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J C Perez-Diaz

Publications and source records attributed to J C Perez-Diaz.

17 recordsLinked to original sources

Macrolide resistance in Peptostreptococcus spp. mediated by ermTR: possible source of macrolide-lincosamide-streptogramin B resistance in Streptococcus pyogenes.

Eighty percent (21 of 26) of macrolide-resistant Peptostreptococcus strains studied harbored the ermTR gene. This methyltransferase gene is also the most frequently found gene among macrolide-lincosamide-streptogramin B-resistant Streptococcus pyogenes strains. Transfer of the ermTR gene from Peptostreptococcus magnus to macrolide-susceptible S. pyogenes strains indicates that this resistance determinant may circulate among gram-positive aerobic and anaerobic species of the oropharyngeal bacterial flora.

Anti-Bacterial Agents↗

New extended-spectrum TEM-type beta-lactamase from Salmonella enterica subsp. enterica isolated in a nosocomial outbreak.

A new extended-spectrum beta-lactamase was detected in a lactose-positive Salmonella enterica subsp. enterica strain that caused a nosocomial outbreak involving eight patients in a pediatric cardiology unit. This strain showed high levels of resistance to ceftazidime and aztreonam and relatively low levels of resistance to cefotaxime and ceftriaxone. Resistance was associated with a conjugative plasmid of 59 kb, which encoded a new beta-lactamase with an isoelectric point of 5.9 that strongly hydrolyzed ceftazidime and to a much lesser extent hydrolyzed cefotaxime. The enzyme activity was inhibited by clavulanate. The corresponding bla gene was cloned and sequenced. The deduced amino acid sequence showed three significant amino acid replacements with respect to the TEM-1 sequence: Arg-164-->His, Glu-240-->Lys, and Thr-265-->Met. This combination is unique among extended-spectrum beta-lactamases and served to characterize the new enzyme, TEM-27.

Amino Acid Sequence↗

In-vitro synergy between aminoglycosides deployed against Staphylococcus spp. harbouring a 6'-aminoglycoside acetyltransferase, 2"-aminoglycoside phosphotransferase enzyme.

In-vitro synergistic effects between commercial aminoglycosides are described for gentamicin resistant Staphylococcus strains harbouring 6'-aminoglycoside acetyltransferase activity. Seventy eight strains were studied using the double-disc test and synergy was observed with combinations in which at least one of the components has a garosamine-like 6'-aminosugar. These results were confirmed in chequerboard titrations (sigma FIC < or = 0.5) carried out on Staphylococcus epidermidis strains RYC13036 and RYC4904. Additionally, a clear reduction in tobramycin acetylating activity was observed in the presence of gentamicin or netilmicin in crude extracts of these strains. These experiments suggest that the observed synergy is due to inhibition of the aminoglycoside-modifying enzyme by aminoglycosides with a garosamine like 6'-aminosugar component.

Acetyltransferases↗

Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants.

Klebsiella pneumoniae BA32, a clinical isolate from Buenos Aires, Argentina, was found to produce a plasmid-encoded beta-lactamase (FOX-1) which conferred resistance to broad-spectrum cephalosporins and cephamycins. Resistance could be transferred by conjugation or transformation into Escherichia coli K-12 via a 48.5-kb plasmid (pGLK1) that produced two FOX-1 molecular variants with isoelectric points of 6.8 and 7.2 and apparent molecular sizes of 37 and 35 kDa, respectively. The kinetic study revealed that the two variants had very similar substrate and inhibition profiles. These values resemble those of chromosomally mediated class C (group 1) cephalosporinases. The structural gene of FOX-1 (blaFOX-1) was cloned into a 2,270-bp PstI-PstI fragment and was expressed in E. coli TG1. The deduced 382-amino-acid sequence of FOX-1 exhibited a high degree of similarity with chromosomally encoded AmpC beta-lactamases of Pseudomonas aeruginosa, Serratia marcescens, Enterobacter cloacae, E. coli, and Citrobacter freundii. These findings suggest that FOX-1 is a plasmid-mediated AmpC-type beta-lactamase that is encoded by a single gene and that has two molecular variants.

Aged↗

Comparison of serotype, biotype and bacteriocin type with rDNA RFLP patterns for the type identification of Serratia marcescens.

Variations in rDNA gene loci in DNA digests of 209 clinical isolates of Serratia marcescens were determined with an Escherichia coli rRNA probe. Forty-one restriction fragment length polymorphism patterns (ribotypes) were identified, based on the size of 4-14 (mean 7.5) hybridization bands. The patterns differed by more than a single band in 98% of pair-wise comparisons. On a subset of 76 isolates, ribotyping proved to be marginally more discriminating than biotyping (discrimination index 0.92 v. 0.89) followed by serotyping (0.87) and bacteriocin typing (0.74). About one-third of isolates belonged to unique ribotypes and only two ribotypes exceeded 5% in frequency (23.0 and 6.4% respectively). A combination of serotype or biotype with ribotyping defined a similar number of strains, although none of the methods alone was sufficiently discriminatory to identify strains. We conclude that due to the accessibility of biotyping and the lack of commercially available antisera for S. marcescens, the biotype and ribotype together provide reliable markers of strain identity.

Bacterial Typing Techniques↗

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

Animals↗

A genetic approach to demonstrate the role of listeriolysin O in the virulence of Listeria monocytogenes.

The locus of insertion of a transposon previously used to obtain a non-haemolytic avirulent mutant was identified: it is the structural gene encoding lisreriolysin O, the thiol-dependent haemolysin, now called hlyA. The gene was completely sequenced. The preliminary structural and functional study of the chromosomal region containing the gene indicates that hlyA belongs to a monocistronic transcriptional unit. If it is the case, the transposon insertion would have no major polar effect on downstream genes and would only affect hlyA expression. These results emphasize the importance of the haemolysin in the virulence of Listeria monocytogenes.

Amino Acid Sequence↗

Synergistic effect of dosage and bacterial inoculum in TEM-1 mediated antibiotic resistance.

The effect of inoculum size and gene dosage on the level of antibiotic resistance mediated by TEM-1 beta-lactamase was measured. From the results it seemed that gene dosage is a more efficient mechanism than inoculum size for increasing TEM-1 mediated resistance to beta-lactam antibiotics. It also seemed that the two mechanisms for enhancing antibiotic resistance are synergistic. The clinical implications of these results are discussed.

Anti-Bacterial Agents↗

Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

Amino Acid Sequence↗

A protoplast transformation system for Listeria sp.

We describe protoplast transformation of members of the genus Listeria with plasmid DNA. Shuttle vectors able to replicate in both Escherichia coli and Listeria have been constructed by fusing the E. coli plasmid pUC8 with the Bacillus subtilis plasmids pBD9 and pBD10.

DNA Replication↗

Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli.

Plasmid deoxyribonucleic acid (DNA) isolated from each of three antibiotic-resistant clinical strains of Escherichia coli producing the same microcin showed multiple bands upon agarose gel electrophoresis. Transformants selected either for microcin resistance or ampicillin resistance yielded plasmid DNA corresponding in size to only one of the multiple bands. Plasmids, isolated from all three hosts, which determined microcin resistance and microcin production measured about 4 megadaltons by sucrose density, restriction enzyme, and contour length analyses; cleavage of the DNAs by each of eight restriction enzymes showed the same response, and DNA-DNA hybridization indicated complete homology. The antibiotic resistance plasmids of the three host strains were uniformly larger, were of different sizes, and showed different restriction enzyme cleavage patterns. One of these R plasmids (pCP106) also determined the synthesis of the same microcin, and DNA-DNA hybridization studies indicated an approximate 2.4-megadalton homology with the 4-megadalton microcin plasmid pCP101. The microcin plasmids were present at approximately 20 copies per genome equivalent and were nonconjugative, whereas the R plasmids had a copy number of about 1, were conjugative, and could mobilize the microcin plasmid. Microcin plasmid pCP101 showed replication properties similar to those of a number of small multicopy plasmids such as ColE1.

Bacteriocins↗