الفهرس 
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Abstract
Pseudomonas aeruginosa is a ubiquitous organism present in many diverse environmental settings and it can be isolated from various living sources, including humans. The ability of P. aeruginosa to survive on minimal nutritional requirements and to tolerate a variety of physical conditions has allowed this organism to persist in both community and hospital settings.
P. aeruginosa presents a serious therapeutic challenge for treatment of both community and hospital acquired infections. Also, selection of the appropriate antibiotic to initiate therapy is essential for optimizing the clinical outcome.
The beta lactam antibiotic family includes penicillins and penicillin derivatives, cephalosporins, carbapenems, monobactams and beta lactamase inhibitors. Bacterial resistance against beta lactam antibiotics is increasing at a significant rate and has become a common problem. There are several mechanisms of antimicrobial resistance to beta lactam antibiotics. The most common and important mechanism by which bacteria can become resistant to beta lactams is to express beta lactamases.
The most widely used classification of beta lactamases is the Ambler classification, whichdivides beta lactamases into four classes (A, B, C and D) based upon their amino acid sequences; where, class A, the active-site serine beta lactamases; and class B, the metallo-beta-lactamases. Later a new class of serine beta lactamases was designated class C, its members are also known as the ‘AmpC’ beta lactamases. Another class of serine beta lactamases, familiarly known as the OXA beta lactamases, was designated class D.
Carbapenem resistance has been observed frequently in P. aeruginosa. Resistance to carbapenem is in part due to carbapenem hydrolyzing enzymes, known as carbapenemases.
The aim of this study was to determine the phenotypic resistance pattern to beta lactam antibiotics among P. aeruginosa clinical isolates and to identify the genetic determinants responsible for beta lactam antibiotic resistance.
A total of 33 P. aeruginosa isolates obtained from clinical samples submitted to the Microbiology Department of Medical Research Institute, Alexandria University, were included in this study.
The isolates were identified as P. aeruginosa using biochemical tests. Strains fulfilling P. aeruginosa profile were further identified, by Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometer (MS), which was carried out on Ultraflex TOF/TOF (Bruker Daltonics).
Identification score criteria were used as recommended by the manufacturer A score of 2.000 indicated species-level identification, a score of 1.700 to 1.999 indicates identification to the genus level, a score of less than 1.700 was interpreted as no identification
Susceptibility of P. aeruginosa isolates was determined by disk diffusion method.
Phenotypic detection of ESBL was performed by the disk combination test,using one disk of ceftazidime or cefotaxime alone and in combination with clavulanic acid.
Phenotypic detection of carbapenemases was carried out by Modified Hodge test.The cloverleaf-shaped indentation of growth of the test strain versus the susceptible indicator strain Klebsiella pneumoniae was interpreted as a positive result for carbapenemases production by the tested strain.
Phenotypic detection of metallo-beta-lactamase (MBL) was performed by the disk combination testusing one disk of imipenem alone and one with imipenem-EDTA.
Phenotypic detection of AmpC was performed by disk potentiation test using onedisk of cefotaxime or ceftazidime alone and one in combination with3-aminophenyl boronic acid (APB).
Genotypic detection of beta lactamase genes was done by conventional Polymerase Chain Reaction (PCR).
Bacterial DNA was extracted using boiling method.PCR amplification of the extracted DNA was carried out on Veriti Thermal Cycler (Applied Biosystems, California,USA). The PCR master mixMyTaq HS Red Mix was supplied by (BioLine, London, UK).The primers were purchased from (Biosearch Technologies, California,USA)
Class A beta lactamase (ESBL) genes:blaTEM, blaSHV, blaPER,blaCTX-M, blaVEB, blaGES, blaPSE-1, metallo-beta-lactamases (MBL) genes:blaVIM, blaIMP, blaNDM-1,blaSPM-1, blaGIM-1 and class D beta lactamase genes blaOXA-1, blaOXA-2, blaOXA-10.
Thirty-five lactose non-fermenter, oxidase positive isolates obtained from the Microbiology Department of Medical Research institute, Alexandria University, were examined for their colonial morphology, presence of characteristic pigments and ability to grow at 42°Cand were accordingly diagnosed as P. aeruginosa.
The 35 isolates presumably identified as P. aeruginosa were tested by MALDI TOF mass spectrometry, 33 strains were diagnosed as P. aeruginosa, while the other two strains were found to be Stenotrophomonas maltophilia and Pseudomonas resinovorans. Twenty-five (75.8%) of the 33 P. aeruginosa had a score value higher than 2, allowing genus and species identification. Eight (24.2%) strains had a lower score ranging from (1.7 to 1.99). They were diagnosed as P. aeruginosa as they showed protein pattern matching only with this organism.
Twelve (36.3%) of the 33 P. aeruginosa isolates were isolated from wound swab samples followed by 11 (33.3%) of the isolates obtained from urine samples and 7 (21.3%) of the isolates were obtained from respiratory tract infections.
The majority of the P. aeruginosa strains (93.94 %) were resistant to both carbapenems and to the third and fourth generation cephalosporin (ceftazidime and cefepime) (90.91%), on the other hand a much lower resistance was observed to aztreonam, piperacillin and piperacillin/ tazobactam combination (21.21%, 42.42%, 42.42% respectively).
Phenotypically, the prevalence of ESBLs producing isolates was 11 (36.7%) out of the 30 P. aeruginosa strains resistant to the third and fourth generation cephalosporins.
The combined disk tests with CAZ-CAZ/CLA and CTX-CTX/CLA were positive for 10 and 5 isolates respectively.
Detection of ESBL genes were carried for the 30 P. aeruginosa isolates resistant to the third and/or fourth generations cephalosporins. Twenty-four (80%) out of the 30 Pseudomonas isolates were positive for ESBL genes. blaGES was detected in 15 (50%) of the isolates, followed by blaVEB in 10 (33%) and blaTEM in 9 (30%). Lower detection rate was observed for blaPER,which was detected in 3 (10%), blaPSE-1in 2 (6.6%) and blaSHVin 1 (3.3%). Seven isolates had 3 ESBL genes and six had 2 genes. blaGES was detected alone in 8 isolates, while no ESBL genes were detected in six isolates.
Among our 32 carbapenem resistant P. aeruginosa isolates 23 (71.9%) were phenotypically MBL positive. Four (17.4%) of the 23 phenotypically MBL positive isolates did not harbor any of the tested MBL genes. On the other hand, genotypic screening of the 32 P. aeruginosa isolates showed that 27 (84.4%) of them harbored MBL genes.
Only 9 (28.1%), out of the 32 carbapenem resistant strains, were positive for Modified Hodge test, while 23 (71.9%) were positive byimipenem–EDTA combined disk method (MBL test).
Only blaVIM gene was detected among our 32 carbapenem resistant P. aeruginosa isolates, 27 (84.4%) out of our strains harbored this gene. blaIMP, blaSPM-1, blaGIM-1 and blaNDM-1 genes were not detected among our strains.
None of our 30 strains was AmpC producer since the addition of 3-aminophenyl boronic acid(APB) to either of the two antibiotic disks did not result in the enlargement of the growth inhibitory zone diameter around the disk by ≥ 5 mm.
Induction of AmpC using disk approximation (D−test) assay was not possible since all of our 30 P. aeruginosa isolates were resistant to both cefoxitin and cefotaxime.
blaOXA-10 gene was detected among 16 (48.5%) of the 33 P. aeruginosa strains.
Our study confirmed the multifactorial resistance of P. aeruginosato beta lactam antibiotics.