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A study on emerging resistance to carbapenem antibiotics in gram-negative bacteria isolated from clinical samples
* Corresponding author: Dr. Jubanlak Mary Pohsnem, Assistant Professor, Department of Medical Laboratory Technology, Assam Down Town University, Guwahati, Assam, India. pohsnemmary@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Das MJ, Pohsnem JM, Singh SK, Kera V, Resuh V. A study on emerging resistance to carbapenem antibiotics in gram-negative bacteria isolated from clinical samples. Ann Natl Acad Med Sci (India). doi: 10.25259/ANAMS_24_2025
Abstract
Objectives
Gram-negative bacteria are a major cause of public health concern due to their resistance to different antibiotics. Numerous reports on carbapenem resistance were published by different authors in different places in India, but there are very few studies on the prevalence of carbapenem resistance in northeast India.
Material and Methods
Pathogens isolated from urine, pus, respiratory, etc., samples were identified & tested with the antibiotic sensitivity test. The modified Hodge's test was performed to detect carbapenemase production. The carbapenemase producers were tested with the synergy test using a checkerboard assay with two antibiotics, meropenem and colistin.
Results
Bacterial isolates (144) from urine, pus, respiratory samples, etc., were collected from the Downtown Hospital, Guwahati, Assam. Klebsiella spp. (54.86%) and Escherichia coli (36.81%) were the most common isolates, followed by Pseudomonas spp. (3.47%), Enterobacter spp. (2.78%), Proteus spp.(0.69%), Citrobacter spp. (0.69%), and Morganella spp. (0.69%). We observed resistance against meropenem (27.8%), ertapenem (25.7%), fosfomycin (16.7%), ceftazidime (45.8%), ceftriaxone (53.5%), cefoxitin (54.9%), ampicillin (61.8%), colistin (52.1%), gentamycin (26.4%), tetracycline (27.8%), chloramphenicol (18.8%), and ciprofloxacin (38.9%).
Conclusion
The study reported high resistance against third-generation cephalosporins, such as ceftriaxone and ceftazidime, including penicillin and colistin. We found carbapenam resistance very common among the isolates, however combination drug showed lower levels of minimum inhibitory concentration (MIC). More studies with bigger sample sizes needs to be conducted in future.
Keywords
Antibiotic sensitivity test
Carbapenem resistance
Gram-negative bacteria (GNB)
Minimum inhibitory concentration (MIC)
INTRODUCTION
Gram-negative bacteria (GNB) are a major public health problem because they are resistant to many types of antibiotics. Most of the clinical isolates come from two main groups, Enterobacteriaceae and non-fermenters.1 GNB cause serious infections such as urinary tract infection, skin, wounds, and blood flow infections.2 In the last decade, resistance to many first-line agents (such as ciprofloxacin and cephalosporin) of GNB has increased.3 These organisms are also resistant to carbapenem.4 Enterobacteriaceae that are resistant to Carbapenem are classified as critical groups and develop antibiotic-resistant infections. According to the Center for Disease Control (CDC) description of antimicrobial-resistant pathogens, carbapenem resistant enterobacterales (CRE), such as Klebsiella, Escherichia coli, and Enterobacter are the most important emerging threats of antimicrobial resistance globally.5
Infections caused by carbapenemase-producing organisms are associated with a worrying mortality rate.6 GNB cause infections, such as pneumonia, blood flow infections, wounds or surgical sites, and meningitis in healthcare environments. Infection of the surgical site is an infection that occurs after an operation on a part of the body. Meningitis is an inflammation of the protective membrane covering the brain and spinal cord.5 The World Health Organization (WHO) and the CDC have defined A. baumannii, which is resistant to carbapenem, as one of the most urgent health threats of this century.5,7
In Enterobacteriaceae, carbapenem resistance is mainly due to the production of carbapenemase enzymes.8 Carbapenem are powerful members of the β-lactam family, inhibiting bacterial biosynthesis & cell wall. It treats infections caused by both gram-negative and gram-positive bacteria.9 Carbapenem-hydrolyzing class D β-lactamase is an important source of resistance to these last-line beta-lactam antibiotics.10 The mechanism of carbapenem resistance includes the production of β-lactamase, the efflux pump, and changes in the expression and function of porin and penicillin-binding proteins.11
CRE proliferation is currently a threat to the health of the global public.12 As per the WHO, carbapenem-resistant GNBs are one of the most important pathogens causing serious infections in humans. They are capable of fatal infections and possess drug-resistant genes that can be transferred from bacteria to bacteria. In many cases, they also resist aminoglycosides, polymyxins, and tetrachloride, and alternative drugs eventually become more effective using new antibiotics and synergistic combinations.13 Carbapenemase among Enterobacteriaceae has been reported increasingly across the world. Most of these strains have demonstrated a multidrug resistant trait.14 Since the 1990s, β-lactamases have been discovered to destroy carbapenems and pose health risks. Carbapenemase are the most diverse family of β-lactamases capable of hydrolyzing carbapenems and many other β-lactams.15 Carbapenemases are β-lactamases with a variety of hydrolytic properties. They can hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria that produce these lactamases can cause serious infections, and many β-lactams are ineffective due to the activity of carbapenemase.16
MATERIAL AND METHODS
Sample collection
Gram-negative isolates were collected from urine, pus, respiratory samples, etc. A total of 144-gram GNB isolates were collected from the Downtown Hospital, Guwahati, Assam. The isolates were transported in a protective sample transfer box. All the clinical isolates were sub-cultured on Macconkey agar and blood agar and stored at 20°C. After that, cultured clinical samples were identified by biochemical tests like catalase, oxidase, MR-VP test, Motility test, TSI agar test, etc.
Antibiotic sensitivity test
The antibiotic sensitivity test was performed by Kirby Bauer disc diffusion method using 12 antibiotics, including Meropenem, Ertapenem, Fosfomycin, Ceftazidime, Ceftriaxone, Cefoxitin, Ampicillin, Colistin, Gentamycin, Tetracycline, Chloramphenicol, and Ciprofloxacin, following Clinical and Laboratory Standards Institute (CLSI) guidelines.17
Identification of carbapenemase producers
The Modified Hodge Test was carried out to detect carbapenemase-producing isolates with Escherichia coli ATCC 25922 (indicator organism), Klebsiella pneumonia ATCC BAA-1705 (positive control) and Klebsiella pneumonia ATCC BAA-1706 (negative control). The test bacteria were inoculated in Mueller-Hinton broth and turbidity adjusted to the 0.5 McFarland standards. Then, the indicator organism was placed in Mueller-Hinton Agar plates and dried for 10 minutes. The meropenam antibiotic discs (10 μg) were placed in the center of the agar plate. The test bacteria were sliced in a straight line from the edge of the disc to the edge of the plate. The plates were then incubated for 18 hours at 37°C and the results were interpreted as per CLSI guidelines.18
Synergy test
The synergistic effect of the two antibiotic combinations has been studied by the checkerboard method. Synergies were assessed using the two last-line antibiotics Colistin and Meropenem. The test bacteria were diluted to 0.5 McFarland standards; the two antibiotics were diluted in separate microtiters and merged into a single plate. In short, meropenem (A) is vertically diluted from column A to column H (1 g/mL to 256 g/mL) in 96-well microtiter plates marked A, while colistin (B) is horizontally diluted from row 1 to 12 in 96-well microtiter plates marked B (1 g/mL to 256 g/mL). The diluted antibiotics from plate A were transferred to plate B in their respective wells. The selected test bacteria were inoculated, and the plates were incubated at 37°C for 18 hours. The synergistic effect is measured by calculating the single and combined antibiotic fractional inhibition concentration index (FIC).19 The FIC was calculated using the universal mathematical expression FIC = (FIC A/=FIC B) and FIC A = (MIC A combined with MIC A alone) and FIC B = ([Minimum inhibitory concentration (MIC) B combined with MIC B alone]). Based on FIC values, the results are interpreted as synergy (FIC ≤ 0.5), indifference (2 >FIC ≤ 0.5), and antagonistic (FIC ≤ 2).
RESULTS
Bacterial isolates
In this study, a total of 144 bacterial isolates were collected. Out of 144 samples, the isolates include: Klebsiella spp.(54.86%) and Escherichia coli (36.81%), which were the most common isolates, followed by Pseudomonas spp. (3.47%), Enterobacter spp. (2.78%), Proteus spp., Citrobacter spp., and Morganella spp. (0.69%) [Figure 1].
- Distribution of isolated gram-negative bacteria.
Age and gender distribution
Out of 144 isolates, the maximum number of GNB were isolated from the age group of >60. In <15 and >60 age groups, collected isolates were found to be more in males as compared to females. Whereas, in case of 16–30, 31–45, and 46–60 age groups, collected isolates were more in females rather than in males [Figure 2].
- Age and gender distribution.
Distribution of gram-negative bacterial isolates from clinical samples
As shown in Table 1, it is inferred that Escherichia coli accounted for 29.2% of the organisms from urine samples followed by 3.5% from pus samples and 4.9% from other samples. 6.94% of Klebsiella spp. were isolated from urine, followed by 3.5% from pus, 41.7% from respiratory, and 2.1% from other samples. 3.5% of Pseudomonas spp. were isolated from pus, 0.69% from respiratory. 0.69% of Enterobacter spp. were isolated from pus, and 4% from respiratory. 0.69% of Citrobacter spp. and Morganella spp. were isolated from urine samples.
| Organism | Source | Tota | |||
|---|---|---|---|---|---|
| Urine | Pus | Respiratory | Others | 144 | |
| Escherichia coli | 29.16% | 3.42% | 0% | 4.86% | 37.5% |
| Klebsiella spp. | 6.94% | 3.47% | 41.66% | 2.08% | 54.16% |
| Pseudomonas spp. | 0% | 3.47% | 0.69% | 0% | 4.16% |
| Enterobacter spp. | 0% | 0.69% | 1.38% | 0% | 2.08% |
| Citrobacter spp. | 0.69% | 0% | 0% | 0% | 0.69% |
| Proteus Spp. | 0% | 0% | 0.69% | 0% | 0.69% |
| Morganella Spp. | 0.69% | 0% | 0% | 0% | 0.69% |
| Total | 37.5% | 11.12% | 44.44% | 6.94% | 144 (100%) |
The bold number represents the total percentage of individual organisms.
Distribution of organisms from outpatient and inpatient departments
Table 2 shows that the total percentage of the collected isolates from OPD was 49.3% and IPD was 50.7%, in which the following organisms were detected, such as Escherichia coli(OPD -19.4%; IPD - 17.4%), Klebsiella spp.(OPD-23.6%; IPD-31.2%), Pseudomonas spp.(OPD -3.5%; IPD - 0%), Enterobacter spp.(OPD -2.1%; IPD - 0.7%), Proteus spp.(OPD -0.7%; IPD - 0%), Citrobacter spp., and Morganella spp.(OPD -0%; IPD - 0.7%).
| Organisms | Out patient department | In-patient department | Total |
|---|---|---|---|
| Escherichia coli | 28 (19.4%) | 25(17.4%) | 53(36.8%) |
| Klebsiella spp. | 34 (23.6%) | 45(31.2%) | 79(54.8%) |
| Pseudomonas spp. | 5(3.5%) | 0(0%) | 5(3.5%) |
| Enterobacter spp. | 3(2.1%) | 1 (0.7%) | 4(2.8) |
| Proteus spp. | 1(0.7%) | 0(0%) | 1(0.7%) |
| Citrobacter spp. | 0(0%) | 1(0.7%) | 1(0.7%) |
| Morganella spp. | 0(0%) | 1(0.7%) | 1(0.7%) |
| Total | 71 (49.3%%) | 73(50.7%) | 144(100%) |
Antibiotic sensitivity and resistance pattern of GNB
Figure 3 shows the Antibiotic sensitivity and resistance pattern of GNB that were tested against 12 antimicrobials. The isolated GNB showed maximum resistance towards third-generation Cephalosporins, such as Ceftriaxone (54.9%), Ceftazidime (45.8%), followed by Ciprofloxacin (38.9%), Penicillin class Ampicillin (61.8%), second generation Cefoxitin (54.9%) and last line drug Colistin (52.1%). The isolates showed maximum sensitivity towards Fosfomycin (77.1%), Flouroquinolone class Chlforamphenicol (76.4%), carbapenem class antibiotic such as Meropenem (70.1%), Ertapenem (68.8%), aminoglycoside class antibiotic Gentamycin (67.4%), and Tetracycline (64.6%).
- Antibiotic sensitivity and resistance pattern of gram-negative bacteria tested against 12 antimicrobials.
Distribution of isolates according to resistance pattern (MDR, XDR, PDR)
Among the 144 isolates, Escherichia coli showed 25.7% MDR, 6.3% extensive drug resistance (XDR), and 1.4% pan drug resistance (PDR). Klebsiella spp. showed 25.69% MDR, 4.9% XDR, and 6.3% PDR. Pseudomonas aeruginosa showed 2.8% MDR. Proteus spp. showed 0.7% (MDR), whereas, Enterobacter spp. showed 1.38% MDR [Figure 4].
- Distribution of isolates according to resistance pattern (MDR, XDR, PDR). MDR: Multidrug-resistant, XDR: Extensively drug-resistant, PDR: Pandrug-resistant
Carbapenemase producer among Gram negative bacilli (GNB) isolates
Results of the screening test for carbapenemase with meropenam disc have been shown in Table 3. The test showed that 17.4% of the isolates were carbapenemase producers, of which 3.5% were IPD and 13.9% were OPD. Among these, 13.2% isolates were Klebsiella spp. and 4.2% isolates were Escherichia coli.
| Organisms | Carbapenemase producers | Non-carbapenemase producer | Total | ||
|---|---|---|---|---|---|
| Out patient department | In-patient department | Out patient department | In-patient department | ||
| Escherichia coli | 2 (1.4%) | 4 (2.8%) | 26 (18.1%) | 21 (14.6%) | 53(36.8%) |
| Klebsiella spp. | 3 (2.1%) | 16 (11.1%) | 31 (21.5%) | 29 (20.1%) | 79(54.8%) |
| Enterobacter spp. | 0 (0%) | 0(0%) | 3 (2.1%) | 1(0.7%) | 4(2.8) |
| Citrobacter spp. | 0 (0%) | 0 (0%) | 0 (0%) | 1 0.7%) | 1(0.7%) |
| Morganella spp. | 0 (0%) | 0 (0%) | 0 (0%) | 1 (0.7%) | 1(0.7%) |
| Proteus spp. | 0 (0%) | 0 (0%) | 1(0.7%) | 0 (0.7%) | 1(0.7%) |
| Pseudomonas spp. | 0 (0%) | 0 (0%) | 5 (3.5%) | 0 (0%) | 5(3.5%) |
| Total | 5 (3.5%) | 20 (13.9%) | 66 (45.8%) | 53 (36.8%) | 144 (100%) |
Synergy test by checkerboard assay
The graph in Figure 5 shows the result of the synergy test for two antibiotics, Colistin (drug A) and Meropenem (drug B). The test shows that the MIC of drug A alone was 64 μg/mL, drug B was 16 μg/mL, drug A in combination was 4 μg/mL, and drug B in combination was 0.125 μg/mL. The MIC value of drug A and B alone was found to be greater than the MIC value of drug A in combination and drug B in combination.
- Minimum inhibitory concentration of Colistin (drug A), Meropenem (drug B) and Combination drug
DISCUSSION
The emergence and spread of carbapenemase is a major public health problem.2 GNB resistant to carbapenem are the most important microorganisms that cause serious infections, and they carry drug resistance genes that are easily transmitted, especially in environments with selective pressure on antibiotics.5 Additionally, resistance against other antibiotics like aminoglycosides, polymyxins, and tigecycline is common too. To avoid treatment failure, new antibiotics are used in combinations for an efficient synergistic effect.13 Namrata Kumari et al., (2022), reported 16.8% carbapenem resistance in GNB in their study.20 Whereas in our study, we found a total of 29.5% resistance to carbapenem.
In this study, among all the 144 isolates tested by Modified Hodge's test, 25 were carbapenemase producers and 119 isolates were non-carbapenemase producers. All carbapenemase producers have a clinical history, and they are resistant to more than one class of antibiotics. MHT test is performed using the indicator and control organisms for carbapenemase producers, which include an indicator organism (Escherichia coli ATCC 25922), a positive control (Klebsiella pneumoniae ATCC BAA-1705), and a negative control (Klebsiella pneumonia ATCC BAA-1706). Pooja G Shah et al., (2015) found that the positivity of Modified Hodge Test (MHT)21 was 92.9%.
In our study, the synergistic effect between colistin and meropenem combination showed 3/5 (60%) isolates of Klebsiella pneumoniae, which was similar to the findings of Anitha Gunalan et al., (2021), where the synergistic effect for Acinetobacter baumannnii was 18/25 (72%) and P.aeruginosa was 6/25 (24%).22
CONCLUSION
We observed an increased rate of carbapenem resistance among the Enterobacteriaceae. Most of the strains were resistant to major antibiotics like ceftazidime, ciprofloxacin, ampicillin, penicillin, and colistin. Multidrug resistance makes it difficult to treat infections with existing antibiotics. More studies conducted in different geographical regions provide a clear picture of the situation. Based on the findings of our study, it can be concluded that with the existing rise in antibiotic resistance among Enterobacteriaceae, the development of newer drugs and strategies is the need of the hour.
Authors' contributions
MJD: Conceptualization, design of the study, sample analysis and interpretation, writing the manuscript, review of manuscript; JMP: Sample collection, data analysis and Interpretation, review of the manuscript; SKS: Review of manuscript, sample collection, sample analysis and Interpretation; VK: Design of the study, sample analysis and interpretation, writing the manuscript, review of manuscript; VR: Sample collection, data analysis and Interpretation, review of the manuscript.
Ethical approval
The research/study approved by the Institutional Review Board at Assam down town University, number AdtU/Ethics/stdnt-lett/2023/008, dated 16th June, 2023.
Declaration of patient consent
Patient's consent not required as there are no patients in this study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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