Mohammed Salim Ammor

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.

The tetracycline uptake kinetics of 35 Bifidobacterium longum strains isolated from the human gastrointestinal tract were examined by fluorescence spectroscopy, and the suitability of the technique as a screening tool of tetracycline resistance or susceptibility was determined. The strains were first grouped into three classes based on their corresponding minimum inhibitory concentrations (MICs) of tetracycline, as established by the microdilution method: susceptible (MICs or=32 microg mL(-1)). The kinetics of tetracycline uptake for the strains in each resistance group were then analyzed over a 20 min period by fluorescence spectroscopy (absorbance wavelength 524 nm, excitation wavelength 400 nm) in a buffer system containing 100 microg mL(-1) tetracycline. Principal component analysis and factorial discriminant analysis of the results showed excellent distinction among susceptible, semi-resistant, and resistant strains. The proposed method provides a powerful and convenient means of rapidly screening tetracycline resistance in B. longum.