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Abstract With the increased use of antibiotics to treat bacterial infections, pathogenic strains have acquired antibiotic resistance, causing a major problem in modern therapeutics. Among the medical problems being greatly affected by the problem of the widespread of antimicrobial resistance is the urinary tract infection (UTI). UTI is a very common infection both in the community and among hospital patients. Escherichia coli is the most common cause of uncomplicated UTI followed by Staphylococcus saprophyticus then other aerobic gram negative rods, such as Klebsiella, Pseudomonas and Proteus species. Aminoglycoside antibiotics (AGAs) are aminocyclitols that kill bacteria by inhibiting protein synthesis. They are among the antibacterials that are highly recommended for treatment of UTIs. As with other drugs, their overuse and misuse lead to development of resistance in many important pathogens. Three mechanisms are known to be responsible for bacterial resistance to aminoglycoside antibiotics: first, decreased intracellular accumulation by outer membrane permeability alteration, diminished inner membrane transport, or active efflux; second, target modification by mutation of 16S rRNA or ribosomal proteins coding genes or by 16S rRNA methylation, a mechanism newly identified in clinical isolates; and third, enzyme-mediated drug modification resulting in compromised binding to target site, the most prevalent in clinical setting. The current study aimed at determination of resistance percentages and patterns of uropathogens of the genera Escherichia, Klebsiella, and SUMMARY 133 Pseudomonas, isolated from hospitalized UTI patients, to aminoglycoside antibiotics and to detect the most prevalent plasmid-mediated aminoglycoside modifying enzymes (AMEs) and 16S rRNA methyltransferases. Atotal of 150 uropathogenic isolates were recovered from urine specimens of hospitalized UTI patients and identified by microbiological methods. Of these isolates, nine isolates (6.0%) were found to be Gram positive cocci and 141 isolates (94.0%) were found to be Gram negative rods. Of these Gram negative rods, 73 E. coli isolates and 24 Klebsiella spp. isolates were identified by testing for indole production, citrate utilization and reactions on TSI agar. Thirteen isolates showed growth with green pigment production on cetrimide agar and positive oxidase test and consequently identified as Pseudomonas species. All the recovered uropathogens were tested for their susceptibility to gentamicin, tobramycin, amikacin, neomycin, netilmicin, and kanamycin by disc diffusion method. An overall resistance percentage of 57.2% was detected to at least one of the tested AGAs. Of which, 17.4% were resistant to one AGA, 31.7% were resistant to two AGAs, and 50.7% were resistant to three or more AGAs. Of all tested AGAs, the highest resistance percentage was detected to kanamycin (53.6%) and lowest resistance percentage was to amikacin (7.2%). Resistance percentages of 33.6%, 23.6%, 24.5%, and 14.5% were detected to gentamicin, tobramycin, neomycin and netilmicin, respectively. Antibiogram analysis of E. coli isolates showed 56.1% resistance to at least one of the tested AGAs. The highest resistance was to kanamycin followed by neomycin, gentamicin, tobramycin, netilmicin then amikacin with resistance SUMMARY 134 percentages of 52.0%, 31.5%, 28.7%, 16.4%, 6.8% then 4.1%, respectively. In Klebsiella spp. isolates, 41.6% were resistant to at least one of the tested AGAs. About 37.5% of all Klebsiella spp. isolates were resistant to kanamycin, 33.3% were resistant to gentamicin, 20.8% were resistant to tobramycin, 12.5% were resistant to netilmicin, 4.1% were resistant to neomycin and 4.1% were resistant to amikacin. A significantly high resistance to AGAs was detected in Pseudomonas spp. isolates. About 92.3% of them were resistant to at least one of the tested AGAs. Pseudomonas spp. isolates had shown highest resistance (92.3%) to kanamycin and lowest resistance (23.0%) to neomycin, 69.2%, 61.5%, 61.5% and 30.7% resistance to tobramycin, gentamicin, netilmicin and amikacin, respectively. A total of 17 resistance phenotypes were identified from the antibiogram analysis of the tested isolates. E. coli isolates exhibited thirteen resistance phenotypes. Seven resistance phenotypes were identified in Klebsiella spp. isolates while six were identified in Pseudomonas spp. isolates. Plasmids were extracted from isolates having reduced susceptibility to at least one of the tested aminoglycoside antibiotics. Detection of two plasmid-mediated resistance mechanisms, enzyme modification and 16S rRNA methylation, was carried out by PCR assay using plasmids as DNA templates. Five sets of primers were designed for PCR detection of the AMEscoding genes: aph(3’)-I, aac(6’)-I, aac(3)-I, aac(3)-II and ant(2’’)-I in all resistant isolates. AMEs-coding genes were detected on the plasmids of 93.6% of all resistant isolates. Individual AMEs-coding genes were SUMMARY 135 detected in 44.4% of isolates, while combinations of two and three AMEs-coding genes were detected in 20.6% and 28.5% of isolates, respectively. A total of nine different AMEs-coding genes combinations were detected in all resistant isolates. Of all tested AMEs-coding genes, ant(2’’)-I was the most frequently encountered gene (53.9%) followed by aac(6’)-I and aac(3)-II each were found in 24 isolates (38%). Twenty-one isolates (33.3%) were found to carry aph(3’)-I, while aac(3)-I gene was not detected in any of the tested resistant isolates. Regarding the distribution of the four AMEs-coding genes in resistant isolates of different genera, ant(2’’)-I was detected in 46.3% of E. coli resistant isolates, aph(3’)-I in 44.1%, while 34.1% and 31.5% of them carried aac(3)-II and aac(6’)-I, respectively. In resistant Klebsiella spp. isolates, ant(2’’)-I was the most commonly detected (70.0%), followed by aac(3)-II (60.0%), aac(6’)-I (40.0%), while aph(3’)-I was the least commonly detected in 20.0% of Klebsiella spp. resistant isolates. The prevalence percentages of the four AMEs-coding genes in resistant Pseudomonas spp. isolates were 66.6%, 58.3, 33.3% and 16.6% for ant(2’’)-I, aac(6’)-I, aac(3)-II and aph(3’)-I, respectively. None of the aforementioned genes was detected on the plasmids of only four resistant isolates (6.3%) despite showing resistance to at least one of the tested AGAs. Four PCR products representing the four genes (aph(3’)-I, ant(2’’)-I, aac(6’)-I and aac(3)-II) were sequenced from both directions by Macrogen company, Korea. Analysis of the translation product of the final sequences showed 100% similarities with their homologous enzymes from different bacterial species. SUMMARY 136 16S rRNA methylation was the second resistance mechanism screened in only the six isolates that showed resistance to all tested members of the 4,6 disubstituted 2DOS-containing AGAs. 16S rRNA methylases were detected phenotypically by testing the susceptibility of the multiresistant isolates to both streptomycin and apramycin. None of the six multiresistant isolates exhibited the N1-A1408 16S-RMTases resistance phenotype and only one of them exhibited that of N7-G1405 16S-RMTases. Two isolates were resistant to all tested AGAs. As the resistance phenotype may be complicated by co-existance of other resistance mechanisms, all isolates that showed resistance to all tested 4,6 disubstituted 2DOS-containing AGAs were screened for 16S RMTasescoding genes by PCR assay. Screening the plasmids of the six multiresistant isolates for the two 16S RMTases-coding genes armA and rmtA showed that, none of them carried any of the tested genes. Finally, plasmid localization of resistance genes and their ability to transfer was tested by transformation assay using E. coli DH5α as a recipient strain. Transformation experiments were successful in 11.1% of resistant ones. The transformation was confirmed by antimicrobial susceptibility testing and by PCR assays. The resistance profiles were identical in six transformants and donor clinical isolates. In only one transformant (116T), resistance to gentamicin was lost as compared to the donor clinical isolate resistance profile. ant(2’’)-I gene was not detected in this transformant (116T) despite being detected in the donor isolate (116). SUMMARY 137 This study demonstrated a high dissemination of plasmid-mediated aminoglycoside resistance in relevant uropathogens. The threat of horizontal gene transfer necessitates the implementation of proper infection control measures in hospitals. It is also recommended that new guidelines have to be undertaken in Egypt to limit or prevent the misuse and abuse of antimicrobial agents. |