الفهرس 
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Abstract
In the current study, a total of 239 Gram negative isolates were recovered from different clinical specimens of infected cancer patients attending the NCI, Cairo, Egypt, in the period from November 2014 to July 2015. One hundred and fifty isolates (62.8%) were recovered from pediatric patients while 89 (37.2%) were recovered from adult patients. The majority of isolates were from blood (57.3%) followed by wound swabs (10.5%), pus (9.6%), sputum (7.5%), stools (4.6%), urine (4.2%), drain (3.3%), bronchoalveolar lavage (1.7%) and chest tubes (1.3%). The collected isolates were identified using the conventional microbiological techniques and confirmed by Vitek GN cards. The recovered isolates were identified as E. coli (39.7%), K. pneumoniae (34.3%), A. baumannii (10%), P. aeruginosa (7.9%) and other Gram negative isolates (7.9%) comprising Ent. cloacae (3.3%), E. aerogenes (2.1%), K. oxytoca (1.3%), P. mirabilis (0.8%) and C. freundii (0.4%).
All the recovered isolates were tested for their susceptibility to four FQ antimicrobial agents, including nalidixic acid, norfloxacin, CIP and LEV, using the disc diffusion method. An overall resistance percentage of 82.0% was detected to at least one of the tested FQs. Of all tested FQs, the highest resistance percentage was detected to nalidixic acid (81.2%), followed by CIP (70.7%), norfloxacin (69.5%) and LEV (67.4%). The same method was also used for testing their susceptibility to other classes of antimicrobial agents including the aminoglycosides, GEN and AMK, the cephalosporin, CRO and the carbapenem, IMP. Among which the highest resistance was detected to CRO (91.6%) and the least was to AMK (34.7%), while 51.0% of the isolates were resistant to GEN and IMP. CIP resistance was significantly associated with resistance to each of the mentioned antimicrobial agents (p < 0.001). The MICs of CIP and LEV were determined using the broth microdilution method. An MIC range of (0.006-256 mg/L) were found for both agents with a significantly different distribution of MIC values (p < 0.001). Lower MIC50 was shown for LEV than CIP among the isolates (16 versus 64 mg/L).
PCR was used for screening the most common PMQR genes. For this purpose, specific primers for each were designed. A multiplex PCR was used for simultaneous amplification of the genes, qnrA, qnrS, qnrB and aac(6’)-Ib, while
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the plasmid-mediated efflux genes, oqxAB and qepA were screened by monoplex PCRs. Amplification products of the aac(6’)-Ib gene were digested by the restriction enzyme, BseGI, whose recognition site is found in the wild type gene but not in the aac(6’)-Ib-cr gene variant. At least one PMQR determinant was detected in 53.6% of the clinical isolates. The aac(6’)-Ib-cr gene was the most frequently detected (36.8%), as shown by restriction analysis of PCR products of aac(6’)-Ib (114). The qnr genes were detected in 31.0% of the isolates. The qnrS gene was more common than qnrB (24.3% versus 7.1%), while only one P. mirabilis isolate was positive for the qnrA gene. qepA gene was not detected in the tested isolates. Despite their absence in other species, oqxA and oqxB were detected in 92.5% of Klebsiella spp. isolates (95.1% of K. pneumoniae and 100% of K. oxytoca). Among PMQR-positive isolates, 32(24.8%) carried 2 PMQR genes while combinations of 3 genes were detectable in 1.6% of isolates. No significant association was found between qnr genes and aac(6’)-Ib-cr. In comparison to isolates recovered from adult patients, a significantly higher PMQR prevalence was found among pediatric patients (43.8% versus 59.3%, p = 0.02). Significantly higher CIP non-susceptibility was detected among PMQR-positive isolates than those without PMQR genes (76.6% versus 64.0%, p = 0.03). Nevertheless, 23.4% of PMQR-positive isolates were susceptible to CIP. Furthermore, among the CIP-resistant isolates, the distribution of CIP MICs of PMQR-positive and PMQR-negative isolates were not significantly different (Mann Whitney U test, p = 0.140). Notably, PMQR determinants were detected in isolates showing CIP MIC as low as 0.125 mg/l that is 16 folds lower than the cut off value set by the CLSI for CIP reduced susceptibility (2 mg/l for intermediate susceptibility and ≥ 4 mg/l for full resistance). PMQR determinants were significantly correlated to CRO and GEN resistance, as revealed by Chi-square test (p = 0.002 and 0.006, respectively). Representative amplicons of the genes, qnrA, qnrS, qnrB, aac(6′)-Ib-cr, oqxA and oqxB were sequenced. Analysis of the translation product of the final sequences showed 100% similarities with their homologous enzymes from different bacterial species. Plasmid DNA was extracted from 74 qnr-positive isolates and plasmid bands were successfully demonstrated by AGE for 86.5% of them.
To investigate the clonal relationship of the qnr-positive isolates of the same species, Two PCR-based fingerprinting techniques; RAPD-PCR and ERIC-PCR, were used. Some of isolates, their RAPD profile showed 85% similarity or more,
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could be differentiated by ERIC-PCR and were reallocated into different ERIC-types. Similarly, isolates of the same ERIC type could also be differentiated by RAPD-PCR. Accordingly, dendrograms based on the combined fingerprints generated by ERIC and RAPD PCRs were constructed. The dendrograms obtained suggest that the dissemination of PMQR-positive isolates among K. pneumoniae and E. coli was unlikely to be the spread of specific clones. Instead, PMQR genes were widely distributed among genetically unrelated isolates in different clusters.
Fifteen isolates of different species, including five K. pneumoniae, three E. coli, three A. baumannii, three P. aeruginosa and one P. mirabilis isolate, were selected for analysis of mutations in the QRDRs of the gyrA and parC genes. Specific primers for amplification of gene fragments encompassing the QRDR and flanking nucleotide sequences in different species were designed. All PCR products were sequenced and the predicted amino acid sequences were analysed for amino acid changes by comparison to the wild-type GyrA and ParC sequences of the corresponding standard strains deposited in the GenBank. Target site mutation analysis revealed at least one missense mutation, including a gyrA mutation, in all isolates. Codon 83 of the gyrA gene had the highest incidence of such mutations (100%) with the resulting substitutions: Ser/Leu, Ile, Tyr or Phe in all species except P. aeruginosa where threonine was substituted by isoleucine. This was accompanied by another mutation at the codon 87 in 40% of isolates (Asp/Asn, Tyr or Ala). Amino acid substitutions in ParC were evident in 93.3% of isolates at codon 80 (86.7%) including: Ser to Ile or Leu substitutions and codon 84 where Lys was substituted for Glu in one isolate (6.7%). Double mutations (1 gyrA/ 1 parC) were the most frequently encountered among the tested isolates (53.3%) followed by three mutations (2 gyrA/1 parC) found in 40%, while only 6.7% showed a single gyrA mutation. With respect to the species type, three mutations (2 gyrA/1 parC) were found in all tested E. coli isolates. All non-fermenter isolates showed two mutations (1 gyrA/1 parC). A combination of two gyrA and one parC mutations were observed in 60% of K. pneumoniae isolates, while two mutations (1 gyrA/1 parC) and one gyrA mutation each were detected in one K. pneumoniae isolate (20%).
The distribution of CIP MIC across the groups of isolates carrying different numbers of target site mutations was found to be significantly different (p = 0.005). The highest MIC50 (128 mg/L) was shown by the group of isolates carrying
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3 target site mutations while those having 2 mutations had an MIC50 value of 32. A single isolate was found to carry one target site mutation and showed CIP MIC of 32 mg/L. Such isolate was also found to co-harbor the PMQR genes, qnrS, qnrB, aac(6′)-Ib-cr and oqxAB. A wide variability in CIP MIC was shown by the isolates carrying two target site mutations. The nucleotide sequences of gyrA and parC genes of some clinical isolates analyzed in the current study were submitted to GenBank nucleotide sequence database.
To investigate the role of efflux pumps in the development of FQs resistance, the MIC of CIP was determined in the presence of sub-inhibitory concentrations of the efflux pump inhibitor; CCCP. Subsequently, the MIC decrease factor (MDF) of each isolate was calculated. An MDF value of 4 or more was considered as a significant effect due to efflux pump inhibition via CCCP. Among the tested isolates, efflux pump inhibition reduced CIP MIC four fold or more (MDF value of more ≥ 4) in 18.3% of the isolates, most commonly in non-fermenters (50.0%). On the other hand, 66 isolates (39.1%) exhibited a MDF value of 2, while CIP MIC of 72 isolates (42.6%) did not show any difference by combining CCCP. Although efflux activity was associated with higher CIP MIC50 (128 versus 64 mg/L), the distribution of CIP MIC was found to be the same across the groups of isolates showing positive efflux activity and others (Mann Whitney U test, p = 0.406).
In an attempt to reduce the level of FQs resistance, the effect of combining CIP with various antimicrobial agents, including COL, TIG, AMK, CRO and MER, was tested in eight isolates of different species by the checkerboard assay. The tested combinations showed indifferent effect on CIP activity on most of the tested isolates (52.5%), additive effect in 42.5% of them. Synergism was evident in only two isolates (5.0%) where CIP was combined with either of AMK and CRO.
The effect of combining subinhibitory concentrations of silver oxide NPs (5 mg/L) with CIP was also tested by the broth microdilution method and showed variable effect on CIP MIC among the isolates selected for the assay. A four fold reduction of CIP MIC was achieved by such combination in two isolates (25.0%) while 37.5% showed two fold CIP MIC reduction. On the other hand, combining silver oxide NP did not make any difference in CIP MIC of two isolates (25.0%)