الفهرس 
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Abstract
Dissemination of carbapenem resistance among Gram-negative pathogens poses a considerable threat to public health. Acquired resistance to carbapenems in pathogens such as K. pneumoniae and P. aeruginosa is a growing problem worldwide. This resistance is mediated by carbapenemases such as plasmid-mediated Ambler class A β-lactamases as K. pneumoniae carbapenemases (KPCs) as well as Ambler class B metallo-β-lactamases (MβLs) including IMP, VIM and NDM types. Laboratory identification of carbapenemase-harboring clinical isolates would be necessary to implement contact precautions and for outbreak detection. The aim of this work was to determine the incidence of carbapenem resistance among clinical isolates of K. pneumoniae and P. aeruginosa from hospitalized patients at Menoufia University hospitals (MUHs) and to detect the production of K. pneumoniae carbapenemases and metallo- β-lactamases among K. pneumoniae and P. aeruginosa clinical isolates by phenotypic and molecular methods with evaluation of different methods used for detection. This study started with 128 K. pneumoniae and 77 P. aeruginosa isolates collected from clinical specimens of 295 patients (one specimen from each patient) admitted to different departments at MUHs. The selected isolates were tested against imipenem, meropenem and ertapenem by disk diffusion method and imipenem MIC assay (agar dilution method) as screening methods for suspected class A and class B carbapenemase production. This was followed by performing the phenotypic confirmatory tests for carbapenemase detection. The modified Hodge test (MHT) was evaluated as a phenotypic confirmatory test to detect both class A and class B carbapenemases production. Novel boronic acid-based method (a combined-disk synergy test) using imipenem in combination with phenylboronic acid as an inhibitor, was used as a confirmatory tool for class A (KPC) detection. A combineddisk test using imipenem in combination with EDTA was also applied to confirm class B (MβLs) production. By using this methodology, isolates producing class A and class B carbapenemases were successfully distinguished from those producing other classes of β-lactamases. Carbapenemase gene detection by molecular methods is the gold standard. Therefore, a multiplex PCR assay was performed (as the molecular confirmatory test) using specific primers for blaKPC, blaVIM and blaIMP genes. Amplified products were detected by agarose gel electrophoresis. Results of multiplex PCR were compared with those of MHT and combined disk synergy tests to evaluate the sensitivity and specificity of each. The results obtained in this study revealed that, regarding the screening tests, the disk diffusion method revealed that, 80/128 (62.5%) of K. pneumoniae isolates were imipenem-resistant compared to 71/128 (55.6%) imipenem-resistant isolates detected by agar dilution method. Statistical analysis revealed almost perfect agreement between the two methods (Kappa test = 0.82). Compared to PCR results, all PCRpositive K. pneumoniae isolates (n=40) (harboring class A and/or class B carbapenemase genes) were imipenem-resistant by disk diffusion test, while 95 % (38/40) of PCR- positive isolates were imipenem- resistant by MIC method and 5 % (2/40) of PCR- positive isolates were reported as imipenem- susceptible by MIC method. As regards P. aeruginosathe disk diffusion screening test revealed that, 40/77 (51.9%) of P. aeruginosa isolates were imipenem-resistant compared to 34/77 (44.2%) imipenem-resistant isolates detected by agar dilution method. Statistical analysis revealed almost perfect agreement between the two methods (Kappa test = 0.85). In relation to PCR results, all PCR- positive isolates (n=26) were imipenem-resistant by both disk diffusion and MIC methods. Regarding distribution of imipenem-resistant isolates among different hospital departments, the highest percentage of imipenemresistant K. pneumoniae isolates (32.5% ; 26/80), was found among ICUs isolates. This was followed by chest department (21.3% ; 17/80). Regarding P. aeruginosa, the highest percentage of imipenem resistant isolates was also found among ICUs isolates (37.5% ; 15/40). In performing the phenotypic confirmatory tests for class A and class B carbapenemase detection, the MHT revealed that 58/80 (72.5%) of imipenem-resistant K. pneumoniae isolates were MHT-positive compared to only 28/80 (35%) isolates detected by combined imipenem/boronic acid synergy test and 39/80 isolates (48.8 %) detected by combined imipenem/EDTA synergy. Statistical analysis revealed a statistically significant difference (P<0.05) between MHT and imipenem/boronic acid synergy test as confirmatory tests for class A carbapenemase detection. There was also a statistically significant difference (P<0.05) between MHT and imipenem/EDTA synergy test as confirmatory tests for class B carbapenemase detection. For P. aeruginosa, the MHT detected only 8/40 (20%) of imipenem-resistant isolates compared to 16/40 (40%) isolates detected by combined imipenem/boronic acid synergy test and 25/40 isolates (62.5 %) detected by combined imipenem/EDTA synergy. Statistical analysis revealed a statistically significant difference (P<0.05) between MHT and imipenem/boronic acid synergy test as confirmatory tests for class A carbapenemase detection. There was also a statistically significant difference (P<0.05) between MHT and imipenem/EDTA synergy test as confirmatory tests for class B carbapenemase detection. Molecular assessment by multiplex PCR revealed that, 8/80 (10%) of imipenem- resistant K. pneumoniae isolates were positive for blaKPC, 27.5 % (22/80) were positive for blaVIM and 1.25 % (1/80) were positive for blaIMP genes. Seven percent and half of isolates (6/80) had both blaKPC and blaVIM genes and 3.75 % (3/80) of isolates had both blaIMP and blaVIM genes. Total class A- positive isolates (14/80) comprised 17.5 % and total class B- positive isolates (35/80) comprised 43.75 % of imipenem resistance in K. pneumoniae. As regards P. aeruginosa, molecular assessment by multiplex PCR revealed that, 5/40 (12.5%) of imipenem- resistant isolates were positive for blaKPC, 25% (10/40) were positive for blaVIM and 10% (4/40) were positive for blaIMP genes. Ten percent of isolates (4/40) co- expressed blaKPC and blaVIM, 5 % (2 /40) co-expressed blaIMP and blaVIM and 2.5 % of isolates (1 /40) co- expressed blaKPC and blaIMP genes. Total class A- positive isolates (10/40) comprised 25% and total class Bpositive isolates (23/40) comprised 57.5 % of imipenem resistance in P. aeruginosa. In comparing the phenotypic and molecular confirmatory tests for detection of class A and class B carbapenemases, our results revealed that- considering PCR as the gold standard- the MHT detected 31 isolates (77.5%) out of total 40 PCR- positive K. pneumoniae isolates with a sensitivity of 77.5 %, specificity 32.5 % and diagnostic accuracy 55 %. Regarding P. aeruginosa, the MHT detected 6 isolates (23.1%) out of total 26 PCR- positive isolates with a sensitivity of 23.1 %, specificity 85.7 % and diagnostic accuracy 45%. The MHT exhibited high falsepositive rate (67.5%) with K. pneumoniae and high false-negative rate (76.9%) with P. aeruginosa isolates. In relation to PCR results, the imipenem/boronic acid synergy test detected 12/14 isolates (85.7 %) of blaKPC- positive K. pneumoniae isolates, thus the sensitivity of imipenem/boronic acid synergy test was 85.7 %, specificity 75.8 % and diagnostic accuracy 77.5 %, considering PCR as the gold standard. For P. aeruginosa, imipenem/boronic acid synergy test detected 8/10 isolates (80 %) of blaKPC- positive isolates, thus the sensitivity was 80 %, specificity 73.3 % and diagnostic accuracy 75.0 %, compared to PCR results. Regarding imipenem/EDTA synergy test, it detected 33/35 (94.3%) isolates of blaVIM- and blaIMP- positive K. pneumoniae isolates, thus the sensitivity of imipenem/EDTA synergy test was 94.3 %, specificity was 86.7 % and diagnostic accuracy was 91.5% (considering PCR as the gold standard). For P. aeruginosa, imipenem/EDTA synergy test detected 22/23 (95.7 %) isolates of blaVIM- and blaIMP- positive isolates, thus the sensitivity was 95.7 %, specificity was 82.4 % and diagnostic accuracy was 93.0 %. Referral to PCR, exposure to invasive procedures among imipenemresistant K. pneumoniae and P. aeruginosa isolates was statistically significant (P < 0.05). For K. pneumoniae isolates, 95 % of PCR- positive infected cases were subjected to invasive procedures such as cannula insertion, mechanical ventilation, urinary catheterization and CVL. For P. aeruginosa isolates, all PCR- positive infected cases were also subjected to invasive procedures. Previous administration of β-lactams especially carbapenems was a significant risk factor for carbapenemase production (P < 0.05) in cases infected with either imipenem- resistant K. pneumoniae or P. aeruginosa. For K. pneumoniae, 97.5 % of cases infected with PCR- positive isolates had history of β-lactam intake of which 82.5 % involved carbapenems and for P. aeruginosa, 84.6% of cases infected with PCR- positive isolates had history of β-lactam intake of which 73.1% involved carbapenems.