates. Thus, distant relationships can be determined by comparing highly conserved regions, whereas closely related species are studied by using more variable (i.e., polymorphic) regions (16).Currently, 16s rRNAs are the principal source of phylogenetic information, and about 3,500 complete 16s rRNA sequences have been determined. A comparison of the sequences of members of each Listeria species (5) demonstrated that this genus is composed of closely related species. The lack of mutations suggested that these species diverged very recently.Analysis of the corresponding 23s rRNA sequences may provide more information concerning phylogenetic relationships. 23s rRNA is about twice as big as 16s rRNA, so we assumed that 23s rRNA contains more variable regions and hence is more discriminative than the smaller 16s rRNA. However, analysis of 23s rRNA has been hampered because of its greater size (about 2.9 kb) and the presence of marked secondary structures absent in 16s rRNA. Recently, the 23s rRNA sequences of two strains of Listeria monocytogenes were deter-*
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