Modified Carbapenem Inactivation Method and Ethylenediaminetetraacetic Acid (EDTA)-Carbapenem Inactivation Method for Detection of Carbapenemase-Producing Enterobacterales and Pseudomonas aeruginosa

Introduction: The rising incidence of carbapenem resistance in Enterobacterales and Pseudomonas aeruginosa is a concern. Since carbapenemase production is the primary resistance mechanism, detecting and identifying the genes responsible for it is crucial to effectively monitor its spread. Objective: This study aims to detect positivity for the modified carbapenem inactivation method (mCIM) and ethylenediaminetetraacetic acid (EDTA)-carbapenem inactivation method (eCIM) for the detection of carbapenemase-producing Enterobacterales and Pseudomonas aeruginosa. Methods: Methods: A cross-sectional study was carried out at a tertiary care hospital, including 250 clinical isolates of Enterobacterales and Pseudomonas aeruginosa. These isolates exhibited resistance to at least one of the carbapenems as determined by the VITEK AST 2 System (bioMérieux, USA). The isolates were subjected to mCIM testing, and those that tested positive were further tested using eCIM. The results were interpreted in accordance with the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) 2023. Results: Out of the total 250 carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa isolates, 151 (60.4%) were Klebsiella pneumonia, 44 (17.6%) were Escherichia coli, 10 (4.0%) were Enterobacter cloacae, 6 (2.4%) were Providencia spp., 4 (1.6%) were Serratia marcescens, 4 (1.6%) were Proteus mirabilis and 31 (12.4%) were Pseudomonas aeruginosa. Positivity for the mCIM was observed in 96% (240 out of 250) of the isolates. Of the mCIM-positive isolates, 234 (97.5%) also tested positive for eCIM, indicating metallo-β-Lactamase (MLB) production. A statistically significant association was found between both mCIM and eCIM positivity and the degree of resistance to carbapenem (p<0.05). Conclusion: This study shows that the inexpensive method, a combination of mCIM and eCIM assists in differentiating between serine carbapenemase producers and MLB producers, thereby guiding the selection of appropriate therapy and useful in infection control in resource-limited settings.


Introduction
Antibiotic-resistant bacteria are emerging as a significant public health issue worldwide, leading to recurring patient infections.These drug-resistant microorganisms cause infections in hospitalized patients, limiting the available treatment options and resulting in higher infection and mortality rates [1].Identifying these drug-resistant organisms promptly is essential to ensure patients receive timely and effective treatment [2].
Carbapenems, including meropenem, imipenem, ertapenem, and doripenem, are effective in treating multidrug-resistant (MDR) infections.However, the incidence of carbapenem resistance (CR) has increased over the last few decades.CR is commonly observed in Enterobacterales such as Escherichia coli and Klebsiella pneumonia, as well as in Pseudomonas aeruginosa and Acinetobacter spp.[3,4].
According to the Centers for Disease Control and Prevention (Atlanta, Georgia, USA), a carbapenemresistant organism is an organism that is resistant to one or more carbapenem antibiotics or produces the carbapenemase enzyme [5].The development of carbapenem resistance is associated with the production of

Procedure
Primary isolates were stored in tryptic soya broth to prevent changes in bacterial properties.The identification and antibiotic sensitivity tests of these clinical isolates were conducted in a microbiology laboratory.These isolates were found to be resistant to at least one of the carbapenems (meropenem, imipenem, or ertapenem), as determined by the VITEK AST 2 System (bioMérieux, USA) using Antimicrobial Susceptibility Testing (AST) panels 405 and 406.
Modified carbapenem inactivation method in conjugation with the EDTA-carbapenem inactivation method: The mCIM and eCIM methods were employed to detect CPE, following the CLSI-2023 guidelines.
For each strain under test, two tubes, each containing 2 mL of Trypticase Soy Broth (TSB), were used concurrently.One tube was supplemented with 20 µL of 0.5 M EDTA (Sigma), while the other tube was kept EDTA-free.A fresh colony from the strain under test was transferred to each tube using a 1 µL inoculating loop.A 10-mg meropenem disk (HiMedia, India) was then incubated with the bacterial suspension from the tested strain for 2-4 hours at 35°C.Following this, meropenem disks from both tubes were placed on Mueller Hinton agar plates, which were then inoculated with the E. coli ATCC 25922 indicator strain.The mCIM was considered to have yielded a positive result when the diameter of the inhibition zone was between 6 and 15 mm, or between 16 and 18 mm but with small colonies present within the inhibitory zone.Interpretation of eCIM results should be undertaken only when the mCIM result indicates the presence of a carbapenemase.If the zone diameter for eCIM exceeds that of mCIM by 5 mm, it suggests a potential production of metallocarbapenemase. E. coli ATCC 25922 was used as a quality control measure.
According to the data provided by CLSI, the mCIM's sensitivity and specificity are greater than 99% for Enterobacterales and range from 97-100% for Pseudomonas aeruginosa.In contrast, the eCIM method demonstrates sensitivity and specificity greater than 95% and 92%, respectively, for Enterobacterales.In this study, we conducted mCIM on all 250 isolates and performed eCIM on those isolates that tested positive for mCIM.

Statistical analysis
The statistical analysis was conducted using GraphPad Prism software (version 10) (GraphPad Software, Boston, MA).The association between the carbapenem results, as determined by Vitek MIC, mCIM, and eCIM, was compared using a 2x2 Fisher's exact test.A p-value of less than 0.05 was considered statistically significant.

Results
Out of the 250 isolates analyzed, 219 (87.6%) were classified as part of the Enterobacterales family, and 31 (12.4%) were identified as Pseudomonas aeruginosa isolates.The majority of carbapenem-resistant isolates (61.2%) were from males, accounting for 153 isolates, while 97 (38.8%) isolates were from females.Patients over 60 years of age made up 134 (53.6%) of the 250 cases.The most common source of isolates was urine, with 88 instances (35%) (Figure 1).The majority of isolates were obtained from the ICU, accounting for 153 (61.2%), followed by in-patient wards with 101 (40.4%), and the OPD with 29 (11.6%).
The distribution of carbapenemase in CRGNB varied from 91.4% to 100% across different samples.Among the three most common samples, carbapenemase production was most prevalent in urine samples 85 (96.6%), followed by respiratory secretions 56 (96.5%), and blood 32 (91.4%) (Table 1).Out of the 250 isolates, 207 (82.8%) demonstrated resistance to all carbapenems tested using the automated VITEK AST 2 system.However, 24 (9.6%) isolates displayed either sensitivity or moderate susceptibility to one or more of the tested carbapenems.Notably, meropenem and imipenem were the drugs most frequently showing moderate susceptibility, with an MIC value of 2 µg/mL for 22 (9.4%) isolates (

TABLE 3: Investigation of the pattern of susceptibility to carbapenems in relation to the positivity of mCIM and eCIM
There is a statistically significant association of modified carbapenem inactivation method (mCIM) positivity with the degree of resistance to carbapenem p-value: 0.0027; There is a statistically significant association of ethylenediaminetetraacetic acid (EDTA)-carbapenem inactivation method (eCIM) positivity with the degree of resistance to carbapenem p-value: 0.0001.
Out of 240 mCIM-positive isolates, 234 (97.5%) were eCIM-positive, indicating the presence of metallo-βlactamase genes (such as NDM, IMP, VIM, KPC, and IMI).The remaining six isolates could be serine βlactamases, or there might be a co-occurrence of serine β-lactamases with MBL genes.Among the 234 eCIMpositive isolates, 22 (9.4%) were sensitive or moderately sensitive to at least one of the carbapenems.
The analysis between mCIM and eCIM results is presented in Table 3.A 2x2 Fisher's exact test was performed to examine the association between mCIM positivity and eCIM.The correlation between these variables showed statistical significance, with a p-value of 0.0001.
In our study, the majority of isolates were obtained from the ICU, accounting for 153 (61.2%), followed by inpatient wards with 101 (27.2%), and the OPD with 29 (11.6%).This distribution is similar to the findings of a study by Baskaran et al. [7].Our study found that the prevalence of carbapenemase production among the carbapenem-resistant isolates screened by the VITEK AST 2 System was 96% (95% CI: 92.77-98.07).This was particularly true for metallo β-lactamases, with 96% mCIM positivity (i.e., 234 out of 240 were positive for EDTA-carbapenem inactivation method (eCIM).This is consistent with a study by Gallego et al., which showed that the prevalence of mCIM was between 96% and 98% [8].A study in Lucknow by Rahman et al. recorded a 100% prevalence of NDM genes in all the carbapenem-resistant Enterobacterales [9].According to a study conducted by Roopashree et al., the mCIM method revealed a prevalence rate of 45.09% [10].
Research conducted in the Coimbatore region of Tamil Nadu revealed a prevalence rate of 73.33% between 2011 and 2015 and 82% in 2017 [11].The majority of these studies focused on isolates from samples taken from patients with specific co-morbid conditions.Furthermore, a study by Gromski et al. found a higher prevalence of positive bile culture in patients who had not previously undergone biliary stent placement [12].The results showed that 86.1% of patients without a prior biliary stent had positive bile cultures, compared to only 55% of patients in an earlier study who had a positive bile culture without a previous biliary stent.However, some studies were limited to the detection of a specific group of genes responsible for carbapenemases using PCR [11][12][13][14].
In our study, the combined prevalence of mCIM and EDTA-carbapenem inactivation method (eCIM) was 97.5%, which aligns with the study by Li and co-workers have reported that the combined incidence of mCIM with eCIM and CDT was 97.5% and 96.2%, respectively [15].Similarly, Koul et al. stated that the combined prevalence of mCIM and eCIM was 58.5% and 58.4%, respectively [16].
Among the carbapenem-resistant Enterobacterales, Klebsiella pneumonia and E. coli are the top two carbapenem-resistant organisms, accounting for 60.4% and 17.6%, respectively.This is confirmed by various studies, such as the one by Gao et al., which showed that Klebsiella pneumonia was the most common CRE organism (44.8%), followed by E. coli (25.8%), and Enterobacter cloacia (13.8%) [18].
In this study, Pseudomonas aeruginosa was found to be most prevalent in respiratory secretions, followed by urine, and pus/wound swabs, with prevalence rates of 25.8%, 22.5%, and 22.5%, respectively.These findings are similar to the study by Khater et al., which reported prevalence rates of 37.2% in urine, 30.2% in sputum, 25.6% in pus samples, and 4.66% in blood [19].In our study, the prevalence of carbapenem-resistant Pseudomonas aeruginosa was found to be 12.4%.This is comparable to the study by Cheemala et al., which reported a carbapenem resistance rate of 24% in Pseudomonas aeruginosa [20].Another study by Vamsi et al. reported a carbapenem resistance rate of 10.9% in Pseudomonas spp [21].
According to a report from the Antimicrobial Resistance (AMR) Surveillance Network of India, the prevalence of carbapenem resistance in Enterobacterales was 40%, and the average number of MBL genes in these isolates was 30%.The occurrence of NDM was relatively high in certain parts of the country.The AMR Surveillance Network also reported that the increase in the prevalence of resistance in E. coli and Klebsiella pneumonia was 5% and 17%, respectively, over a five-year period in India (2017-2022) [22].
This study has many limitations and challenges.We have used a combination of mCIM and eCIM for phenotypic detection of carbapenem-producing Enterobacterales and Pseudomonas aeruginosa.Further molecular detection of serine beta-lactamases genes and metallo beta-lactamases by real-time PCR would have been useful in discriminating specific enzymes However, we could not differentiate between the cooccurrence of genes with MBL genes and serine ß-lactamases.However, this will not impact the broad findings of the study regarding the use of eCIM and mCIM to detect and distinguish carbapenemase in resource-limited settings.

Conclusions
The prevalence of infections caused by Gram-negative, carbapenem-resistant pathogens is increasing at an alarming rate.With its high sensitivity and specificity, the mCIM proves to be an effective test for detecting these pathogens.However, for the mCIM to yield consistent and accurate results, it must be conducted as a phenotypic test under standardized conditions, as described by the CLSI guideline.The eCIM test shows a positive result irrespective of the presence or absence of the serine carbapenemase genes in combination with metallo-β-lactamase (MBL).Therefore, this method does not distinguish between the co-occurrence and presence of MBL genes in combination with carbapenemases and those of MBL genes alone.The significant occurrence of MBL in eCIM cases implies that the ideal combination for mCIM-positive isolates in this study region is ceftazidime-avibactam plus aztreonam.Implementing phenotypic methods (eCIM and mCIM) to detect and distinguish carbapenemases would be useful in guiding empirical antibiotic therapy, especially in and useful in infection control in resource-limited settings.

Table 3
presents an analysis comparing the results from the VITEK AST 2 system and the mCIM test.A 2x2 Fisher's exact test was performed to investigate the relationship between carbapenem susceptibility testing using the automated minimum inhibitory concentration (MIC) method and mCIM positivity.The analysis revealed a statistically significant correlation between the variables, with a p-value of 0.0027.It was observed that isolates showing resistance to all three carbapenems through MIC determination are more likely to be carbapenemase producers, compared to isolates that are sensitive or moderately sensitive to one or more carbapenems.