Genotypic and phenotypic studies on resistance to macrolides in certain bacteria involved in respiratory tract infection
Amr Shaker Meselhy;
Abstract
One hundred and eighty nine bacterial isolates were recovered from sputum and bronchoalveolar lavage specimens of respiratory tract infected patients. Testing the susceptibility of the recovered isolates to erythromycin, clarithromycin, azithromycin and clindamycin showed that, out of the collected isolates, 64 isolates (33.9%) showed resistance to at least 3 of the tested antibiotics. Gram staining showed that, 52 out of 64 resistant bacterial isolates (81.3%) were found to be Gram-negative, while the remaining 12 isolates (18.7%) were Gram-positive organisms. The resistant 64 isolates were categorized and identified, fourty isolates were lactose fermenters (LF) including 19 isolates Klebsiella pneumoniae, 13 isolates E. coli, 4 isolates Enterobacter cloacae and 4 isolates of other species of LF, and 12 isolates were non-lactose fermenters (NLF) including 9 isolates Pseudomonas spp., one isolate Acinetobacter baumannii and 2 isolates of other species of NLF. Eight Staphylococcus spp. isolates were identified among the 12 Gram-positive resistant isolates.
Agarose gel electrophoresis of plasmid extracts of the MAC resistant isolates showed that 50 out of 64 resistant isolates (78.1%) harbored plasmids. Using PCR, it was found that MAC reistance-coding genes were detected on the plasmids of 14 Klebsiella pneumoniae isolates, 12 E. coli isolates, 9 Pseudomonas spp. isolates and 7 Staphylococcus spp. isolates.
PCR analysis, verified by DNA sequencing of PCR products, revealed that all resistant isolates harboring plasmids carried at least one of the tested MAC resistance-coding genes (mph, ere, erm and msr), on their plasmids. Thirty-two isolates (64%) were found to harbor a single macrolide resistance-coding gene while combinations of two, three and four macrolide resistance-coding genes were detected in 15 isolates (30%), 2 isolates (4%) and one isolate (2%), respectively. Of all tested MAC resistance-coding genes, mph was the most frequently encountered gene (78%), followed by erm gene which was detected in 22 isolates (44%). Nine isolates (18%) were found to carry ere gene, while only two isolates (4%) were found to harbor msr gene. Regarding the distribution of the four MAC resistance-coding genes in resistant isolates of different genera, mph, erm and ere genes were detected in 83.3%, 41.6% and 8%, respectively, of tested E. coli resistant isolates, while msr gene was not detected in any of the tested E. coli isolates. Different combinations of two MAC resistance-coding genes could be detected in 33.3% of resistant E. coli isolates. In case of Klebsiella pneumoniae isolates, mph was the most commonly detected (64.3%), followed by erm (50%). However, both ere and msr genes were completely absent from this genus. About 14% of Klebsiella pneumoniae isolates carried a combination of (mph+erm) genes. The prevalence percentages of the four MAC resistance-coding genes in the tested Pseudomonas spp. isolates were 55.6% for each of ere and erm genes, 44.4% for mph gene, while msr gene was totally absent. Combined two and three MAC resistance-coding genes were carried by 33.3% and 11.1%, respectively, of tested Pseudomonas spp. isolates. In plasmid-harboring resistant Staphlococcus spp. isolates, mph was the most commonly detected (100%), followed by erm (57.1%), while each of ere and msr genes were present at equal prevalence percentages of 28.6%. It was found that 71.5% of tested Staphlococcus spp. isolates carried combinations of two or more of MAC resistance-coding genes.
Detection of MLS (Macrolides, lincosamides and streptogramine B) resistance phenotype in Staphylococcus spp. isolates using double diffusion disc method (Dtest) showed that, four resistant Staphylococcus spp. isolates (50%) showed constitutive MLS resistance, while the other isolates showed inducible MLS resistance. PCR products representing the four genes (mph, ere, erm and msr) were sequenced from both directions. The obtained sequence files were assembled using Staden Package program, and the final contigs were obtained, and their corresponding ORF were analysed, annotated and submitted to GeneBank data base. These deposited genes could be reached under the accession codes of KJ710360, KJ710359, KJ710358, KJ652913 and KJ710361 for two mph genes, erm, ere and msr genes, respectively.
The plasmids extracted from 92.8% of Gram-negative resistant isolates could be transformed into E. coli DH5α. The resultant transformants showed resistance phenotypes identical to those of parent donor isolates.
Agarose gel electrophoresis of plasmid extracts of the MAC resistant isolates showed that 50 out of 64 resistant isolates (78.1%) harbored plasmids. Using PCR, it was found that MAC reistance-coding genes were detected on the plasmids of 14 Klebsiella pneumoniae isolates, 12 E. coli isolates, 9 Pseudomonas spp. isolates and 7 Staphylococcus spp. isolates.
PCR analysis, verified by DNA sequencing of PCR products, revealed that all resistant isolates harboring plasmids carried at least one of the tested MAC resistance-coding genes (mph, ere, erm and msr), on their plasmids. Thirty-two isolates (64%) were found to harbor a single macrolide resistance-coding gene while combinations of two, three and four macrolide resistance-coding genes were detected in 15 isolates (30%), 2 isolates (4%) and one isolate (2%), respectively. Of all tested MAC resistance-coding genes, mph was the most frequently encountered gene (78%), followed by erm gene which was detected in 22 isolates (44%). Nine isolates (18%) were found to carry ere gene, while only two isolates (4%) were found to harbor msr gene. Regarding the distribution of the four MAC resistance-coding genes in resistant isolates of different genera, mph, erm and ere genes were detected in 83.3%, 41.6% and 8%, respectively, of tested E. coli resistant isolates, while msr gene was not detected in any of the tested E. coli isolates. Different combinations of two MAC resistance-coding genes could be detected in 33.3% of resistant E. coli isolates. In case of Klebsiella pneumoniae isolates, mph was the most commonly detected (64.3%), followed by erm (50%). However, both ere and msr genes were completely absent from this genus. About 14% of Klebsiella pneumoniae isolates carried a combination of (mph+erm) genes. The prevalence percentages of the four MAC resistance-coding genes in the tested Pseudomonas spp. isolates were 55.6% for each of ere and erm genes, 44.4% for mph gene, while msr gene was totally absent. Combined two and three MAC resistance-coding genes were carried by 33.3% and 11.1%, respectively, of tested Pseudomonas spp. isolates. In plasmid-harboring resistant Staphlococcus spp. isolates, mph was the most commonly detected (100%), followed by erm (57.1%), while each of ere and msr genes were present at equal prevalence percentages of 28.6%. It was found that 71.5% of tested Staphlococcus spp. isolates carried combinations of two or more of MAC resistance-coding genes.
Detection of MLS (Macrolides, lincosamides and streptogramine B) resistance phenotype in Staphylococcus spp. isolates using double diffusion disc method (Dtest) showed that, four resistant Staphylococcus spp. isolates (50%) showed constitutive MLS resistance, while the other isolates showed inducible MLS resistance. PCR products representing the four genes (mph, ere, erm and msr) were sequenced from both directions. The obtained sequence files were assembled using Staden Package program, and the final contigs were obtained, and their corresponding ORF were analysed, annotated and submitted to GeneBank data base. These deposited genes could be reached under the accession codes of KJ710360, KJ710359, KJ710358, KJ652913 and KJ710361 for two mph genes, erm, ere and msr genes, respectively.
The plasmids extracted from 92.8% of Gram-negative resistant isolates could be transformed into E. coli DH5α. The resultant transformants showed resistance phenotypes identical to those of parent donor isolates.
Other data
| Title | Genotypic and phenotypic studies on resistance to macrolides in certain bacteria involved in respiratory tract infection | Authors | Amr Shaker Meselhy | Issue Date | 2014 |
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