الفهرس 
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Abstract
A. baumannii is a Gram-negative human pathogen. A major clinical impact of A. baumannii is hospital-acquired infections including VAP, skin and soft-tissue infections, wound infections, urinary-tract infections, secondary meningitis, and blood stream infections. The incidence of severe infection caused by MDR and pandrug resistance A. baumannii has been increasing worldwide. Crude mortality rates of 30 – 75% have been reported for nosocomial pneumonia caused by A. baumannii.
Nowadays, bacterial resistance against β-lactam antibiotics is increasing at a significant rate and has become a common problem. There are several mechanisms of antimicrobial resistance to β- lactam antibiotics. The most common and important mechanism through which bacteria can become resistant against β-lactams is by expressing β-lactamases.
The aim of the present study was to determine the phenotypic resistance pattern to β-lactam antibiotics among Acinetobacter clinical isolates and to identify the genetic determinants responsible for β-lactam antibiotics resistance.
The study was carried out in the department of Microbiology from July 2014 to September 2015. After the approval of the Ethical Committee of the Medical Research Institute (MRI), 52 Acinetobacter isolates were collected from patients confined to ICU in different Alexandria hospitals.
Bacterial identification and antimicrobial susceptibility testing were carried out by the automated colorimetric system (VITEK 2 system, BioMerieux) using the GN identification card and AST-GN71 card respectively.
o Phenotypic detection of ESBL was performed by the combined disk test.
o Phenotypic detection of carbapenemases was carried out by MHT.
o Phenotypic detection of MBL was done by the combined disk test.
o Phenotypic detection of AmpC by disk potentiation test.
Genotypic detection of β- lactamase genes was done by conventional PCR for the following:
o Class A β-lactamase (ESBL) genes, blaTEM, blaSHV, blaCTX-M, blaPER and blaVEB
o Class B MBL blaVIM, blaIMP and blaNDM
o Class C AmpC genes blaADC, ISAba1
o Class D β-lactamase genes blaOXA-23, blaOXA-24, blaOXA-51 and blaOXA-58, ISAba1
Fifty two Gram negative non lactose fermenting, oxidase negative isolates collected from patients confined to ICU in different Alexandria hospitals were identified using biochemical VITEK 2 system as A. baumannii complex. VITEK 2 did not permit reliable identification of Acinetobacter isolates to species level. Members of the A. baumannii group, i.e. A. baumannii, A. nosocomialis and A. pittii are identified a A. baumannii complex“, but correct species identification is not possible.
The blaOXA-51 -like gene has been proposed as a marker for A. baumannii and was found in the 38 (100%) Acinetobacter isolates tested for this gene, confirming the value of this gene as a genetic marker for the diagnosis of A. baumannii.
Twenty six (49.9%) of the Acinetobacter strains were isolated from the respiratory tract including endotracheal tube (11.5%) and bronchoalveolar lavage (9.6%) followed by wounds (30.7%). Strains isolated from urine constitute only (9.6%) of cases.
Fifty one (98.1%) out of the 52 Acinetobacter isolates tested by VITEK 2 system were MDR. Resistance to aminoglycoside group ranged from 67.2% tobramycin to 80.6% gentamicin, while resistance to ciprofloxacin was 98.1%. Only 55.7% of Acinetobacter isolates were resistant to trimethoprim/sulfamethoxazole. Acinetobacter isolates showed an exquisite sensitivity to tigecycline.
On the other hand, resistance of Acinetobacter isolates ranged from 100% to ampicillin and cefazolin, 98.1% to ceftriaxone and azteronam and 96.2% to cefepime. Resistance to carbapenem group ranged from 94.2% to meropenem to 88.4% imipenem.
The 49 isolates which were resistant to carbapenem were tested by MHT and inoculated on chromID®CARBA agar. 48 (98%) out of the 49 carbapenem resistant isolates were able to grow on the selective agar, but only 39 (79.6%) were positive for MHT.
MBLs were detected in 43 (87.8%) out of the 49 carbapenem resistant isolates as tested by the imipenm-EDTA combined disk method.
blaVIM was detected in an extremely high percentage 42 (97.7%) out of phenotypically MBL producers. On the other hand, blaIMP was not detected among our isolates.
blaNDM was the second MBL produced among our isolates with percentage of 37.2% out of 43 phenotypically MBL producers.
The 38 A. baumanii isolates that grew on chrom ID CARBA and were positive for MHT were positive for blaOXA-51 (100%) while 37(97.4%) out of them were positive for blaOXA-23. Only one isolate was positive for blaOXA-24, while blaOXA-58 was not detected among our isolates
ISAba1 was detected upstream the blaOXA-23 gene in 35(94.6%) of the 37 isolates which were blaOXA-23 gene positive, while it was not found upstream the blaOXA-51.
All the 51 ceftriaxone resistant Acinetobacter isolates were able to grow on chromID®ESBL agar but only 64.7% were positive for combined disk test.
blaTEM was detected in 33 (100%) among the 33 phenotypically ESBL producings
blaCTX-M was detected in 14 (42.4%) and blaPER in 11(33.3%) among them.
blaVEB and blaSHV genes were not detected among our isolates.
AmpC was detected phenotypically in 16 (31.4%) out of our 51 ceftriaxone resistant Acinetobacter isolates.
blaADC and ISAba1 were detected in all phenotypically AmpC positive isolates, while ISAba1 was detected upstream the blaADC in 87.5% of the isolates.
65.4% out of the 52 Acinetobacter isolates had a number of resistance genes ranging from 3-5 while only 19.2% carried a higher number of genes ranging from 6-8 and 15.4% carried only 1-2 genes.
36.6% out of the 52 Acinetobacter isolates had ESBL, MBL and OXA β-lactamase classes followed by MBL-OXA β-lactamase classes in 15.4% and ESBL-AmpC-MBL-OXA in 11.5% of the isolates.
In Conclusion
1. Fifty two Gram-negative non lactose fermenting oxidase negative isolates collected from ICU in different Alexandria hospitals were identified using biochemical VITEK 2 system as A. baumannii complex. VITEK 2 does not permit reliable identification of Acinetobacter isolates to species level. Members of the A. baumannii group, i.e. A. baumannii, A. nosocomialis and A. pittii are identified as “A. baumannii complex“,
2. The blaOXA-51 -like gene has been proposed as a marker for A. baumannii and was found in the 38 (100%) Acinetobacter isolates tested for this gene confirming the value of this gene as a genetic marker for the diagnosis of A. baumannii.
3. 26 (49.9%) of the Acinetobacter strains were isolated from the respiratory tract.
4. 51 (98.1%) of the 52 Acinetobacter isolates tested by VITEK 2 system were MDR.
5. 88.4% - 94.2% of the 52 Acinetobacter were resistant to carbapenem group.
6. 48 (98%) out of the 49 carbapenem resistant isolates were able to grow on chromID®CARBA agar, but only 39 (79.6%) were positive for MHT.
7. MBLs were detected in 43 (87.8%) out of the 49 carbapenem resistant isolates as tested by the imipenm-EDTA combined disk method.
o blaVIM was isolated in 42 (97.7%) out of phenotypically MBL producers.
o blaIMP was not detected among our isolates.
o blaNDM was detected in 16 (37.2%) out of 43 phenotypically MBL producer.
8. blaOXA-51 was detected in 100% of the 38 A baumanii isolates that grew on chrom ID CARBA.
o blaOXA-23 was detected in 37 (97.4% )
o blaOXA-58 was not detected among them
o ISAba1 was detected upstream the blaOXA-23 gene in 35(94.6%) of the 37isolates which were blaOXA-23 gene positive, while it was not found upstream the positive blaOXA-51.
9. All the 51 ceftriaxone resistant Acinetobacter isolates were able to grow on chromID®ESBL agar but only 64.7% were positive for combined disk test.
o blaTEM was detected in 33 (100%) among the 33 phenotypically ESBL producers
o blaCTX-M was detected in 14 (42.4%) and blaPER in 11(33.3%) among them.
o blaVEB and blaSHV genes were not detected among them.
10. AmpC was detected phenotypically in 16 (31.4%) out of the 51 ceftriaxone resistant Acinetobacter isolates.
o blaADC and ISAba1 were detected in all phenotypically Amp C positive isolates,
o ISAba1 was detected upstream the blaADC in 87.5% of the isolates.
11. 65.4% out of the 52 Acinetobacter isolates had a number of resistance genes ranging from 3-5 while only 19.2% carried a higher number of genes ranging from 6-8 and 15.4% carried only 1-2 genes, indicating that resistance of Acinetobacter isolates is multifactorial.