الفهرس 
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Abstract
Carbapenem resistance in Pseudomonas aeruginosa and other Gram negative bacteria becomes a worldwide problem.
Among 22 CRPA isolates enrolled in PCR, 12 (54.5%) isolates were harboring carbapenemase genes. This prevalence is high in comparison with other researches.
In contrast to other studies, which detected only VIM as P. aeruginosa mettallo- ß-lactamase variants in Kuwait, in the current study IMP and VIM genes were detected in CRPA from Kuwait hospitals as mettalo-ß-lactamase genes, in addition to KPC gene. But in the current study NDM variants were not detected in Kuwait hospitals. However, SME, NDM-1(using two pairs of primers) and OXA genes were not detected in any of the tested CRPA isolates in the current study. In another Middle East country, VIM-2 and NDM-1 genes were reported in Egypt among isolates of P. aeruginosa.
It was noticed that KPC was the most frequent detected gene in this study and it may be found with VIM gene only in the presence of IMP gene, while IMP could not be detected alone. KPC gene is carried on plasmid only, making these elements highly transferable and explaining their rapid dissemination in hospital environment. The carbapenem resistance was associated with resistance to all the tested antibiotics except colistin. Resistance to carbapenem antibiotics in this study was associated with resistance to other antibiotics including penicillins, flouroquinolones, third and fourth generations of cephalsporins.
PCR detection of carbapenemase genes, antibiotic sensitivity and MIC determination of selected strains before and after induction of mutation showed that loss (or mutation) of the three genes VIM, KPC and IMP result in loss of carbapenem resistance while loss of VIM gene alone was not associated with loss of carbapenem resistance (although MIC was decreased). This result proves that presence of VIM alone was not the mechanism for carbapenem resistance in this strain, although it may participate. On the other hand, presence of KPC and IMP together was enough for carbapenem resistance in the tested strains.
Molecular methods including multiplex PCR and real time PCR were developed by many authors, however, in the present study specific multiplex PCR to detect a set of carbapenemase encoding genes in one reaction was also tested. This was restricted by the PCR product sizes and accordingly it was successful with the selected carbapenemase encoding genes IMP {PCR product: 488 bp}, VIM {PCR product: 280 bp}, SME {PCR product: 820 bp}. This can facilitate and speed the detection of carbapenemase encoding genes.
CRPA strains that were induced for mutation by exposure to UV radiation were also treated with SDS and heat to enhance plasmid curing and the resultant strains were tested for antibiotic sensitivity and carbapenemase encoding genes by nanosphere-verigene technique. The sensitivity of the microarray based technique nanosphere-verigene was tested against PCR results in all the 12 strains harboring carbapenemase encoding genes. Only three isolates were positive for VIM gene among the known 5 VIM harboring strains by PCR test. On the other hand, no IMP or KPC genes were detected by nanosphere-verigene among known 11 KPC and 4 IMP harboring strains. Accordingly the results of SDS and heat treatment for CRPA did not add additional information more than that provided by UV induced mutation.
A lot of significant sequence alignments confirmed PCR results and supported that carbapenemase encoding genes are distributed among species of Gram negative bacteria. Sequencing of such genes provide a powerful evidence for its transfer as recently reported draft genome sequences of four blaKPC-containing bacteria identified as Klebsiella aerogenes, Citrobacter freundii, and Citrobacter koseri. Additionally, they reported the draft genome sequence of a K. aerogenes strain that did not contain a blaKPC gene but was isolated from the patient who had the blaKPC-2containing K. aerogenes strain.
On the other hand, KPC gene nucleotide sequence alignment between PCR product (Reverse primer) of PCR strain 16 (Parent strain 52) and its UV mutant 42 shows 99% identity with 4 nucleotide difference. For forward primer, it shows 98% identity and 9 nucleotide difference. While, IMP gene nucleotide sequence alignment between PCR product of PCR strain 16 (Parent strain 52) and its UV mutant 42 shows 99% (3 nucleotide difference) and 98% (6 nucleotide difference) for reverse and forward, respectively. It was not confirmed that these differences are due to exposure to UV radiation. In general, the capacity of prokaryotes to withstand significant UV radiation requires a wide array of physiological responses, including transcriptional regulation and cellular repair of irradiation-induced damage. So, in mutation induction by UV exposure VIM gene was lost, while IMP and KPC genes were preserved with repaired damage or little modification of their nucleotide sequence in the PCR amplified segments and the carbapenem resistance was preserved.
In conclusion, Multidrug-resistant Gram-negative bacteria such as Acinetobacter and Pseudomonas species are the main organisms spreading into the community. Despite efforts to control carbapenem resistance, a definite solution to the problem is still far from achievement.