الفهرس 
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Abstract
Lower respiratory tract infections are considered among the most common diseases in humans. They account for about two thirds of antimicrobials prescribed. Globally, they are the fourth overall leading cause of death. This study aimed to elucidate both the phenotypic and genotypic bases of antimicrobial resistance of some multiple drug-resistant lower respiratory tract pathogens against the most common antimicrobial agents used in treatment. It aimed to develop an accurate local periodic report of antimicrobial resistance to guide Egyptian healthcare practitioners to more effective prescription strategies.
A total of 231 clinical bacterial isolates were recovered from sputum and bronchoalveolar lavage specimens from lower respiratory tract infected patients from which 84% were found to be Gram-negative and 16% were Gram-positive. The isolates were K. pneumoniae (39%), E. coli (20%), P. aeruginosa (25%) and S. aureus (16%). They were tested against several classes of antimicrobials commonly used to treat respiratory tract infections according to the international guidelines. The antimicrobial susceptibility testing revealed that the lowest resistance was observed to doxycycline (14%); only 32 isolates were resistant. On the other hand, the highest resistance was observed to amoxicillin (74%); 171 isolates were resistant.
Gram-negative pathogens are particularly worrisome because they are becoming resistant to nearly all available antibiotic options. Among the Gram-negative isolates, the lowest resistance was observed to amikacin (15%); only 30 isolates were resistant. On the other hand, the highest resistance was also observed to amoxicillin (79%); 154 isolates were resistant. Among the Gram-positive isolates, none of the isolates were resistant to linezolid nor vancomycin. The lowest resistance was observed to doxycycline (3%); only 1 isolate was resistant. On the other hand, the highest resistance was observed to ciprofloxacin (51%); 19 isolates were resistant.
The study focused on the carbapenem-resistant Gram-negative isolates and the MRSA isolates as the WHO considered them high priority pathogens that pose the greatest threat to human health. All of these isolates collected in our study were multiple drug resistant. Among the carbapenem-resistant K. pneumoniae isolates, the lowest resistance was observed to doxycyline (4%); only one isolate was resistant. On the other hand, all of these isolates were resistant to amoxicillin, co-amoxiclav, cefadroxil, cefuroxime and cefotaxime. Among the carbapenem-resistant P. aeruginosa isolates, the lowest resistance was observed to aztreonam (42%). On the other hand, all of these isolates were resistant to amoxicillin, co-amoxiclav, cefadroxil, cefuroxime, cefotaxime, cefepime, ciprofloxacin and levofloxacin. None of the E. coli isolates were carbapenem- resistant. All the tested MRSA isolates were sensitive to vancomycin and linezolid. The lowest resistance was observed to doxycyline (6%); only one isolate was resistant. On the other hand, all of these isolates were resistant to amoxicillin, co-amoxiclav, cefuroxime, cefotaxime, cefepime, ciprofloxacin, levofloxacin, gentamicin and tobramycin.
None of the carbapenem-resistant Gram-negative isolates harbored the blaKPC nor the blaIMP genes. Among the carbapenem-resistant K. pneumoniae isolates, the most prevalent carbapenemase-coding gene detected was blaNDM (64%), followed by blaOXA-48 (50%) and finally blaVIM (18%). Among the carbapenem-resistant P. aeruginosa isolates, both blaOXA-48 and blaVIM were detected in 25% of the isolates, followed by blaNDM which was detected in only 8% of the isolates. High level of extended spectrum β-lactamases (ESBLs) were also detected. Among the K. pneumoniae isolates the most prevalent ESBL-coding gene detected was blaCTX-M (68%), followed by blaSHV (45%) and blaTEM (45%). Among the P. aeruginosa isolates the most prevalent ESBL-coding gene detected was blaSHV (92%), followed by blaCTX-M (67%) and finally blaTEM (58%).
All of the MRSA isolates harbored the mecA gene responsible for the production of altered PBP2a; this strongly highlights its importance to develop resistance against methicillin and the other members. None of the carbapenemase-coding genes were detected. Approximately, 62% of the isolates harbored one or more of the ESBL-coding genes; the most frequently detected gene was blaCTX-M (62%), followed by blaSHV (25%) and finally blaTEM (12%).
The presence of one aminoglycoside modifying enzyme was studied; the AAC(6’)-Ib/AAC(6′)-Ib-cr. All the carbapenem-resistant Gram-negative isolates, as well as the MRSA isolates, were resistant to either fluoroquinolones or one of the aminoglycosides studied. Approximately, 86% of them carried the aac(6’)-Ib/aac(6′)-Ib-cr gene. The highest prevalence of the gene was detected in the K. pneumoniae isolates (91%), followed by P. aeruginosa isolates (83%) and finally S. aureus isolates (81%).
The presence of genes coding for efflux pumps was also studied. The AcrA subunit from the AcrAB-TolC multidrug efflux pump was detected in 86% of the carbapenem-resistant K. pneumoniae isolates, while the MexA subunit from the MexAB-OprM multidrug efflux pump was detected in all the carbapenem-resistant P. aeruginosa isolates. On the other hand, 75% of the MRSA isolates harbored the MepA multidrug efflux pump. The wide prevalence of multidrug efflux pumps suggests their role in the development of resistance.
For the purpose of cloning and expression, the polymerase chain reaction (PCR) was carried out using especially designed primers containing restrictions sites, which are also found in the multiple cloning site (MCS) of the used plasmid vectors (pUCpu21 and pET-22b). Fifty-five different trials were carried out using variable conditions. Only white colonies of the correct transformant E. coli DH5α could be obtained. Unfortunately, ligation of the DNA inserts and the pET-22b vector could not be successfully accomplished.
In conclusion, multiple drug resistance is a major health concern limiting the use of common antimicrobials in therapy. Therefore, antimicrobials should only be prescribed judiciously as the selective pressure of antimicrobial use inevitably leads to increased resistance in the community. Besides, antimicrobial misuse, the unnecessary prophylactic use of antimicrobials, imprecise diagnosis, lack of regulations over the dispensing of antimicrobials and the spread of counterfeit drugs are among the major reasons of spread of antimicrobial resistance in developing countries.
Accurate local periodic reports of the resistance pattern are of great importance to provide the healthcare practitioners with a clear picture and to guide them for prescription of more effective antimicrobial therapy. Guided prescription policies must be implemented nationwide to limit the further spread of multiple drug resistant organisms. Public awareness should also be raised to limit the high level of antimicrobials misuse and to highlight the importance of hygienic practices. Last but not least, investment to incorporate the rapid identification techniques in the Egyptian hospitals should become a medical priority to allow an improved routine-care.