73 research outputs found
Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium
No full textHigh-level ampicillin resistance in Enterococcus faecium has been shown to be associated with the synthesis of a modified penicillin-binding protein 5 (PBP 5) which had apparently lost its penicillin-binding capability (R. Fontana, M. Aldegheri, M. Ligozzi, H. Lopez, A. Sucari, and G. Satta. Antimicrob. Agents Chemother. 38:1980-1983, 1994). The pbp5 gene of the highly resistant strain E. faecium 9439 was cloned and sequenced. The deduced amino acid sequence showed 77 and 54% homologies with the PBPs 5 of Enterococcus hirae and Enterococcus faecalis, respectively. A gene fragment coding for the C-terminal part of PBP 5 containing the penicillin-binding domain was also cloned from several E. faecium strains with different levels of ampicillin resistance. Sequence comparison revealed a few point mutations, some of which resulted in amino acid substitutions between SDN and KTG motifs in PBPs 5 of highly resistant strains. One of these converted a polar residue (the T residue at position 562 or 574) of PBP 5 produced by susceptible and moderately resistant strains into a nonpolar one (A or I). This alteration could be responsible for the altered phenotype of PBP 5 in highly resistant strains
VanA gene cluster in a Vancomycin-resistant clinical isolate of Bacillus circulans
No full textWe report on the cloning and sequencing of the vanA gene cluster present in the glycopeptide-resistant clinical isolate Bacillus circulans VR0709 (R. Fontana, M. Ligozzi, C. Pedrotti, E. M. Padovani, and G. Cornaglia, Eur. J. Clin. Microbiol. Infect. Dis. 16:473-474, 1997). The presence of a vanA- related gene in VR0709 was demonstrated in a PCR assay which permitted the specific amplification of an internal segment of vanA. Southern blotting suggested that the vanA gene was located in the chromosome in a 7.6-kb EcoRI fragment. DNA sequence analysis revealed the presence of all seven genes of the vanA cluster (vanR, vanS, vanH, vanA, vanX, vanY, and vanZ). The degree of identity between homologous proteins encoded by Tn1546 and the chromosome of B. circulans VR0709 ranged from 87 to 95%. Neither PCR nor Southern blotting with specific primers and probes, respectively, showed the presence of open reading frames (ORFs) 1 and 2 which encode the transposase and the resolvase of Tn1546, respectively, the transposon found to carry the vanA gene cluster in enterococci. Determination of the sequences of the flanking regions of the van gene cluster of B. circulans revealed perfect inverted repeats of 10 bp which delineated a 9.2-kb region containing the van gene cluster and an ORF which encoded a putative protein (178 residues) which displayed a low level of identity (28%) to the resolvase of Tn1546. These results suggest that glycopeptide resistance in B. circulans VR0709 is associated with the acquisition of a vanA gene cluster which shows a high degree of homology with that of enterococci. In B. circulans, however, the cluster is not carried by Tn1546 and is borne by the chromosome
VanA gene cluster in a Vancomycin-resistant clinical isolate of Bacillus circulans
No full textWe report on the cloning and sequencing of the vanA gene cluster present in the glycopeptide-resistant clinical isolate Bacillus circulans VR0709 (R. Fontana, M. Ligozzi, C. Pedrotti, E. M. Padovani, and G. Cornaglia, Eur. J. Clin. Microbiol. Infect. Dis. 16:473-474, 1997). The presence of a vanA- related gene in VR0709 was demonstrated in a PCR assay which permitted the specific amplification of an internal segment of vanA. Southern blotting suggested that the vanA gene was located in the chromosome in a 7.6-kb EcoRI fragment. DNA sequence analysis revealed the presence of all seven genes of the vanA cluster (vanR, vanS, vanH, vanA, vanX, vanY, and vanZ). The degree of identity between homologous proteins encoded by Tn1546 and the chromosome of B. circulans VR0709 ranged from 87 to 95%. Neither PCR nor Southern blotting with specific primers and probes, respectively, showed the presence of open reading frames (ORFs) 1 and 2 which encode the transposase and the resolvase of Tn1546, respectively, the transposon found to carry the vanA gene cluster in enterococci. Determination of the sequences of the flanking regions of the van gene cluster of B. circulans revealed perfect inverted repeats of 10 bp which delineated a 9.2-kb region containing the van gene cluster and an ORF which encoded a putative protein (178 residues) which displayed a low level of identity (28%) to the resolvase of Tn1546. These results suggest that glycopeptide resistance in B. circulans VR0709 is associated with the acquisition of a vanA gene cluster which shows a high degree of homology with that of enterococci. In B. circulans, however, the cluster is not carried by Tn1546 and is borne by the chromosome
Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium
No full textFive ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance. Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 micrograms/ml) but not in highly resistant strains (MIC, 128 micrograms/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability
Development of a rapid method for quantitative evaluation of Mycobacterium tuberculosis growth based on competitive polymerase chain reaction
No full textA competitive polymerase chain reaction (cPCR) assay for the quantitative evaluation of Mycobacterium tuberculosis growth was developed based on co-amplification of genomic DNA and a modified DNA fragment derived from a well-conserved region of the 16S rRNA gene. There was a good correlation between the number of DNA copies in the sample, indicated by competitive PCR, and the number of colony forming units determined by conventional culture methods
Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction
No full textA rapid method for the detection of methicillin resistance in staphylococci was developed. The method was based on the polymerase chain reaction (PCR) and primers that targeted the internal region of the coding frame of the mec gene. The amplification reaction was carried out with crude cell lysates as a source of target DNA and provided data in less than 5 h. Seventy-four isolates of coagulase-negative staphylococci were tested by PCR, DNA hybridization with a probe derived from the mec gene, and an agar dilution susceptibility assay. PCR results showed a 100% correlation with the susceptibility assay carried out with high inocula (10(8) CFU) and incubation at 32 degrees C for 48 h. PCR was more sensitive and specific than DNA hybridization in detecting methicillin resistance in coagulase-negative staphylococci. The former technique identified the mec gene in all the strains which were phenotypically resistant but which did not hybridize with the probe. Identification of methicillin-resistant strains by PCR offers a very specific, sensitive, and rapid alternative to traditional susceptibility tests and DNA hybridization as a guide for the treatment of infections caused by staphylococci
Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5
No full textEnterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized
Affinities of cephalosporins for penicillin-binding proteins and their antibacterial activities in the presence of human serum albumin
No full textDevelopment of a real-time quantitative polymerase chain reaction assay for the detection of congenital human cytomegalovirus infection in urine samples
No full textA TaqMan real-time quantitative PCR (qPCR), based on amplification of HCMV UL54 gene specific sequence, was developed and compared with shell vial viral culture assay, the gold standard technique for the diagnosis of congenital HCMV infection, using urine samples collected from 110 newborns. The results indicate that this qPCR is slightly more sensitive than shell vial assay suggesting that qPCR may be considered a useful alternative for diagnosing congenital HCMV infection
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