In vitro selection of glycopeptide-resistant variants of Enterococci.

In order to study the possible phenotypic and genotypic changes related to glycopeptide pressure on enterococci, a study was undertaken using stepwise in vitro exposure to achieve the following objectives: (i) to evaluate the development of resistance and cross-resistance between vancomycin and teicoplanin; (ii) to determine the stability of the acquired level of resistance; (iii) to determine the phenotypic and genotypic changes related to glycopeptide pressure; and (iv) to assess the spectrum of antibiotic-susceptibility of all strains. Our results showed that no variants resistant to glycopeptides could be selected after in vitro glycopeptide exposure experiments. However some strains showed increased MIC values: 8 mg/l to vancomycin in eight strains selected by vancomycin itself, while teicoplanin produced intermediate values to vancomycin in only three strains. The phenotypes were stable in vitro after numerous passages in antibiotic-free medium and three out of nine strains with a changed MIC level, showed 40, 42 and 43 kDa proteins in cell membrane preparations. The profile of antibiotic resistance was comparable in all isogenic strains tested with the exception of three selected strains that became susceptible to penicillin G. The pressure produced by glycopeptides, particularly vancomycin has contributed to an increased level of MIC that can influence the acquisition and/or full expression of this resistance.

Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.

In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.

Molecular epidemiology of enterococci with high-level resistance to aminoglycosides.

DNA-based methodologies are considerably more powerful than other phenotype-based typing systems, providing a finer level of epidemiological discrimination, differentiating both closely and distantly related independent isolates that otherwise may appear as identical. In this study, plasmid analysis and pulsed-field gel electrophoresis were used to compare 28 isolates of Enterococci (respectively 13 strains of Enterococcus faecalis and 15 strains of Enterococcus faecium) with high-level resistance to aminoglycosides, isolated in Catania (Italy). Plasmid profile analysis resolved 20 different patterns among 24 plasmid harboring strains; many isolates showed one or two plasmids of the same size, but different plasmid content. Analysis of the PFGE-based RFLP patterns after SmaI digestion of genomic DNA resolved 26 different clones from 28 isolates: particularly, it resolved two different clones from three isolates showing identical plasmid profiles, and it identified as a single clone two isolates exhibiting different plasmid profiles. Thus, on the basis of our PFGE-based RFLP analysis data, we concluded that all the strains included in the study were genetically unrelated with two exceptions.

Observations on the tolerance and the paradoxical effect in enterococci.

Enterococci, already know to be relatively unaffected by several antibiotics due to their inheritant characteristics, are increasingly resistant to some very important groups of drugs, by means of acquisition or exchange of new genetic traits of resistance. Resistance or moderate susceptibility towards penicillin is an interesting characteristic of enterococci, whose low degree of susceptibility to this drug is due to a low affinity for penicillin-binding proteins (PBP). Some strains of enterococci are not killed by the action of this drug but are "tolerant" (MIC/MBC > 32 mg/l). This kind of "resistance", in which the probability of surviving under selective pressure of the drug is increased, is probably linked to the deficiency of the cell's autolytic system. Only rarely does another form of resistance called the "paradoxical effect" appear, in which higher numbers of cells survive at high concentrations than at lower concentrations. In our study the degree of bactericidal activity of some beta-lactams was considered. Our results demonstrate that: i) the paradoxical effect appears more in cultures in exponential phase compared to aged cultures; ii) mutated strains show an increased number of cells that respond paradoxically (the behavior is genetically determined); iii) different beta-lactams induced different degrees of autolysis.

Microbiological considerations of the etiological agents of lower respiratory tract infections.

One hundred eight-four sputum specimens from the same number of patients with lower respiratory tract infections were examined to determine the bacterial count and the relationship between the microorganisms isolated and the presumptive pathology. The sputa were subdivided into three groups; "high probability", "low probability", and "contaminated sputa", following the criteria of the microscopic readings: sputum with more than 25 white cells and low numbers of squamous epithelial cells represents true lower respiratory tract infections (high probability); those with fewer than 25 white cells represent non-bacterial infections or non-infected sputa (low probability) while sputa with more than 25 squamous cells per field represent contaminated specimens (contaminated sputa). Statistical analysis was carried out to correlate these data. Haemophilus influenzae, Haemophilus parainfluenzae, Streptococcus pneumoniae, and Streptococcus pyogenes showed significant differences in the three groups considered.