|Year : 2022 | Volume
| Issue : 4 | Page : 295-300
Genotypic characterization of carbapenem-resistant enterobacterales and their antibiotic susceptibility pattern in a tertiary care hospital
Savitha B Hiremath1, BV Renushree2
1 Department of Microbiology, Sri Venkateshwaraa Medical College Hospital and Research Centre, Puducherry, India
2 Department of Microbiology, Sri Siddhartha Medical College, Tumkur, Karnataka, India
|Date of Submission||23-Sep-2021|
|Date of Acceptance||06-Feb-2022|
|Date of Web Publication||17-Mar-2023|
Dr. Savitha B Hiremath
Department of Microbiology, Sri Venkateshwaraa Medical College Hospital and Research Centre, Puducherry
Source of Support: None, Conflict of Interest: None
Background: Carbapenems have been considered as a robust antibiotic to treat extended-spectrum β-lactamases (ESBLs) in the past ten years. Carbapenems, among the β- lactams, are the most effective against gram-positive and gram-negative bacteria presenting a broad spectrum of antibacterial activity. Detection of carbapenemase-producing isolates by clinical microbiology laboratories is essential to provide targeted therapy, antimicrobial stewardship, and to update local antibiotic guidelines for clinicians.
Objectives: To identify and characterize carbapenem-resistant Enterobacterales (CRE) by phenotypic and genotypic methods, and the antibiotic sensitivity patterns of CRE isolated from the different clinical samples.
Methods: The samples were collected for a period of one year. Enterobacterales strains were identified through standard biochemical reactions and subjected to phenotypic screening for detection of carbapenemase, and confirmed with multiplex PCR. Antibiotic sensitivity patterns were also studied.
Results: A total of 447 strains of Enterobacterales species were isolated from various clinical samples over a period of one year. Out of these, 38 (8.5%) of them showed decreased susceptibility to carbapenems including 12 Escherichia coli, 11 Klebsiella pneumonia, 1 Klebsiella oxytoca, 6 Proteus mirabilis, 2 Proteus vulgaris, and 6 Citrobacter freundii. 20 isolates of them showed the presence of carbapenemase genes in Multiplex PCR, isolates included NDM, IMP, OXA-48. Two strains showed simultaneous co-existence of two genes including NDM with OXA-48 in Klebsiella pneumonia, and NDM with IMP in Proteus mirabilis. No KPC genes were detected in our study. 97% of isolates showed sensitivity to fosfomycin (for urine samples only), 73.68% of them to tigecycline, 36.84% of them to polymyxin B, 50% to colistin, 26.31% to amikacin, 18.42% to aztreonam, 21.05% to piperacillin-tazobactam, and 10.52% to cefoperazone + sulbactam combinations. There were seven isolates of urine sample, and the sensitivity of nitrofurantoin to these isolates was 42.85%.
Conclusion: The frequency of CRE was found to be low. E. coli and K. pneumoniae were the most common isolates. NDM was the commonest gene detected. Highest sensitivity was found with fosfomycin and tigecycline.
Keywords: Antibiotic sensitivity pattern, CRE, genotypic detection, phenotypic detection
|How to cite this article:|
Hiremath SB, Renushree B V. Genotypic characterization of carbapenem-resistant enterobacterales and their antibiotic susceptibility pattern in a tertiary care hospital. J NTR Univ Health Sci 2022;11:295-300
|How to cite this URL:|
Hiremath SB, Renushree B V. Genotypic characterization of carbapenem-resistant enterobacterales and their antibiotic susceptibility pattern in a tertiary care hospital. J NTR Univ Health Sci [serial online] 2022 [cited 2023 Apr 1];11:295-300. Available from: https://www.jdrntruhs.org/text.asp?2022/11/4/295/371747
| Introduction|| |
Carbapenems have been considered as a robust antibiotic to treat the bacterial strains producing extended-spectrum β-lactamases (ESBLs) in the past ten years. ESBLs are one of the most common β-lactamases encoding resistance genes distributed among gram-negative bacilli through plasmids and transposons. The novel β-lactamases with direct carbapenem-hydrolyzing activity has contributed to an increased prevalence of carbapenem-resistant Enterobacteriaceae (CRE), which is causing therapeutic failure worldwide. In recent times, development of antimicrobial resistance is rapidly changing, and the impending public health challenges these may cause in many health sectors need worldwide coordinated interventions.
Carbapenems, among the β-lactams, are the most effective against gram-positive and gram-negative bacteria presenting a broad spectrum of antibacterial activity. Their unique molecular structure is due to the presence of a carbapenem together with the β-lactam ring. This combination confers exceptional stability against most β-lactamases (enzymes that inactivate β-lactams) including ampicillin and carbenicillin (AmpC), and the extended-spectrum β-lactamases (ESBLs).
At this time, there are a limited selection of treatment options for CRE infections. Clinicians have been forced to re-evaluate the use of agents, which have been historically rarely used due to efficacy and/or toxicity concerns, such as polymyxins, fosfomycin, and aminoglycosides. Additional CRE treatment strategies include optimization of dosing regimens and combination therapy. The following antibiotics are often the only ones that can be used to treat quadruple multiresistant gram-negative strains (MRGN) pathogens: colistin, aminoglycosides, tigecycline, fosfomycin, ceftazidime/avibactam, and ceftolozane/tazobactam. Carbapenems, too, may still be an option in certain situations. There is also evidence that combinations of antibiotics against which the pathogen is resistant individually can sometimes be a valid treatment option; these include combinations of colistin with one or two carbapenems.
Detection of carbapenemase-producing isolates by clinical microbiology laboratories is essential to provide targeted therapy, antimicrobial stewardship, and update local antibiotic guidelines for clinicians. Furthermore, screening of resistance mechanisms by the use of antimicrobial susceptibility test in addition to detection of genes associated by phenotype and molecular analysis provides confirmation of clinically observed treatment failure.
We, in our study intend to focus on identification of the CRE, detect the genes belonging to classes A, B, and D. We also intend to look for sensitivity patterns of selected antibiotics of the CRE isolated from clinical samples.
| Materials and Methods|| |
The samples included in the study were prospectively collected over a period of one year. All the Enterobacterales strains isolated from various clinical samples were identified by standard biochemical tests. The approval from IEC was obtained and the date of approval is 6.11.2014.
Screening for reduced susceptibility to carbapenems
All the identified Enterobacteriaceae species were screened for reduced susceptibility to carbapenems using meropenem (10 μg) and ertapenem (10 μg) discs by Kirby Bauer disc diffusion method according to CLSI guidelines and further confirmed using meropenem E strips. The strains which showed MIC ≥4 μg for meropenem were considered as carbapenem-resistant Enterobacteriaceae. For quality control, well-characterized strains were used. E. coli ATCC 25922 was used as a susceptible strain.
Phenotypic confirmation for production of carbapenemases
The strains of reduced susceptibility to carbapenems were subjected to Modified Hodge Test (MHT). Mueller Hinton Agar was lawn cultured with standard 0.5 McFarland suspension of E. coli ATCC 25922. A single meropenem disc was placed on the plate. Three to five colonies of test organism grown overnight were inoculated in a straight line out from the edge of the disc with at least 20–25 mm in length. Following incubation, enhanced growth showing clover leaf model indicates carbapenemase production and no enhanced growth indicates negative for carbapenemase production.,
Genotypic detection of carbapenemases
The strains with reduced susceptibility to carbapenems were subjected to PCR for the detection of the following genes: KPC, NDM, VIM, IMP, OXA-48.
The primers were reconstituted according to the manufacturer's instructions. DNA was extracted with initial denaturation at 98°C for 10 minutes followed by thermocentrifugation for 10 minutes at 10,000 rpm. The supernatant was collected and used for multiplex PCR amplification. The initial step of initial denaturation at 94°C for 10 minutes was followed by annealing temperature at 55°C for 2 minutes and extension temperature at 70–72°C for 1 minute. The amplicons were subjected to gel electrophoresis and the bands read in gel electrophoresis.
Antibiotic susceptibility testing
All the isolates were tested for sensitivity patterns to all classes of antibiotics, including penicillins, cephalosporins, fluoroquinolones, cotrimoxazole, fosfomycin, tigecycline, colistin, polymyxin B, aztreonam, and aminoglycosides.
| Results|| |
A total of 447 strains of Enterobacterales species were isolated from various clinical samples over a period of one year.
Identification of isolates
Out of these, 38 (8.5%) of them showed decreased susceptibility to carbapenems including 12 Escherichia More Details coli, 11 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 6 Proteus mirabilis, 2 Proteus vulgaris, 6 Citrobacter Freundii [Table 2]. The distribution of genes in various isolates of CRE is as shown in Table 3.
Phenotypic confirmation of carbapenemases production
Of the 38 isolates showing reduced susceptibility of carbapenemase production, 33 (86.84%) of them showed enhanced growth with clover leaf pattern, indicating carbapenemase production.
Genotypic detection of carbapenemases
38 isolates of Enterobacterales species were subjected to genotypic detection of carbapenemase genes. 20 isolates of them showed the presence of carbapenemase genes in multiplex PCR. The various genes isolated included NDM, IMP, OXA-48. Two strains showed simultaneous co-existence of NDM with OXA-48 in Klebsiella pneumonia, and NDM with IMP in Proteus mirabilis. No KPC genes were detected in our study [Table 1].
Antibiotic susceptibility testing
The 38 isolated strains of Enterobacterales species were checked for susceptibility to various classes of antibiotics. 97% of isolates showed sensitivity to fosfomycin, 73.68% of them to tigecycline, 36.84% of them to polymyxin B, 50% to colistin, 26.31% to amikacin, 18.42% to aztreonam, 21.05% to piperacillin-tazobactam, and 10.52% to cefoperazone + sulbactam combinations. There were seven isolates of urine sample and the sensitivity of nitrofurantoin to these isolates was 42.85% [Table 4] and [Table 5].
| Discussion|| |
Carbapenem-resistant Enterobacterales (CRE) is a considerable health problem globally and are associated with increased mortality, and thus, rapid detection of carbapenem resistance. Adequate treatment of such cases is mandatory in a healthcare setting.
Our results revealed 38 (8.5%) of them showed decreased susceptibility to carbapenems of 447 strains of Enterobacterales. A study conducted by Elbadawi et al. revealed that the prevalence of carbapenemase production among different gram-negative isolates is increasing (up to 83%). Another study done by Okoche et al. has revealed a prevalence of the carbapenemase phenotype of 22.4%.
In our study, of the 38 isolates showing reduced susceptibility of carbapenemase production, 33 (86.84%) of them showed carbapenemase production. 20 isolates of them showed the presence of carbapenemase genes in multiplex PCR. The various genes isolated included NDM, IMP, OXA-48. Two strains showed simultaneous co-existence of two genes, including NDM with OXA-48 in Klebsiella pneumonia, and NDM with IMP in Proteus mirabilis. No KPC genes were detected in our study.
In another study conducted by Gupta et al., out of 50, 18 strains which were resistant to carbapenems (MIC) failed to show MBL or KPC production by phenotypic tests since another important cause of carbapenem resistance among Enterobacteriaceae could be overproduction of ESBL or ampC enzyme with porin changes. Thus, in this study, CTX-M type of ESBLs and NDM type of MBLs were the predominant types of enzymes.
In a study conducted by Irmak Baran, when 181 CRE isolates were analyzed, 5% of the isolates tested positive with E-test MBL strips for metallo-β-lactamase production. In 47.51% of the isolates, the blaOXA-48 gene was detected by multiplex PCR. 3.33% were blaNDM-1 gene positive and 0.56% blaVIM gene was found positive.
The study conducted by Elbadawi et al. reveals that out of 206 isolates, 171 were positive by phenotypic analysis, including isolates with resistance to carbapenems. The genotypic analysis detected 121 positive isolates.
The 38 isolated strains of Enterobacterales species were checked for susceptibility to various classes of antibiotics. All the isolates exhibited multidrug resistance to antibiotics tested. 97% of isolates showed sensitivity to fosfomycin, 73.68% of them to tigecycline, 36.84% of them to polymyxin B, 50% to colistin, 26.31% to amikacin, 18.42% to aztreonam, 21.05% to piperacillin-tazobactam, and 10.52% to cefoperazone + sulbactam combinations. There were seven isolates of the urine sample and the sensitivity of nitrofurantoin to these isolates was 42.85%.
All members of carbapenem-resistant Enterobacteriaceae showed high resistant rate for all or most commonly used penicillins, cephalosporins, monobactams, and quinolones as reported by many studies. The antibiotics which have good activity on most CRE isolates were fosfomycin, tigecycline, polymyxin, amikacin, gentamycin, and colistin. As we found the use of combined therapies up to day is only known appropriate and highly effective choice for treatment of CRE infections. But the use of combined therapies knock down the beneficial flora in the body and may lead to many serious diseases or unexpected complications. So looking for other alternative therapies may be one of the solutions to this problem.
There are signs that combination treatment is beneficial in some constellations. Some studies indicate that colistin should preferably be administered together with another antibiotic. This is controversial, however, and it remains uncertain which preparation constitutes the best colistin combination partner for which pattern of antibiogram. Retrospective studies have indicated that double carbapenem treatment is beneficial, but due to the various limitations of these studies, there is no robust evidence.
However, in the study by Mushi et al., of the 227 isolates, 76 were K. pneumoniae as the most predominant species followed by E. coli (56) and P. aeruginosa (41. 22) This might be due to the limited number of genes targeted in our study as well as due to other mechanisms of resistance such as porin loss/mutations. They also detected a low prevalence of NDM gene among multidrug-resistant gram-negative bacteria.
In Time-kill studies conducted by Corvec et al., the test strain was highly susceptible to fosfomycin, tigecycline, and colistin, as well as to gentamicin. However, a reduction of 99.9% CFU/ml was achieved only with fosfomycin and colistin, both in logarithmic and stationary growth (at concentrations which are achievable in the clinical setting). In contrast, tigecycline exhibited bacteriostatic activity and does not seem to be effective as a single agent against implant-associated infections due to gram-negative bacilli.
The study conducted by Saeed et al. shows a significant decrease of CRE incidence in the second half of the study period (2015–2017) compared to the rise during the first half (2013–2015), which was related to the intense CRE control program through development and strict implementation of new CRE policy. K. pneumoniae accounted for the largest proportion of CRE (87.0%), followed by E. coli (only 7.9%). Most of CRE isolates were sensitive to both colistin and tigecycline, which make them the best combination for empiric frontline therapy for suspected serious CRE infection in our facility.
It is obvious that novel antibiotics are urgently needed. However, this is not easy. Pharmaceutical companies have to invest a great deal of money and time into scientific research and, even when they do, only one out of five drugs that reach the initial phase of testing on humans will receive approval from the U.S. Food and Drug Administration.
Financial support and sponsorship
Management of Sri Siddhartha Medical College.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]