الفهرس 
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Abstract
This study was concerned with screening clinical bacterial isolates, from patients suffering from signs and symptoms of urinary tract infections (UTIs), for resistance against different antimicrobial agents commonly used in the treatment of such infections. The aim was to determine the prevalence of resistance among uropathogens to different antimicrobials and to detect the prevalence of multidrug-resistant (MDR) uropathogens. The study was also focused on the detection of plasmid-mediated antimicrobial resistance, as this type of resistance can spread easily and rapidly among the microbial population.
In the present study, a total of 150 clinical bacterial isolates were obtained from the Microbiology laboratory of El-Demerdash Hospital, Cairo, Egypt. All the isolates were recovered at the hospital’s laboratory from urine specimens collected from UTI patients. Of these isolates, 34 isolates (22.7%) were Gram-positive and 116 isolates (77.3%) were Gram-negative. Of the 34 Gram-positive isolates; 23 (67.6%) were coagulase-negative Staphylococcus spp., 6 (17.6%) were S. aureus, and 5 (14.7%) were Enterococcus spp. Of the 116 Gram-negative isolates; 107 (92.2%) were identified as members of Enterobacteriaceae, 7 (6%) were Pseudomonas spp., and 2 (1.7%) were Acinetobacter spp.
The susceptibilities of the recovered isolates to antimicrobial agents belonging to ten classes of antimicrobials commonly used to treat UTIs were tested. The antimicrobial susceptibility of the Gram-positive cocci (GPC) collected in this study (n=34) showed that the lowest resistance rates were observed to doxycycline, vancomycin, and imipenem (5.9%, 8.8%, 10.3%). On the other hand, the highest resistance rates were observed to cefoxitin and ceftazidime (44.8% each); ampicillin/sulbactam and cefepime (41.4% each); and ciprofloxacin and erythromycin (38.2% each). The antimicrobial susceptibility of the Gram-negative bacilli (GNB) collected in the present study (n=116) showed that the lowest resistance rates were observed to doxycycline (11.0%) and imipenem (16.4%). The highest resistance rates were observed to ampicillin/sulbactam (52.3%); co-amoxiclav (49.5%); ceftazidime (42.2%); cefepime (41.4%); and ciprofloxacin (40.5%).
Among the 150 collected isolates; 65 isolates (43.3%) were found to be resistant to three or more classes of antimicrobial agents, thus they were considered MDR isolates; 14 (21.5%) of which were GPC including S. aureus (4/14; 28.6%), S. haemolyticus (3/14; 21.4%), S. xylosus (3/14; 21.4%), S. epidermidis (2/14; 14.3%), and S. lentus (2/14; 14.3%); while 51 (78.5%) isolates were GNB including E. coli (24/51; 47.1%), K. pneumoniae (15/51; 29.4%), K. terrigena (4/51; 7.8%), P. mirabilis (5/51; 9.8%), A. baumannii (2/51; 3.9%), and P. aeruginosa (1/51; 2%).
For MDR GPC, the highest resistance rates were observed to ceftazidime and cefepime (85.7% each); ampicillin/sulbactam, ciprofloxacin, and clindamycin (78.6% each); and co-amoxiclav, cefoxitin, levofloxacin, azithromycin, and erythromycin (71.4% each). On the other hand, the lowest resistance rates were observed to vancomycin and doxycycline (14.3% each); and imipenem (21.4%). Regarding MDR GNB, the highest resistance rates were observed to ampicillin/sulbactam (88%); ceftriaxone (86%); ceftazidime and cefepime (80.4% each); and ciprofloxacin (74.5%). The lowest resistance rate was observed to doxycycline (16%).
from the MDR isolates, plasmids were extracted and detected in 39 (60%) out of the 65 isolates; 17 of which were extracted from E. coli, 17 from Klebsiella spp., 3 from Staphylococcus spp., 1 from P. mirabilis, and 1 from P. aeruginosa isolates. After PCR amplification, one or more extended spectrum β-lactamase (ESBL) genes were found on the plasmids of the 39 MDR isolates; the blaSHV gene was found on the plasmids of 16 (41%) of the 39 MDR isolates, blaCTX-M gene was detected in plasmids of 35 (89.7%) isolates, and blaTEM gene was found in the plasmids of 33 (84.6%) isolates. The gene conferring resistance to aminoglycosides and its variant conferring resistance to ciprofloxacin (aac(6ʹ)-Ib/aac(6ʹ)-Ib-cr gene) was found on the plasmids of 36 (92.3%) isolates. As for the plasmid-mediated quinolone resistance (PMQR) genes; qnrA gene was found on the plasmids of 3 (7.7%) isolates, qnrB gene was detected on the plasmids of 18 (46.2%) isolates, and qnrS gene was found on the plasmids of 36 (92.3%) isolate. Quinolone efflux pump genes were also detected; only 4 (10.3%) of the 39 MDR isolates carried the qepA gene on their plasmids, 8 (20.5%) MDR isolates carried the oqxA gene on their plasmids, and 15 (38.5%) isolates carried the oqxB gene on their plasmids. Amplification of some carbapenem resistance genes was carried out by PCR using the proper primers, and the plasmids of 12 carbapenem resistant MDR isolates as PCR templates. Out of the 12 isolates, 9 (75%) carried blaOXA gene.
Plasmids extracted from the MDR isolates were used to transform competent E. coli DH5α for detection of their resistance phenotypes in this host strain. In case of plasmids harboring ESBL coding genes (n=39), successful transformation was achieved with plasmids extracted from 28 isolates (71.8%) harboring such plasmids. These included plasmids extracted from 15 Klebsiella spp. isolates (n=17; 88.2%), 10 E. coli isolates (n=17; 58.8%), 2 Staphylococcus spp. isolates (n=3), and 1 P. aeruginosa isolate (n=1). None of the P. mirabilis isolates (n=1) were successfully transformed. Out of 36 MDR isolates that carried the aminoglycoside resistance gene aac(6ʹ)-Ib/aac(6ʹ)-Ib-cr, the plasmids extracted from 15 isolates (41.7%) were successfully transformed. These included extracted plasmids from 9 Klebsiella spp. isolates (n=16; 56.3%), and 6 E. coli isolates (n=15; 40%). Isolates that carried any of the PMQR genes (aac(6ʹ)-Ib-cr, qnrA, qnrB, qnrS, qepA, oqxA, and oqxB) were found to be 38 out of the 39 tested MDR isolates (97.4%), plasmids extracted from 10 of which (26.3%) were successfully transformed. As for plasmids extracted from carbapenem-resistant MDR isolates carrying the blaOXA gene (n=9), only 2 were successfully transformed, and both of which were extracted from Klebsiella spp. isolates.
Some selected PCR products of amplified genes were sent for sequencing at GATC, Germany using ABI 3730 xl DNA Sequencer. The assembly of the obtained forward and reverse sequence files into the final consensus was done using BioEdit v7.2.5 software. Sequence analyses proved the existence of the tested genes on the extracted plasmids.
The efflux pump inhibitor CCCP was used to elaborate the extent of contribution of efflux pumps to the resistance to fluoroquinolone antimicrobial agents. Thirteen (86.7%) of the 15 isolates, which were found to carry one or more of the quinolone efflux pump genes, had no change in ciprofloxacin MIC values; only 2 isolates (44S, S. aureus; and 135E, E. coli) showed a 2-fold decrease in MIC upon the addition of CCCP; indicating the minor contribution of efflux pumps to the resistance against fluoroquinolones.
In conclusion, multidrug resistance is currently a major health threat limiting the use of common antimicrobials in treatment. The unnecessary prophylactic use of antimicrobials, inaccurate diagnosis, and lack of regulations over the dispensing of antimicrobial agents are among the main causes of rapid spread of antimicrobial resistance in developing countries. Accordingly, the misuse of antimicrobials should be avoided to prevent increased resistance in the community. Finally, it is recommended to report MDR isolates, and bear in mind the rational prescription of antimicrobial agents to limit the emergence and prevalence of the resistance mechanisms.