الفهرس 
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Abstract
With the increased use of antibiotics in the treatment of bacterial infections, pathogenic strains have acquired antibiotic resistance, causing a major problem in treatment and therapeutic outcomes. Among the medical concerns being greatly affected by the problem of the widespread of antimicrobial resistance are the respiratory tract infections (RTIs). RTIs are very common infections both in the community and among hospitalized patients. The most common type of bacteria which cause community-acquired pneumonia (CAP) is Streptococcus pneumoniae. Atypical pneumonias are caused by pathogens such as Mycoplasma pneumoniae, Chlamydia spp., Legionella spp., Coxiella burnetti and viruses. Nosocomial pneumonia is caused by a variety of bacteria that originate from the patient flora or the health care environment as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella spp., E. coli, Acinetobacter spp., and Enterobacter spp. Macrolide antibiotics (MACs) are polyketides that stop bacterial growth by inhibiting protein synthesis. They are among the antibacterials that are highly recommended for treatment of RTIs. As with other drugs, their overuse and misuse lead to development of resistance in many important pathogens.
The current study aimed at determination of resistance percentages of bacterial respiratory pathogens recovered from hospitalized RT infected patients to MAC antibiotics and to detect the most prevalent plasmid-mediated resistance mechanisms.
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. Categorization and identification of the resistant 64 isolates revealed that 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.
Antibiogram analysis data showed that all tested isolates each of E. coli, Klebsiella pneumoniae, and Pseudomonas spp were resistant to three macrolide antibiotics (ERY, CLA and CLI) while the resistance percentages against AZI for these three microbial species were 92.3, 78.9 and 44.4%, respectively. In case of Staphylococcus spp., all tested isolates showed resistance to the three MAC antibiotics (ERY, CLA and AZI), while 50% of these isolates were resistant to CLI.
Plasmids were extracted from isolates having reduced susceptibility to the tested MAC antibiotics. Detection of genes involved in different plasmid-mediated resistance mechanisms like target site modification, enzymatic inactivation of MACs and active-efflux of the drug, could be achieved by PCR assay using the extracted plasmids as DNA templates.
Four sets of primers were used for PCR detection of the MAC resistance-coding genes: mph, ere, erm and msr in all plasmid-harboring resistant isolates. MAC resistance-coding genes were detected on the plasmids of 78.1% of all resistant isolates. Single MAC resistance-coding genes were detected in 64% of isolates, while combinations of two, three and four MAC resistance-coding genes were detected in 30%, 4% and 2% of isolates, respectively. A total of six different MAC resistance-coding genes combinations were detected in all tested resistant isolates.
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 resistance phenotype in Staphylococcus spp. isolates using double diffusion disc method (D-test) 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 ORFs were analysed, annotated and submitted to GeneBank. These deposited gene sequences 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.