الفهرس 
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Abstract
Carbapenems are considered the most effective antimicrobial agents for the treatment of MDR Enterobacterales isolates. However, with the widespread use of carbapenems, carbapenem resistance has been increasingly reported among Enterobacterales and has become a serious threat to public health. The early detection of CPE is essential in the treatment of clinical infections.
The aim of this study was the phenotypic and genotypic detection of carbapenemase-producing Escherichia coli and Klebsiella spp. isolated from clinical samples and their susceptibility to the novel combination β-lactam/ β-lactamase inhibitor Ceftazidime/ Avibactam with or without the addition of Aztreonam.
This study was carried out on Klebsiella species and E. coli strains isolated from clinical specimens referred to the Microbiology department of the Medical Research Institute, Alexandria University from June 2021 to September 2022.
Bacterial isolation and identification were carried out by culturing on Blood and MacConkey agar. The isolated lactose fermenting colonies were then further identified as E. coli or Klebsiella by Gram staining and biochemical tests.
AST, including carbapenem group, was done to all isolated strains. Twenty E. coli carbapenem resistant strains and 80 Klebsiella carbapenem resistant strains were further subjected to identification and AST using the BioMerieux VITEK® 2 system.
Phenotypic detection of carbapenemase enzymes was carried out by MASTDISC Combi Carba plus. Five cartridges were used for the detection of carbapenemase enzymes and OXA-48 enzyme production in Enterobacterales (Cartridge A: Penem discs, Cartridge B: Penem + MβL inhibitor, Cartridge C: Penem + KPC inhibitor, Cartridge D: Penem + AmpC inhibitor, and Cartridge E: Temocillin + MβL inhibitor).
Bacterial DNA was extracted using Thermo Scientific Gene JET Genomic DNA Purification Kit method. Genotypic detection of carbapenemase genes was performed by using primers for KPC, GES, NDM, VIM, IMP, and OXA-48 genes. SYBR Green Real Time PCR was done using Mx3000P™ apparatus.
CAZ/AVI and ATM synergy was determined using the CAZ/AVI E-test and ATM disc diffusion method. The synergy was demonstrated as inverse D between CAZ/AVI E-test and ATM disc.
Among the 219 Klebsiella species isolates, 80 (36.53%) isolates were found resistant to the carbapenem group, while only 20 (5.13%) out of 390 E. coli isolates were resistant to the carbapenem group. Two E. coli isolates showed intermediate resistance to imipenem. There was a highly statistically significant difference between prevalence of carbapenem-resistance among Klebsiella spp. isolates and E. coli isolates (p-value <0.001).
Among the 80 carbapenem-resistant Klebsiella species identified by BioMerieux VITEK® 2 SYSTEM, 79 Klebsiella species (98.75%) were identified as Klebsiella pneumoniae ssp pneumoniae while 1 Klebsiella species (1.25%) was identified as Klebsiella pneumoniae ssp ozaenae.
The majority of Klebsiella species were isolated from blood cultures (58.8%) followed by wound swabs (16.3%) and urine (8.8%) while the majority of E. coli were isolated from aspirate (35%) followed by urine (30%).
According to the VITEC 2 system AST results, all the carbapenem-resistant Klebsiella isolates were resistant to the penicillin group (Ticarcillin and Piperacillin), β-lactam/ β-lactamase inhibitors (Ticarcillin/Clavulanic Acid and Piperacillin/Tazobactam), Carbapenems (Imipenem and Meropenem), and were highly resistant to cephalosporins group, Ceftazidime (98.75%) and Cefepime (100%), and Aztreonam (98.75%) while resistance to aminoglycosides, quinolones, sulfonamides ranged from (96.25%) for tobramycin to (71.25%) for gentamycin. Colistin had the lowest resistance rate (23.75%).
All the carbapenem-resistant E. coli isolates were resistant to the penicillin group (Ticarcillin and Piperacillin), β-lactam/ β-lactamase inhibitors (Ticarcillin/Clavulanic Acid and Piperacillin/Tazobactam), and Sulfonamides (Trimethoprim/Sulphamethoxazole),
(95%) of the isolates were resistant to ciprofloxacin. Only (75%) were resistant to cefepime and aztreonam, and only (15%) were resistant to colistin, while resistance to aminoglycosides ranged from (15%) for amikacin to (50%) for tobramycin.
Among the carbapenem-resistant Klebsiella isolates, the majority were XDR (76.25%), while (18.75%) were PDR, and only (5.00%) were MDR.
Among the carbapenem-resistant E. coli isolates, the majority were XDR (75.00%), while (20.00%) were MDR, and only (5%) showed PDR.
Among the carbapenem-resistant Klebsiella species isolates, NDM gene was detected in almost all tested isolates (91.25%), followed by OXA-48 gene (72.5%), GES gene (70%), VIM gene (68.75%) and (11.25%) KPC gene, while IMP gene was not detected among the tested isolates.
Among the carbapenem-resistant E. coli isolates, VIM gene was detected in all E. coli isolates (100%), followed by NDM gene (95%), OXA-48 gene (95%), GES gene (90%), and KPC gene (85%). All the tested genes were detected in the 2 E. coli isolates of intermediate resistance to imipenem. As in Klebsiella species isolates, IMP gene was not detected among the tested isolates.
More than one carbapenemase gene was detected among the carbapenem-resistant Klebsiella isolates, 3 – 5 genes were detected in (72.5%), followed by 2 genes in (26.25%). Only 1 Klebsiella isolate carried a single carbapenemase gene.
Ninety percent of E. coli isolates carried 4 – 5 genes, while the remaining (10%) isolates carried 2-3 genes.
The sensitivity of MAST Carba plus for the detection of carbapenemase enzymes among carbapenem resistant Klebsiella species isolates was 85.42%, and among carbapenem resistant E. coli isolates was 69.64%. There was a statistically significant association between MAST Carba plus and PCR results regarding detection of carbapenemase enzymes among carbapenem resistant Klebsiella species (p<0.001), and E. coli isolates (p=0.012).
Among the carbapenem-resistant Klebsiella species isolates, the sensitivity of MAST Carba plus in the detection of MBL enzymes was 93.51%, while among carbapenem-resistant E. coli isolates was 95%.
Among the carbapenem-resistant Klebsiella species isolates, the sensitivity of MAST Carba plus in the detection of OXA-48 enzyme was 79.31%, while among carbapenem-resistant E. coli isolates was 47.37%.
Among the carbapenem-resistant Klebsiella species isolates, the sensitivity of MAST Carba plus to detect KPC enzyme was 55.56%, while among carbapenem-resistant E. coli isolates was 64.71%.
Synergy was observed between CAZ/AVI E- test and ATM disc in (98.75%) of the carbapenem-resistant Klebsiella species isolates.
Among the carbapenem-resistant E. coli isolates, Synergy was also observed between CAZ/AVI E-test and ATM disc in (95%) of isolates.
All the 16 pan drug-resistant bacterial isolates showed synergy between CAZ/AVI E-test and ATM disc.