Phenotypic and genotypic studies on antimicrobial resistance of lower respiratory tract bacterial pathogens

Abstract

The present study was concerned with the isolation of bacteria from Egyptian patients suffering from lower respiratory tract infections (LRTIs), and screening them against different antimicrobials agents commonly used to treat LRTIs. The aim was to know the prevalence of resistance among common lower respiratory tract pathogens in Cairo, Egypt and to know the prevalence of multiple drug resistant (MDR) pathogens. The study was also concerned with the detection of plasmid-mediated antimicrobial resistance and the involvement of certain resistance genes among MDR isolates as this kind of resistance contributes to its rapid spread among microbial population. In this study, a total of 235 bacterial isolates were recovered from sputum and bronchoalveolar lavage specimens collected from lower respiratory tract infected patients. Of which, 118 isolates (50.2%) were Gram-positive and 117 isolates (49.8%) were Gram-negative. The susceptibilities of the recovered isolates to sixteen different antimicrobial agents commonly used to treat lower respiratory tract infections were tested. Results revealed that the lowest resistance was observed to amikacin, doxycycline and meropenem; only 19 isolates (8%) were resistant to each. On the other hand, the highest resistance was observed to clindamycin; 108 isolates (45.9%) were resistant. The antimicrobial susceptibility testing of the Gram-positive bacterial isolates collected in this study (n=118) showed that the lowest resistance was observed to doxycycline; only one isolate (0.8%) was resistant. On the other hand, the highest resistance was observed to clindamycin (16.9%) and erythromycin (16.9%). Regarding the antimicrobial susceptibility testing of the Gram-negative bacterial isolates collected in this study (n=117), the lowest resistance was observed to meropenem and azithromycin; only 13 isolates (11.1%) were resistant to each. Summary MSc Thesis 2015 Page 129 While, the highest resistance was observed to clindamycin; 88 isolates (75.2%) were resistant. Among the 235 collected isolates; 62 isolates (26.4%) were found to be resistant to members related to three or more classes of antimicrobial agents (MDR isolates); 53 isolates were Gram-negative (85.5%) while 9 isolates were Grampositive (14.5%). Out of these 62 MDR isolates; 23 isolates were Klebsiella pneumoniae (37.1%), 15 isolates were Escherichia coli (24.2%), 10 isolates were Pseudomonas aeruginosa (16.1%), 9 isolates were Staphylococcus aureus (14.5%), 3 isolates were Enterobacter cloacae (4.8%) and 2 isolates were Acinetobacter baumannii (3.2%). All K. pneumoniae, E. coli and P. aeruginosa MDR isolates showed resistance to amoxicillin, cefadroxil, cefuroxime, ceftriaxone and cefepime. Moreover, all E. coli and P. aeruginosa MDR isolates also showed resistance to clindamycin. Additionally, all MDR P. aeruginosa isolates showed resistance to co-amoxiclav. Resistance to meropenem was, however, absent in case of the MDR K. pneumoniae and E. coli isolates. Also, none of the MDR P. aeruginosa isolates were resistant to azithromycin. On the other hand, all MDR S. aureus isolates showed resistance to amoxicillin, cefadroxil, ceftriaxone, ciprofloxacin, azithromycin, clarithromycin and erythromycin. While resistance was absent to doxycycline. From the MDR isolates, plasmids were extracted and detected in 60 (96.8%) out of the 62 MDR isolates. Regarding the MDR S. aureus isolates, 7 isolates (77.8%) were found to harbor plasmids. All the tested MDR Gram-negative bacterial isolates (n=53; 100%) were found to harbor plasmids. Nineteen out of the 62 MDR isolates were resistant to amikacin and they all carried the aminoglycoside acetyltransferase gene (aac6’-Ib) reported to be responsible for the plasmid mediated resistance to amikacin. Fourteen (27.5%) of the 51 isolates resistant to ciprofloxacin carried one of the qnr gene variants, of which 10 isolates (19.6%) Summary MSc Thesis 2015 Page 130 carried the qnrA gene, while the remained 4 isolates (7.8%) carried the qnrB gene. These 51 isolates were also screened for the presence of the aac6’-Ib-cr gene and it was found that 32 isolates (62.7%) were found to carry this gene. Fifty out of the 53 Gram-negative isolates (94.3%), resistant to ceftriaxone, were found to carry one or more of the ESBL coding genes. Each of ctx-m and tem genes was found in 46 isolates (86.8%), while shv gene was found in only 41 isolates (71.4%). Thirty-five isolates (66%) carried all three genes together, 13 isolates (24.5%) carried two of the genes together, while only 2 isolates (3.8%) carried only a single gene. All tested resistant K. pneumoniae, E. coli and A. baumannii isolates (100%) showed one or more of the tested ESBL coding genes. While only 8 resistant P. aeruginosa isolates carried at least one of these genes. The double disk synergy test (DDST) was applied to all isolates resistant to ceftriaxone for phenotypic detection of their productivities to ESBLs. However, it gave negative results with P. aeruginosa, S. aureus, E. cloacae and A. baumannii test isolates. However, only 17 K. pneumoniae isolates (73.9%) gave positive DDST. Likewise, only 11 E. coli isolates (73.3%) gave positive DDST. Therefore, genotypic detection of ESBL genes is more sensitive than phenotypic detection. No correlation was detected between the MIC values of both ceftriaxone and cefepime and the number of tested ESBLs-coding genes harbored by test organisms. It was found that eleven isolates (17.7%) harbored one of the qnr variants, one or more of the ESBL-coding genes and the aac(6’)-Ib/aac(6’)-Ib-cr gene simultaneously. Nine isolates carried ctx-m, shv, tem, aac(6’)-Ib/aac(6’)-Ib-cr and qnr genes together, while 2 isolates carried ctx-m, tem, aac(6’)-Ib/aac(6’)-Ib-cr and qnr genes together. In case of plasmids harboring ESBL coding genes, successful transformation into E. coli DH5α was achieved with plasmids extracted from 38 out of 53 isolates harboring such plasmids (71.7%). While all plasmids extracted from Summary MSc Thesis 2015 Page 131 the Gram-negative isolates resistant to amikacin (n=14; 100%) and those harboring the qnr gene (n=14; 100%) were successfully transformed into the E. coli DH5α host strain. PCR products of the four genes suggestive for ctx-m, shv, tem, aac(6’)-Ib, obtained upon using plasmids extracted from certain Gram-negative isolates, were purified and 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 analyzed. This proved the existence of ctx-m, shv, tem, aac(6’)-Ib on the extracted plasmids. These genes were annotated and submitted to GenBank database where they were accepted and reported under the accession codes, KM052216, KM052217, KM052218 and KM052219, respectively. 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, imprecision of 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 MDR organisms. Public awareness should also be raised to limit the high level of antimicrobials misuse and to highlight the importance of hygienic practices.