[Distribution and diversity of conjugative plasmids among some multiple antibiotic resistant E.coli strains isolated from river waters].
In natural bacterial communities the microbial structure and functions are subjected to dynamic environmental and genetic adaptation. Plasmid-mediated horizontal genes transfer has a major impact on the adaptability of bacteria, exemplified by the interspecific and intergeneric transfer of antibioresistance genes in a variety of aquatic media. The high incidence of resistant bacteria has been documented for fresh waters, marine waters and chronically polluted waters. The aim of this study was to establish the distribution and diversity of plasmids and to study the transfer of plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) belong to 12 multiple antibiotic resistant E. coli strains isolated from river waters. Antimicrobial resistance patterns were performed for aminoglycosides (gentamycin, kanamycin), beta-lactams (ampicillin), cephalosporins (ceftazidime and cefotaxime), tetracycline, nalidixic acid and chloramphenicol by disk diffusion method following NCCLS recommendations. Minimum inhibitory concentrations (MICs) were performed using dilution method in Mueller-Hinton broth with a 0.06-64 micrograms/ml concentration range for all antimicrobials and bacterial inoculum corresponding to 0.5 standard of the McFarland scale. For the data analysis NCCLS breakpoints for resistance and sensitivity were used. Bacterial plasmid isolation was performed by an alkaline lysis method. Genetic characterization was performed by agarose gel electrophoresis and spectrophotometric analysis. R-plasmid transfer frequencies were estimated by conjugation of drug-resistant E. coli strains used as donors with E. coli DH5 alpha F recipient marked with chromosomal resistance to nalidixic acid (Nal). The drug resistance markers possessed by a particular donor strain were sequentially used to screen for R+ transconjugants by incorporation the particular drug in the selective media. All E. coli strains are multiple antibiotic resistant, 65% of them being resistant to all 8 antibiotics tested. Plasmid profile analysis revealed the presence of several plasmids ranging from 3.8 kpb to more than 50 kpb. All aquatic R+ strains transferred two or more of their resistance markers to E. coli DH5 alpha F, transfer of resistance to ampicillin and tetracycline being the most frequent and having a frequency of 10(-4) or greater (expressed as transconjugants/donor). The phenotypic data shows the frequency and dynamic flow of multiple antibioresistant E. coli strains in aquatic media. Electrophoretic patterns analysis reflects the high incidence and diversity of plasmids in aquatic E. coli strains. Plasmid-harboring E. coli strains transferred antibiotic resistance and, hence, possessed conjugative R plasmids. Of these, 80% transferred drug resistance at a frequency of about 10(-4).
Cernat
Ramona
R
Facultatea de Biologie, Dep. Microbiologie-Imunologie, Universitatea din Bucureşti.
Lazăr
Veronica
V
Balotescu
Carmen
C
Cotar
Ani
A
Coipan
Elena
E
Cojocaru
Cristina
C
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics*; D045483:Rivers / Q000382:microbiology*; D014871:Water Microbiology
D000900:Anti-Bacterial Agents