الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Enterobacteriaceae are diverse and large group of gram negative basilli bacteria whose humans and animals intestinal tract is the normal habitat. The family contain numerous genera (Shigella, E.coli, Salmonella, Enterobacter, Klebsiella, Proteus, Serratia,and others). Some enteric bacteria, such as E.coli, are causing disease incidentally and are one of the microbiota or normal flora, but others, are regularly humans pathogenic. Significant cause of infections in the urinary tract (UTIs), various intra-abdominal, bloodstream, healthcare- and hospital- associated pneumonias are Enterobacteriaceae family.
Resistance to 3rd-generation cephalosporins among Enterobacteriaceae is typically brought about by production of ESBLs that hydrolyze extended- and wide- spectrum cephalosporins, carbapenem (carbapenemases), and monobactams.
Β-lactamases are enzymes that open the β-lactam ring of β-lactam antibiotics irreversibly. Based on the sequence of amino acid the lactamases enzymes are divided to 4 Classes: ClassA, ClassB, ClassC, and ClassD β-lactamases utilize serine active site for β-lactam ring opening, whereas the Class B enzymes are metallo-β-lactamases.
Carbapenemases enzymes are one of ESBLs group that are capable of hydrolyzing carbapenem antibiotics, in addition to cephalosporins and other β-lactam antimicrobials.
Three major classes of carbapenemases are widespread globally in clinical Carbapenem resistance Enterobacteriaceae (CRE) isolates, including class A mainly Klebsiella pneumoniae carbapenemase (KPC), class B, New Delhi metallo-β-lactamase (NDM),Verona imipenemase (VIM) and Imipenemase (IMP) and class D oxacillinase (OXA-48 and OXA-48 variants, OXA-162 and OXA-181, etc.)
There are many various genes encoding for carbapenemases, like: class A carbapenemases encoded by KPC-1, KPC-2, and also KPC-3, class B carbapenemases encoded by the VIM,NDM and IMP and the OXA genes encoding class D carbapenemases.
The aim of study was to detect the prevalence of resistance to carbapenem among Enterobacteriaceae isolated from Egyptian patients by phenotypic methods and genotypic methods.
One hundred fifty Enterobacteriaceae isolates collected at the microbiology department Medical Research Institute Hospital from different sites, where urine samples (50.7%), sputum (28.0%), wound swab (14.0%), pus (4.0%), blood (2.0%) and wound aspirate (1.3%), the study included 69 (46.0 %) samples from males and 81 (54.0 %) samples from females during the period from July 2019 to March 2020.
Kirby-Bauer method was used to test antimicrobial susceptibility of the 150 Enterobacteriaceae isolates and further tested for detection of carbapenemase enzyme.
Resistance to at least one of the three carbapenems tested (imipenem, meropenem, ertapenem) was found to be that the strains slightly higher resistant to (Etrapenem and Imipenem= 35.3%, compared to Meropenem=34.0%) out of 150 Enterobacteriaceae isolates, 53(35.3%) were carbapenems resistant isolates
Among 53 carbapenem resistant Enterobacteriaceae, resistance to β-lactam antibiotics and β-lactamase inhibitor combinations was high ranging (from 92.5% to 100.0 %).
The resistance to quinolones (Ciprofloxacin, Levofloxacin and Norfloxacin) ranged (from 92.5% to 96.2%). the resistance to aminoglycosides (Gentamycin, Tobramycin and Amikacin) was (62.3%, 79.2% and 64.2% respectively).
Resistance to (Trimethoprim-sulfamethoxazole, Nitrofurantoin, chloramphenicol and Fosfomycin) ranged (from 62.3% to 88.7%).
The 53 Carbapenem resistant Enterobacteriaceae were isotated from samples of urine (47.2%), sputum (30.2%), wound swab and pus (9.4%), blood (3.8%). On the other hand, The 97 Carbapenem Susceptible Enterobacteriaceae were from samples of urine (52.6%), sputum (26.8%), wound swab (16.5%), blood and pus, (1.0%), Wound aspirate (2.1%). Also, 45.3% of out of the 53 Carbapenem resistant Enterobacteriaceae were from male patients and 54.7% were from female patients. No statistically significant was found between CRE isolates and carbapenem susceptible isolates regarding the type of specimen they were isolated from, nor the gender of patients who provided the clinical specimens.
Following the CLSI guidelines for performing phenotypic carbapenemase enzyme detection by the mCIM with eCIM combination method, it was found that among our 53(35.3%) CRE isolates, metallo-β-lactemase was detected in 28(52.8%) while serine carbapenemase was in 16(30.2%) of the isolates. On the other hand, 5(9.4%) of isolates gave inconclusive result, while 4(7.5%) of the isolates were carbapenemase negative by this test. All results were further confirmed by carbapenemase genes detection according to CLSI 2020.
Out of 53 carbapenem resistant Enterobacteriaceae by disk diffusion method, 37 (69.8%) were Klebsiella pneumoniae, followed by 15 (28.3%) E coli, while only one isolate (1.9%) Klebsiella oxytoxa.
The carbapenem resistant Enterobacteriacea distributed to 28 metallo-β-lactamase producing Enterobacteriaceae included 19/28 (67.9%) Klebsiella pneumonia and 9/28 (32.1%) E coli. On the other hand 16 serine carbapenemase where 15/16 (93.8%) were Klebsiella pneumoniae, while 1/16 (6.3%) was Klebsiella oxytoxa.All the five E coli tested inconclusive by eCIM and mCIM. Four isolates out of the 53CRE by disk diffusion tested carbapenemase negative by eCIM and mCIM 3/4 (75.0%) were Klebsiella pneumoniae, 1/4 (25.0%) were E coli. was highly significant difference between Carbapenemase production (phenotypic test) and Enterobacteriaceae in CRE among 53 carbapenem resistant Enterobacteriaceae isolates. (P<0.05)
Out of 53 carbapenem resistant Enterobacteriaceae isolates, 23 (43.4%) carried OXA-48 gene, followed by 18 (34.0%) carried both NDM and OXA-48 genes, 12 (22.6%) carried NDM gene. On the other hand VIM, IMP and KPC genes were not detected in any of the isolates.
The OXA-48 gene was detected in a total of 23/53 CRE isolate; 15 (65.2%) K. pneumoniae, 7 (30.4%) E. coli and 1(4.4) K. oxytoxa, while NDM gene was detect in a total of 12/53 CRE isolates 8 (66.7%) K. pneumoniae and 4 (33.3%) E. coli . While out of 18 from total 53 CRE isolates 14 (77.8%) K. pneumonia and 4 (22.2%) E. coli co-existence the NDM gene with the OXA-48 gene to together. All 53 isolates were negative for genes of the blaKPC, blaVIM, and blaIMP types.
Out of 16 isolates displaying serine carbapenemase phenotypically, 14 (87.5%) were carrying OXA-48 gene and 2 (12.5%) were carrying the metallo-β-lactamase encoding gene NDM. This may be explained by the presence of other genes that encode for serine carbapenemase in these 2 isolates which were preferentially expressed over the NDM gene. In this case the presence of NDMgene without being expressed has resulted in false negative phenotypic result for metallo-β-lactamase enzyme by mCIM and eCIM test. On the other hand out of 28 metallo-β-lactamase isolates, only 8 (28.6%) were carrying NDM gene alone while 2 (7.14%) were carrying only the serine carbapenemase encoding OXA-48gene.This may be explained by the presence of other genes that encode for metallo-β-lactamase in these 2 isolates which were preferentially expressed over the OXA-48gene . The remaining 18 (64.3%) isolates with the metallobetalactamase phenotype were carrying both NDM&OXA-48 genes together. This indicates that the isolates carrying both genes preferentially expressed the metallo-batalactamase phenotype by mCIM and eCIM tests. This finding is contrary to the CLSI 2020 guidelines statement, which states that in case of co-production of a serine carbapenemase and a metallo-β-lactamase by the same oraganism, differentiation between enzymes will not be possible and the eCIM test may give false negative results.
Out of 4 isolates displaying negative phenotypic test results (false negative); one (25.0%) was carrying NDM gene, and 3 (75.0%) were carrying OXA-48 gene. As for the 5 isolates displaying inconclusive results, genotypic testing revealed that 1 (20.0%) was carrying NDM gene, and 4 (80.0%) were carrying OXA-48 gene.
Accordingly, negative results of carbapenemase phenotypic detection test (mCIM and eCIM combination test) according to CLSI 2020 guidelines can not be considered as true negative, and genotypic testing only is reliable in detecting the types of carbapenemases produced by the isolates. This indicates that testing for susceptibility to carbapenem by disk diffusion method is sufficient to indicate the production of carbapenemase, and that the phenotypic tests mCIM and eCIM of the CLSI 2020 for detecting the types of carbapenemase only confounds the results.
Genotypically, the presence of OXA-48 gene is statistically significantly higher among isolates displaying the serine carbapenemase phenotype (P <0.001). On the other hand, the co-existence of both NDM and OXA-48 genes was statistically significantly associated with the metallo-β-lactamase phenotype (P <0.001).
6.2 Conclusions
1. Carbapenem resistant is high among members of Enterobacteriaceae family mostly Imipenem 35.3% following by Meropenem 34.0%, with Etrapenem 35.3% .
2. ß-lactam / ß-lactmase inhibitors have reduced effectiveness in organisms producing carbapenemase enzyme.
3. Most of carbapenem resistant isolates were isolated from female with urinary tract infection.
4. Metallo-β-lactemase was more prevalent among the carbapenem resistant isolates followed by serine carbapenemase
5. Among members of the Enterobacteriaceae family carbapenem resistance is more prevelant among Klebsiella pneumoniae, followed by E coli.
6. Among the carbapenem resistant Enterobacteriaceae isolates the OXA-48 gene was the highest prevalence, followed by both NDM and OXA-48 genes together.
7. There is no added value for the phenotypic test mCIM and eCIM of the CLSI in detection of carbapenemase production among Enterobacteriaceae as phenotypic testing with disc diffusion test is more sensitive in detection of carbapenemase production as confirmed by genotypic tests.
8. Enterobacteriaceae isolates may carry both genes of serine and metallo-β-lactamase; hence phenotypic differentiation between of two type by mCIM and eCIM is not of significant importance.
9. There is no added value for the phenotypic test mCIM and eCIM of the CLSI in detection of carbapenemase production among Enterobacteriaceae as phenotypic testing with disc diffusion test is more sensitive in detection of carbapenemase production as confirmed by genotypic tests.
10. Enterobacteriaceae isolates may carry both genes of serine and metallo-β-lactamase; hence phenotypic differentiation between of two type by mCIM and eCIM is not of significant importance.
6.3 Recommendations
1. We do not recommend using the mCIM with eCIM combination method in screening for carbapenemase production to differentiate between the carbapenemase classes.
2. We recommend using polymerase chain reaction (PCR) for the differentiation between the carbapenemase classes. The PCR considered as the gold standard for detection.
3. Perform detailed studies with greater member of samples, and detection of more carbapenemase genes.
4. Abuse of antibiotics should be prohibited to minimize occurrence of resistance.