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Begum N, Garg A, Agarwal K. Antimicrobial susceptibility testing of colistin: Evaluation of standard broth microdilution method for gram-negative bacilli. mljgoums 2025; 19 (5) :16-20
URL: http://mlj.goums.ac.ir/article-1-1794-en.html
URL: http://mlj.goums.ac.ir/article-1-1794-en.html
1- Department of Microbiology, LLRM Medical College, Meerut, India
2- Department of Microbiology, LLRM Medical College, Meerut, India ,dr.karvi1593@gmail.com
2- Department of Microbiology, LLRM Medical College, Meerut, India ,
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Escherichia coli
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD were 26 (17.93%), 23 (15.86%), and 22 (15.17%), respectively. Out of 26 isolates that were found to be resistant by the BMD method, BD Phoenix detected resistance in only 23 isolates, and the remaining 3 isolates showed discordant results. KBDD detected resistance in only 22 isolates, and the remaining 4 cases failed to detect the resistance.
Klebsiella spp.
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD was 13 (10.48%), 13 (10.48%), and 11 (8.87%), respectively. BD Phoenix successfully detected resistance in all 13 isolates that were found to be resistant by the BMD method. KBDD detected resistance in only 11 isolates and failed to detect resistance in two cases.
Pseudomonas aeruginosa
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD were 5 (15.62%), 3 (9.37%), and 4 (12.50%), respectively. Out of 5 isolates that were found to be resistant by the BMD method, BD Phoenix and KBDD failed to detect resistance in 2 and 1 isolates, respectively.
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Introduction
Multidrug resistance (MDR) among Gram-negative bacilli (GNB) has become a serious public health issue, negatively impacting the clinical outcome of infected individuals. Colistin is an ancient antibiotic that has resurfaced as a last-resort treatment for infections caused by these MDR pathogens. The emergence of colistin resistance (COL-R), whether caused by chromosomal mutations or plasmid-mediated (MCR) mechanisms (Which all result in modifications of the lipopolysaccharides of the outer membrane in GNB), has now been identified in animals, food animal products, and human samples, and it represents a new threat to global public health (1).
For patient management and monitoring of colistin resistance, a reliable, reproducible antimicrobial susceptibility testing (AST) approach is necessary. Since 2016, CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have both recommended the Broth Microdilution Method (BMD) for determining colistin MIC. However, reference BMD, which requires freshly produced or frozen antibiotic solutions, is rarely performed in routine clinical laboratories (2).
Other methods available, such as agar dilution and gradient diffusion, are currently not recommended susceptibility testing procedures. A variety of more user-friendly commercial automated AST systems based on the BMD approach are now available (3). Due to difficulties with polymyxin testing, such as poor diffusion of polymyxins into agar, inherent cationic properties of polymyxins, the occurrence of heteroresistance to polymyxins in many species, and the lack of a reliable reference method that may allow reliable comparisons of commercial tests, polymyxin susceptibility testing presents a significant challenge for a clinical laboratory (4).
The introduction of automated systems like BD Phoenix provides the benefits of speed, efficiency, and accuracy, making them suitable for routine susceptibility testing. However, their ability to accurately determine colistin susceptibility, particularly in the context of MDR organisms, requires comprehensive evaluation (5). The purpose of this study was to assess the performance of BMD against BD Phoenix and routine KBDD to detect colistin resistance in GNB. The results of colistin susceptibility were evaluated and compared with the reference BMD method.
Methods
Ethical approval and study design
The present cross-sectional study was conducted in the Department of Microbiology at Lala Lajpat Rai Memorial (LLRM) Medical College in Meerut, associated with a tertiary care hospital, over six months (September 2023-February 2024). It was approved by the institutional ethics committee and complied with all regulations vide letter No. SC-1/2023/5328. A total of 320 clinical isolates of common Gram-negative bacteria from various clinical specimens of patients received from different departments, after obtaining informed consent from the patients for routine culture sensitivity testing, were processed in the clinical bacteriology laboratory.
Specimen collection and identification of bacteria
Various clinical specimens were obtained from 320 patients from both the in-patient and out-patient departments of the hospital. The specimens included blood, pus/tissue, body fluids, respiratory specimens, and urine. These isolates comprised a mixed population of immunocompetent, immunosuppressed/immunocompromised, and critically ill patients. Upon collection, all the clinical specimens were subsequently processed using standard microbiological methods. The clinical samples were streaked onto appropriate agar plates. Confirmation of the identity and antibiotic susceptibility testing (AST) of the isolates was done by automated BD Phoenix M-50.
Antibiotic susceptibility testing
Three distinct methods were used for colistin susceptibility testing: in-house BMD, routine KBDD, and automated BD Phoenix M-50. Colistin testing was done by reporting the MIC values as per the CLSI 2023 standards. The procedures of the methods used are described as follows:
Kirby-Bauer disk diffusion method
This method involves placing an antibiotic disc onto the Mueller-Hinton agar plate inoculated with the test isolates, and the plates were then incubated for 16-18 hours at 37°C. The zone diameters around each disc were measured, and the results were interpreted as “sensitive” or “resistant” according to CLSI 2023 guidelines.
BMD
The reference in-house BMD was performed according to CLSI 2023 guidelines (6,7). Colistin stock solution (5120 g/ml) was made by dissolving 102.4 mg of colistin sulfate powder (Sigma-Aldrich; Potency = 500 μg/mg) in 10 ml of sterile water. Filter-sterilized colistin stock solution was aliquoted in smaller amounts and stored at -60°C. The cation-adjusted Mueller-Hinton broth (CaMHB) was prepared, and a stock solution of colistin was prepared from colistin sulfate salt. The final bacterial inoculum size of 0.5 McFarland was used. The test was done in triplicate in a polystyrene microtitre plate (Corning CLS3585 flat-bottom 96 wells with lid) and incubated for 16 to 20 hours at 35°C ± 2°C and examined visually, and MIC values were noted. For sterility control, physiological saline was added to wells instead of bacterial inoculum. Because the CLSI does not provide clinical breakpoints for colistin for Enterobacteriaceae, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints were used for interpretation.
Based on the epidemiological cut-off value for Enterobacteriaceae, Klebsiella spp., and Escherichia coli were considered sensitive if the MIC value was ≤ 2 μg/ml and resistant if the MIC value was ≥ 4 μg/ml. For Pseudomonas aeruginosa and Acinetobacter spp., an MIC value of ≤ 2 μg/ml was interpreted as sensitive and an MIC value of ≥ 4 μg/ml was interpreted as resistant (6).
BD phoenix M-50 system
The manufacturer’s instructions were followed to determine the colistin susceptibility of the various test isolates. The probable range of MIC for BD Phoenix was ≤ 1 to > 4 μg/ml.
Quality control
For quality control, the following strains were used: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), and Acinetobacter baumannii (ATCC 17978).
Reconfirmation of discrepant results
Repeat testing was used to confirm discrepant results between BD Phoenix and the BMD methods.
Results
A total of 320 GNB isolates from various clinical specimens were obtained and identified. The mean age of the patients was 45.34 years. The study showed male predominance in the sample group, with 187 males (58.43%) and 133 females (41.56%) and a male-to-female ratio of 1.4. The majority of the patients, accounting for 248 (77.5%) cases, were from the outpatient department, and 72 (22.5%) cases were from the various other wards. Analysis of sample type showed that pus was the most common sample, accounting for 76 (23.75%) cases, followed by urine with 59 (18.43%) cases, blood with 48 (15%) cases, sputum with 43 (13.43%) cases, ascitic fluid with 41 (12.81%) cases, tissue biopsy with 25 (7.81%) cases, pleural fluid with 18 (5.62%) cases, and tracheal aspirate with 10 (3.12%) cases, respectively (Figure 1).
Of the 320 Gram-negative bacteria, Escherichia coli was the most common isolate in 45.31% of cases, followed by Klebsiella pneumoniae in 38.75% of cases, Pseudomonas aeruginosa in 10% of cases, and Acinetobacter baumannii complex in 5.93% of cases, respectively.
Colistin Resistance: The overall resistance to colistin among GNB was found to be 17.18% (55/320) by the gold standard BMD method, of which 58.18% (32/55) were males and 41.81% (23/55) were females. The resistance to colistin was found to be 14.49% (39/269) in Enterobacteriaceae (Escherichia coli and Klebsiella spp.) and 31.37% (16/51) in non-fermenters (Acinetobacter spp. and Pseudomonas aeruginosa), respectively. In comparison, BD Phoenix showed overall resistance to colistin among GNB in 49 isolates, i.e., 15.31%. BD Phoenix failed to detect resistance in 6 cases which were shown resistant by the BMD method. KBDD showed resistance to colistin in 46 cases (14.37%) and showed discordant results in 9 cases (Table 1).
Multidrug resistance (MDR) among Gram-negative bacilli (GNB) has become a serious public health issue, negatively impacting the clinical outcome of infected individuals. Colistin is an ancient antibiotic that has resurfaced as a last-resort treatment for infections caused by these MDR pathogens. The emergence of colistin resistance (COL-R), whether caused by chromosomal mutations or plasmid-mediated (MCR) mechanisms (Which all result in modifications of the lipopolysaccharides of the outer membrane in GNB), has now been identified in animals, food animal products, and human samples, and it represents a new threat to global public health (1).
For patient management and monitoring of colistin resistance, a reliable, reproducible antimicrobial susceptibility testing (AST) approach is necessary. Since 2016, CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have both recommended the Broth Microdilution Method (BMD) for determining colistin MIC. However, reference BMD, which requires freshly produced or frozen antibiotic solutions, is rarely performed in routine clinical laboratories (2).
Other methods available, such as agar dilution and gradient diffusion, are currently not recommended susceptibility testing procedures. A variety of more user-friendly commercial automated AST systems based on the BMD approach are now available (3). Due to difficulties with polymyxin testing, such as poor diffusion of polymyxins into agar, inherent cationic properties of polymyxins, the occurrence of heteroresistance to polymyxins in many species, and the lack of a reliable reference method that may allow reliable comparisons of commercial tests, polymyxin susceptibility testing presents a significant challenge for a clinical laboratory (4).
The introduction of automated systems like BD Phoenix provides the benefits of speed, efficiency, and accuracy, making them suitable for routine susceptibility testing. However, their ability to accurately determine colistin susceptibility, particularly in the context of MDR organisms, requires comprehensive evaluation (5). The purpose of this study was to assess the performance of BMD against BD Phoenix and routine KBDD to detect colistin resistance in GNB. The results of colistin susceptibility were evaluated and compared with the reference BMD method.
Methods
Ethical approval and study design
The present cross-sectional study was conducted in the Department of Microbiology at Lala Lajpat Rai Memorial (LLRM) Medical College in Meerut, associated with a tertiary care hospital, over six months (September 2023-February 2024). It was approved by the institutional ethics committee and complied with all regulations vide letter No. SC-1/2023/5328. A total of 320 clinical isolates of common Gram-negative bacteria from various clinical specimens of patients received from different departments, after obtaining informed consent from the patients for routine culture sensitivity testing, were processed in the clinical bacteriology laboratory.
Specimen collection and identification of bacteria
Various clinical specimens were obtained from 320 patients from both the in-patient and out-patient departments of the hospital. The specimens included blood, pus/tissue, body fluids, respiratory specimens, and urine. These isolates comprised a mixed population of immunocompetent, immunosuppressed/immunocompromised, and critically ill patients. Upon collection, all the clinical specimens were subsequently processed using standard microbiological methods. The clinical samples were streaked onto appropriate agar plates. Confirmation of the identity and antibiotic susceptibility testing (AST) of the isolates was done by automated BD Phoenix M-50.
Antibiotic susceptibility testing
Three distinct methods were used for colistin susceptibility testing: in-house BMD, routine KBDD, and automated BD Phoenix M-50. Colistin testing was done by reporting the MIC values as per the CLSI 2023 standards. The procedures of the methods used are described as follows:
Kirby-Bauer disk diffusion method
This method involves placing an antibiotic disc onto the Mueller-Hinton agar plate inoculated with the test isolates, and the plates were then incubated for 16-18 hours at 37°C. The zone diameters around each disc were measured, and the results were interpreted as “sensitive” or “resistant” according to CLSI 2023 guidelines.
BMD
The reference in-house BMD was performed according to CLSI 2023 guidelines (6,7). Colistin stock solution (5120 g/ml) was made by dissolving 102.4 mg of colistin sulfate powder (Sigma-Aldrich; Potency = 500 μg/mg) in 10 ml of sterile water. Filter-sterilized colistin stock solution was aliquoted in smaller amounts and stored at -60°C. The cation-adjusted Mueller-Hinton broth (CaMHB) was prepared, and a stock solution of colistin was prepared from colistin sulfate salt. The final bacterial inoculum size of 0.5 McFarland was used. The test was done in triplicate in a polystyrene microtitre plate (Corning CLS3585 flat-bottom 96 wells with lid) and incubated for 16 to 20 hours at 35°C ± 2°C and examined visually, and MIC values were noted. For sterility control, physiological saline was added to wells instead of bacterial inoculum. Because the CLSI does not provide clinical breakpoints for colistin for Enterobacteriaceae, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints were used for interpretation.
Based on the epidemiological cut-off value for Enterobacteriaceae, Klebsiella spp., and Escherichia coli were considered sensitive if the MIC value was ≤ 2 μg/ml and resistant if the MIC value was ≥ 4 μg/ml. For Pseudomonas aeruginosa and Acinetobacter spp., an MIC value of ≤ 2 μg/ml was interpreted as sensitive and an MIC value of ≥ 4 μg/ml was interpreted as resistant (6).
BD phoenix M-50 system
The manufacturer’s instructions were followed to determine the colistin susceptibility of the various test isolates. The probable range of MIC for BD Phoenix was ≤ 1 to > 4 μg/ml.
Quality control
For quality control, the following strains were used: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), and Acinetobacter baumannii (ATCC 17978).
Reconfirmation of discrepant results
Repeat testing was used to confirm discrepant results between BD Phoenix and the BMD methods.
Results
A total of 320 GNB isolates from various clinical specimens were obtained and identified. The mean age of the patients was 45.34 years. The study showed male predominance in the sample group, with 187 males (58.43%) and 133 females (41.56%) and a male-to-female ratio of 1.4. The majority of the patients, accounting for 248 (77.5%) cases, were from the outpatient department, and 72 (22.5%) cases were from the various other wards. Analysis of sample type showed that pus was the most common sample, accounting for 76 (23.75%) cases, followed by urine with 59 (18.43%) cases, blood with 48 (15%) cases, sputum with 43 (13.43%) cases, ascitic fluid with 41 (12.81%) cases, tissue biopsy with 25 (7.81%) cases, pleural fluid with 18 (5.62%) cases, and tracheal aspirate with 10 (3.12%) cases, respectively (Figure 1).
Of the 320 Gram-negative bacteria, Escherichia coli was the most common isolate in 45.31% of cases, followed by Klebsiella pneumoniae in 38.75% of cases, Pseudomonas aeruginosa in 10% of cases, and Acinetobacter baumannii complex in 5.93% of cases, respectively.
Colistin Resistance: The overall resistance to colistin among GNB was found to be 17.18% (55/320) by the gold standard BMD method, of which 58.18% (32/55) were males and 41.81% (23/55) were females. The resistance to colistin was found to be 14.49% (39/269) in Enterobacteriaceae (Escherichia coli and Klebsiella spp.) and 31.37% (16/51) in non-fermenters (Acinetobacter spp. and Pseudomonas aeruginosa), respectively. In comparison, BD Phoenix showed overall resistance to colistin among GNB in 49 isolates, i.e., 15.31%. BD Phoenix failed to detect resistance in 6 cases which were shown resistant by the BMD method. KBDD showed resistance to colistin in 46 cases (14.37%) and showed discordant results in 9 cases (Table 1).
.PNG) Figure 1. Distribution of different sample types for colistin susceptibility testing |
Escherichia coli
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD were 26 (17.93%), 23 (15.86%), and 22 (15.17%), respectively. Out of 26 isolates that were found to be resistant by the BMD method, BD Phoenix detected resistance in only 23 isolates, and the remaining 3 isolates showed discordant results. KBDD detected resistance in only 22 isolates, and the remaining 4 cases failed to detect the resistance.
Klebsiella spp.
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD was 13 (10.48%), 13 (10.48%), and 11 (8.87%), respectively. BD Phoenix successfully detected resistance in all 13 isolates that were found to be resistant by the BMD method. KBDD detected resistance in only 11 isolates and failed to detect resistance in two cases.
Pseudomonas aeruginosa
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD were 5 (15.62%), 3 (9.37%), and 4 (12.50%), respectively. Out of 5 isolates that were found to be resistant by the BMD method, BD Phoenix and KBDD failed to detect resistance in 2 and 1 isolates, respectively.
|
Table 1. Colistin resistance shown by different methods
.PNG) |
.PNG)
.PNG)
Acinetobacter baumannii
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD was 11 (37.89%), 10 (52.63%), and 9 (47.36%), respectively. Out of 11 isolates that were found to be resistant by the BMD method, BD Phoenix detected resistance in only 10 isolates and failed to detect resistance in 1 strain. KBDD detected resistance in only 9 isolates, and the remaining 2 cases failed to detect the resistance.
Discussion
The literature demonstrates that the rise of carbapenem-resistant Enterobacteriaceae (CRE) has become a serious problem. In patients with carbapenem-resistant GNB infections, colistin is the mainstay of treatment, and its usage has increased worldwide, particularly in India, after the emergence of CRE. Nonetheless, there are differences in susceptibility test findings obtained using various methodologies. An accurate method is required to test colistin susceptibility, as there are elevated trends of colistin MICs noted worldwide. In the present study, the performance of the gold standard BMD method was evaluated and compared with automated BD Phoenix M-50 and KBDD for colistin susceptibility testing. The study findings shed light on the scope of antibiotic resistance and the reliability of testing methodologies, providing important insights for clinical practice and antimicrobial stewardship.
The mean age of the patients in our study was 45.34 years. Taneja et al., Aggarwal et al., Arjun et al., and Goel et al. (8-11) reported a similar mean age. In terms of sex distribution, our study included 187 males (58.43%) and 133 females (41.56%), indicating a modest male predominance among the sample group. Kumari et al., Taneja et al., Pragasam et al., Aggarwal et al., and Goel et al. all reported a similar pattern of sex distribution (8-10,12,13). The sample type analysis in our study revealed that pus was the most common sample, which was similar to the studies done by Zaki et al. and Pawar et al. (14,15).
Butta et al. found 19.17% (140/730) resistance to colistin among Gram-negative bacilli using the gold standard BMD technique, which was in concordance with our study (16).
In a study by Bernhardt et al., Klebsiella pneumoniae showed higher resistance to colistin when tested by the BMD method (6/10 isolates), and BD Phoenix showed lower resistance to colistin (3/10 isolates), while in our study all the Klebsiella pneumoniae showed equal resistance with both methods (17).
In our study, out of 320 Gram-negative isolates tested, 82.81% were sensitive to colistin when tested by the BMD method. Studies by Arjun et al., Ramesh et al., and Behera et al. reported colistin sensitivity of 70.83%, 55.55%, and 94.23%, respectively, among MDR-GNB (11,18,19).
A recent study by Lai CC et al. and Pfennigwerth et al. also showed unreliable colistin MIC results by an automated method (20,21). It is particularly important to test all MDR isolates for colistin susceptibility using the BMD method because false negative and positive results would place the patient on incorrect antimicrobial therapy. In this study, evaluation of both carbapenem-susceptible (non-MDR) and non-susceptible (MDR) bacterial isolates, and colistin susceptibility testing by BMD, was performed concurrently with the BD Phoenix and routine KBDD method on the same day of isolation as a regular colistin susceptibility testing method.
Although performing AST methods such as BMD for clinical testing is technically demanding, laboratories must train the staff to perform BMD and overcome common challenges such as making initial dilutions, multiple skipped wells, contamination, or other quality control issues that are not present in automated systems.
Abbreviations
MICs: Minimum Inhibitory Concentrations; BMD: Broth Microdilution method; GNB: Gram-Negative Bacilli; CLSI: Clinical Laboratory Standards Institute; (COL-R): Colistin Resistance; MCR: Chromosomal Mutations; MDROs: Multidrug-Resistant pathogens; BMD: Broth Microdilution; AST: Antimicrobial Susceptibility Testing; EUCAST: European Committee on Antimicrobial Susceptibility Testing
Conclusion
Compared to disk diffusion and automated methods like BD Phoenix, the BMD method is the most cost-effective and reliable approach for colistin susceptibility testing. Reporting by automated methods is a simple procedure, and the results in terms of BMD are generally acceptable in Klebsiella spp., Escherichia coli, and Acinetobacter baumannii complex. Automation could be utilized to test colistin susceptibility in low-risk patients. BMD, on the other hand, should be employed in high-risk and immunocompromised patients hospitalized in critical care units. The resistance profiles of Gram-negative organisms in our study highlight the urgent need for novel therapeutic approaches and efficient infection control measures. Strong antimicrobial stewardship initiatives are recommended to counteract the rising rate of resistance.
Acknowledgement
We acknowledge the help of the faculty and technical staff of the Microbiology Department and all the participants for their involvement and feedback in the study.
Funding sources
All authors have declared that no financial support was received from any organization for the submitted work.
Ethical statement
The research study has been approved by the institutional ethics committee and is compliant with all necessary regulations. Ref. No. SC-1/2023/5328.
Conflicts of interest
Authors declare no conflicts of interest.
Author contributions
Dr. Naila Begum performed the laboratory work, drafted the paper, and provided critical inputs; Dr. Amit Garg conceived and designed the study and revised the manuscript for important intellectual content; Dr. Karvi Agarwal rechecked the manuscript.
Data availability statement
The authors can make the data available upon a reasonable request.
The total number of isolates showing resistance to colistin by BMD, BD Phoenix, and KBDD was 11 (37.89%), 10 (52.63%), and 9 (47.36%), respectively. Out of 11 isolates that were found to be resistant by the BMD method, BD Phoenix detected resistance in only 10 isolates and failed to detect resistance in 1 strain. KBDD detected resistance in only 9 isolates, and the remaining 2 cases failed to detect the resistance.
Discussion
The literature demonstrates that the rise of carbapenem-resistant Enterobacteriaceae (CRE) has become a serious problem. In patients with carbapenem-resistant GNB infections, colistin is the mainstay of treatment, and its usage has increased worldwide, particularly in India, after the emergence of CRE. Nonetheless, there are differences in susceptibility test findings obtained using various methodologies. An accurate method is required to test colistin susceptibility, as there are elevated trends of colistin MICs noted worldwide. In the present study, the performance of the gold standard BMD method was evaluated and compared with automated BD Phoenix M-50 and KBDD for colistin susceptibility testing. The study findings shed light on the scope of antibiotic resistance and the reliability of testing methodologies, providing important insights for clinical practice and antimicrobial stewardship.
The mean age of the patients in our study was 45.34 years. Taneja et al., Aggarwal et al., Arjun et al., and Goel et al. (8-11) reported a similar mean age. In terms of sex distribution, our study included 187 males (58.43%) and 133 females (41.56%), indicating a modest male predominance among the sample group. Kumari et al., Taneja et al., Pragasam et al., Aggarwal et al., and Goel et al. all reported a similar pattern of sex distribution (8-10,12,13). The sample type analysis in our study revealed that pus was the most common sample, which was similar to the studies done by Zaki et al. and Pawar et al. (14,15).
Butta et al. found 19.17% (140/730) resistance to colistin among Gram-negative bacilli using the gold standard BMD technique, which was in concordance with our study (16).
In a study by Bernhardt et al., Klebsiella pneumoniae showed higher resistance to colistin when tested by the BMD method (6/10 isolates), and BD Phoenix showed lower resistance to colistin (3/10 isolates), while in our study all the Klebsiella pneumoniae showed equal resistance with both methods (17).
In our study, out of 320 Gram-negative isolates tested, 82.81% were sensitive to colistin when tested by the BMD method. Studies by Arjun et al., Ramesh et al., and Behera et al. reported colistin sensitivity of 70.83%, 55.55%, and 94.23%, respectively, among MDR-GNB (11,18,19).
A recent study by Lai CC et al. and Pfennigwerth et al. also showed unreliable colistin MIC results by an automated method (20,21). It is particularly important to test all MDR isolates for colistin susceptibility using the BMD method because false negative and positive results would place the patient on incorrect antimicrobial therapy. In this study, evaluation of both carbapenem-susceptible (non-MDR) and non-susceptible (MDR) bacterial isolates, and colistin susceptibility testing by BMD, was performed concurrently with the BD Phoenix and routine KBDD method on the same day of isolation as a regular colistin susceptibility testing method.
Although performing AST methods such as BMD for clinical testing is technically demanding, laboratories must train the staff to perform BMD and overcome common challenges such as making initial dilutions, multiple skipped wells, contamination, or other quality control issues that are not present in automated systems.
Abbreviations
MICs: Minimum Inhibitory Concentrations; BMD: Broth Microdilution method; GNB: Gram-Negative Bacilli; CLSI: Clinical Laboratory Standards Institute; (COL-R): Colistin Resistance; MCR: Chromosomal Mutations; MDROs: Multidrug-Resistant pathogens; BMD: Broth Microdilution; AST: Antimicrobial Susceptibility Testing; EUCAST: European Committee on Antimicrobial Susceptibility Testing
Conclusion
Compared to disk diffusion and automated methods like BD Phoenix, the BMD method is the most cost-effective and reliable approach for colistin susceptibility testing. Reporting by automated methods is a simple procedure, and the results in terms of BMD are generally acceptable in Klebsiella spp., Escherichia coli, and Acinetobacter baumannii complex. Automation could be utilized to test colistin susceptibility in low-risk patients. BMD, on the other hand, should be employed in high-risk and immunocompromised patients hospitalized in critical care units. The resistance profiles of Gram-negative organisms in our study highlight the urgent need for novel therapeutic approaches and efficient infection control measures. Strong antimicrobial stewardship initiatives are recommended to counteract the rising rate of resistance.
Acknowledgement
We acknowledge the help of the faculty and technical staff of the Microbiology Department and all the participants for their involvement and feedback in the study.
Funding sources
All authors have declared that no financial support was received from any organization for the submitted work.
Ethical statement
The research study has been approved by the institutional ethics committee and is compliant with all necessary regulations. Ref. No. SC-1/2023/5328.
Conflicts of interest
Authors declare no conflicts of interest.
Author contributions
Dr. Naila Begum performed the laboratory work, drafted the paper, and provided critical inputs; Dr. Amit Garg conceived and designed the study and revised the manuscript for important intellectual content; Dr. Karvi Agarwal rechecked the manuscript.
Data availability statement
The authors can make the data available upon a reasonable request.
Research Article: Research Article |
Subject:
Microbiology
Received: 2024/03/22 | Accepted: 2025/02/23 | Published: 2025/10/8 | ePublished: 2025/10/8
Received: 2024/03/22 | Accepted: 2025/02/23 | Published: 2025/10/8 | ePublished: 2025/10/8
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