الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Ventilator associated pneumonia (VAP) is a complication occurring in ventilator-dependent patients of all ages associated with hospital mortality rates as high as 40%, significantly greater hospital lengths of stay, and increased healthcare costs.
One of the major issues concerning the management of VAP is the increasing prevalence of MDR or XDR pathogens.
The aim of this study was to isolate the bacterial pathogens causing ventilator associated pneumonia (VAP), determine the susceptibility pattern of the isolates, and detect the most common mechanism of resistance for the most commonly isolated organisms.
A total of one hundred endotracheal aspirates collected from patients in ICU at different hospitals in Alexandria were included in this study.
The causative organisms were identified using conventional culture and biochemical tests. Isolated strains were further identified and typed using MALDI-TOF MS, which was carried on Ultraflex TOF/TOF (Bruker Daltonics).
The Susceptibility of the isolated strains was determined by disc diffusion according to CLSI recommendations.
Out of the 100 VAP cases, 45 (45%) were categorized as early-onset, while the remaining 55 (55%) were late-onset VAP. 65% of the patients were males while 35% were females, and 60% of them were above 60 years. Previous antibiotic administration was reported in 90% of the cases.
The most common isolated organism was K.pneumoniae followed by Acinetobacter. The 29 K. pneumoniae isolateswere identified by conventionalmethodsand confirmed by MALDI-TOF MS.
Out of the 29 Klebsiella pneumoniae isolates 24 (82.8%) were identified as MDR.
The isolatedKlebsiella pneumoniae strains were further subjected for various tests to determine phenotypic and genotypic mechanisms of antibiotic resistance.
Phenotypic detection of ESBL was carried out by CDT, DDST and growth on MacConkey’s agar supplemented with CTAZ. Out of 29 Klebsiella pneumoniae 26 (%) were resistant to CAZ and CTX
Only four (15%) isolates out of the 26 isolates resistant to CAZ and CTX. were diagnosed as ESBL producers by DDST
Twenty isolates (77%) out of the resistant 26 isolates were diagnosed as ESBL producers by combined disc test
All 26 isolates (100%) showed growth on MacConkey’s agar supplemented with CTAZ and were identified as ESBL producers.
Phenotypic detection of Carbapenemases was carried out by MHT, growth onMacConkey’s agar supplemented with MEM.Out of 21 Klebsiella pneumoniae resistant to IMP and MEM, 20 (95%) isolates were identified as carbapenamase producer by the MHT.
All 21resistant isolates (100%) were identified as carbapenemase producers by subculturing them on MacConkey agar supplemented with MEM
Phenotypic detection of MBL was carried out by combined disc test using IMP and EIP (IMP+EDTA)Out of the 21 resistant isolates, 14 (66.7%) were positive for metallo- β-lactamase production
AmpC screening: None of the 23 isolates resistant to cefoxitin was AmpC producer
Detection of KPC-producers: Out of the 21 isolates resistant to IPM only 5 (23.8%) isolates were positive for KPC production
Phenotypic detection of Quinolone resistance was carried out by:
MIC determination using micro-dilution broth method: The 24 (82.8%) isolates showing MIC above the breakpoints were resistant by disk diffusion
Phenotypic Detection of quinolone resistance due to efflux pump: The presence of the efflux pump in the examined isolates was determined by at least four fold decreases in the MIC values of ciprofloxacin in the presence of pump inhibitor; phenylalanine arginyl β-naphthylamide (PAβN). Out of the 24 isolates resistant to ciprofloxacin 16 (66.7%) isolates showed ≥ 4 fold decreases in MIC values indicating the presence of efflux pump.
Molecular detection of Quinolone resistance in klebsiella pneumoniae:
Detection of Quinolone resistance due to mutation of chromosomal genes gyrA and parC:
gyrAand parC genes were successfully amplified in 27 out of 29 K. pneumoniae isolates. Amplified gyrA and parC genes were further sequenced for the detection of mutation associated with quinolones resistance. Detection of mutations was done by comparing the nucleotide and amino acid sequences to reference strain using Bioedit program. In gyrAthat base substitution G259A, C248A and C248T were the most commonly detected among our strains, which encoded for Asp87-Asn, Ser83-Tyr and Ser83-Phe respectively. Six isolates showed single mutation at Ser83-Ile or Ser83-Tyr, on the other hand 15 isolates showed 2 point mutations at Ser83 and Asp87.
In parC gene, the most common detected base substitution was G 239 T and resulted in amino acid mutation Ser80-Ile in 17 out of the 29 Klebsiella pneumoniae isolates. Other nucleotide mutations including C 240 T, C 504 T, G 250 A, C 345 T and C 369T.didn’t result in amino-acid substitution (silent mutations).
Detection of plasmid mediated quinolone resistance genes
Out of the 24 isolates examined for plasmid mediated Quinolone resistance, 21 (72.4) were positive for aac(6’)Ibcr, 14 (48.3%) were positive for qnrB , 13(44.8%) were positive for qnrS, 5(17.2%) were positive for qnrA, 1(3.5%) was positive for qnrC and qepA genes. 10 patterns of plasmid mediated quinolone resistance gene distribution were detected among K.pneumoniae isolates.The patterns qnrB, aac(6’)-Ib-cr, with and without qnrS were the most detected in 41.6% of isolates.