Detection Of Colistin Resistance Research Articles

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings

BackgroundColistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT).ResultsThis study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 μg/mL), while broth microdilution identified 48 (MIC = 32–128 μg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357).ConclusionThe global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Performance of modified colistin broth disc elution vis-a-vis broth microdilution method for susceptibility testing of Enterobacterales

Objectives: Recently, Clinical and Laboratory Standards Institute (CLSI) has approved colistin broth disc elution (CBDE) to be a supplemental test. This requires multiple discs and tubes to get the desired concentrations of colistin -1, 2, and 4 µg/mL and 10 mL volume of cation-adjusted Mueller–Hinton broth for a single isolate. The present study was aimed to evaluate the performance of CBDE in a microtiter plate format modified (mCBDE) with the reference method broth microdilution (BMD) for detection of colistin resistance in carbapenem-resistant Enterobacterales (CRE) isolates. Materials and Methods: One hundred and sixty non-duplicate clinical CRE isolates (May 2021–April 2022) were simultaneously subjected for BMD and mCBDE. For mCBDE, colistin 10 µg discs and Mueller–Hinton broth no-2 control cations were procured from HiMedia, Mumbai, and drug concentrations were prepared following CLSI-M100Ed31. Results of mCBDE were compared with reference BMD (Minimum inhibitory concentration [MIC] ≤2 µg/mL – intermediate and ≥4 µg/mL – resistant). Statistical Analysis: The performance of mCBDE was compared with BMD and expressed in terms of Categorical, essential agreement (EA), very major error (VME), and major error (ME). The sensitivity and specificity were calculated using Fisher’s contingency Table. Results: Of the 160 CRE isolates, 152 had exactly the same minimal inhibitory concentration (MIC) in both the tests with four isolates having higher and four having lower colistin MIC by mCBDE, giving a major error of 2.1% and VME of 5.5%. Categorical and essential agreement of mCBDE were 97.5% and 98.7%, respectively. Conclusions: mCBDE is an easy, economical, and reliable alternative test for determining colistin susceptibility for CRE isolates. Further, large-scale study is needed to strengthen our observation.

Detection of colistin resistance via four methods among Gram-negative bacteria

Objective: We aimed to find the most accurate and robust method for the detection of colistin resistance among Gram-negative bacteria by comparing the performance of four methods. Methods: Colistin resistantance was determine by disk diffusion (DD), Etest, colistin broth disk elution (CBDE) and compared with standard broth microdilution (BMD) method. Results: An exploratory study was conducted in a tertiary hospital in Jaipur, India, from November 2021 to November 2022. Of the 384 isolates tested, 41 (10.7%) were resistant against colistin by BMD and CBDE testing. Compared with BMD method, the categorical agreement of DD and Etest methods were 65.9% and 78.1%, respectively, and the rates of very major error were 34.1% and 21.9%, respectively. Conclusions: Due to the high frequency of very major error, the use of DD and Etest are not recommended by Clinical laboratory and standards institute (CLSI) for colistin susceptibility testing while CBDE is the recommended method which yields a categorical agreement of 100% with BMD method. The CBDE method can be used in routine laboratories for the detection of colistin resistance.

Mobile colistin resistance (mcr) genes and recent developments in colistin resistance detection.

The peptide antibiotic colistin has been reserved as a last resort antibiotic treatment option for cases where other antibiotics including carbapenems have failed. Recent emergence of colistin resistance and discovery of mobile colistin resistance (mcr) genes, which encode the cell wall modifying phosphoethanolamine transferase enzyme, complicates the issue. The mcr genes have been associated with conjugative plasmids and can be horizontally transferred between different bacterial species. The global spread of mcr genes has been extensively documented and this warrants surveillance of the resistance genes in the community. However, susceptibility testing of colistin is fraught with practical challenges owing to the chemical nature of the drug and multiple mechanisms of resistance. Although broth microdilution is the current gold standard for colistin susceptibility testing, the method poses technical challenges. Hence alternative detection methods for screening colistin resistance are the need of the hour. Several methods have been studied in the recent times to address this issue. In this review, we discuss some of the recent developments in the detection of colistin resistance.

Evaluation the phenotypic assays for the detection of colistin resistance in Klebsiella pneumoniae isolates from Critical Care Units in Tertiary Care Hospital, Kanpur

Evaluation the phenotypic assays for the detection of colistin resistance in Klebsiella pneumoniae isolates from Critical Care Units in Tertiary Care Hospital, Kanpur

Plasmid-Borne Mobile Colistin Resistant Gene (Mcr-1) Detection and Multidrug Resistant Bacteria Isolated from Some Abattoir Environments in Benin City, Edo State, Nigeria

Antibiotics resistance is an increasing public health challenge globally, and very recently, global attention has focused on colistin, which is termed “last resort antibiotics”. This study was aimed at investigating the prevalence of plasmid-borne mobile colistin resistant and multidrug resistant bacteria from 6 major abattoirs located in Benin City, Edo State, Nigeria. Two hundred and eighty-eight samples from fresh water, wastewater, utensils, and handlers were obtained over a 6-months period. Mean mesophilic aerobic bacteria (MAB) and thermotolerant coliform bacteria (TCB) were determined by pour plate method, while the indoor air of the abattoirs was sampled using passive sedimentation technique. Bacterial isolates were identified by morphological, biochemical and 16S rRNA analysis. Phenotypic detection of colistin-resistance as well as multi-drug resistant profile of all isolates was done by the modified Kirky Bauer method. The presence and/or absence of colistin-resistance gene (mcr-1 to mcr-8) were investigated by polymerase chain reaction. The MAB ranged from 0.3 ± 0.0 cfu/m3 in indoor air from both Holy Ghost B and Bob Izua abattoir to 2.6 ± 0.3 cfu/ml in wash water from Holy Ghost A abattoir, while the TCB ranged from 0.0 ± 0.0 cfu/ml in wastewater from Lawal and Sons abattoir to 0.6 ± 0.1 cfu/ml in wash water from Osazee abattoir. A total of 149 bacterial isolates, belonging to 6 different species (Pseudomonas aeruginosa PA01, Enterobacter ludwigii EN-119, Providencia stuartii PRV00010, Klebsiella quasipneumoniae strain KqPF26, Enterococcus saccharolyticus ATCC 43076 and Provincia rettgeri strain AR_0082) were obtained with the majority (>90%) being multidrug resistant. Seven (4.7%) of the isolates were phenotypically resistant to colistin, while only 3 harbored the mcr-1 gene. This result shows that plasmid-borne colistinresistant and multidrug resistant bacteria are prevalent in abattoir environment located in Benin City, Edo State, Nigeria. This is an indication that abattoir facilities could be a source of human exposure to colistin resistant bacteria, and efforts must be made at reducing the high dependence of antibiotics in farm animals.

Two-site study on performances of a commercially available MALDI-TOF MS-based assay for the detection of colistin resistance in Escherichia coli

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool.

Detection of Colistin Resistance in Gram Negative Pathogens: A One Year Cross-sectional Study in a Tertiary Care Centre in Northeast India

International Journal of Medicine and Public Health,2022,12,4,175-179.DOI:10.5530/ijmedph.2022.4.32Published:January 2023Type:Original ArticleDetection of Colistin Resistance in Gram Negative Pathogens: A One Year Cross-sectional Study in a Tertiary Care Centre in Northeast IndiaDeepayan Biswas, Wihiwot Valarie Lyngdoh, Sheryl Lanong, Clarissa Jane Lyngdoh, Prithwis Bhattacharyya, and Nari Mary Lyngdoh Deepayan Biswas1, Wihiwot Valarie Lyngdoh2, Sheryl Lanong2, Clarissa Jane Lyngdoh2, Prithwis Bhattacharya3, Nari Mary Lyngdoh3 1Department of Microbiology, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA 2Department of Microbiology, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA. 3Department of Anesthesiology and Critical Care, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA. Abstract:Background: Indiscriminate use of colistin for gram-negative infections has led to increase incidence of colistin resistance. The problem of nosocomial infections especially caused by multi-drug-resistant gram-negative bacteria (MDR-GNB), particularly Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae is a matter of great concern. This study was conducted to find out the prevalence of colistin resistant gram-negative isolates from patients attending outdoor patient department (OPD), those admitted in wards and Intensive care unit from a Tertiary care centre in North-East India. Materials and Methods: Clinical samples obtained were processed using standard microbiological methods. The gram-negative isolates showing colistin resistance by Kirby- Bauer’s disc diffusion method were included and further subjected for MIC (minimum inhibitory concentration) testing by VITEK-2 system followed by confirmation by Broth microdilution method. Results: Colistin resistance was observed in 26 isolates out of 1040 gram-negative isolates using Broth microdilution method. The MIC values varied from 8 to ≥ 32 μg/ml. Majority of them belong to Pseudomonas species followed by Acinetobacter species and were highly resistant to ß-lactams, aminoglycosides, fluoroquinolones. Conclusion: This study highlights an increasing trend of colistin resistance amongst multidrug resistant (MDR) gram-negative isolates warranting routine screening for colistin resistance to guide appropriate therapy for future use. Keywords:Broth microdilution, Colistin, Disc diffusion, Multidrug resistant organisms, VITEK-2View:PDF (346.91 KB)

Evaluation of Broth Disk Elution Method to Determine Colistin Resistance in Klebsiella pneumoniae and Escherichia coli Strains.

The reference broth microdilution (rBMD) method for the determination of colistin resistance is very laborious and time consuming, and many manual errors can occur. There are also limitations in detection of colistin heteroresistance. Therefore, alternative methods with satisfactory performance are required for routine laboratory work. In our study, the colistin broth disk elution (CBDE) method recommended by the Clinical and Laboratory Standards Institute (CLSI) for the detection of colistin resistance in routine applications was compared with rBMD. The compatibility and error rates of the method were evaluated and its usability in routine laboratory studies was examined. Eighty-nine multidrug resistant Klebsiella pneumoniae and five Echerichia coli strains isolated from various clinical specimens were included in the study. Identification of strains and antibiotic susceptibility tests were performed with MALDI-TOF MS (bioMerieux, France) and Vitek-2 (bioMerieux) system. Minimum inhibitory concentration (MIC) was studied in 0.125 - 128 mg/L dilution range by using polystyrene microplate and colistin sulfate salt according to ISO-standard (20776-1) recommendations for the reference BMD test. The CBDE method was performed according to the CLSI recommendations. Isolates with MIC ≤ 2 mg/L were considered susceptible, while isolates with MIC > 2 mg/L were considered resistant according to EUCAST recommendations. The performance of the CBDE method was evaluated according to ISO criteria (Categorical agreement > 90%; major error and very major error rates < 3%). Categorical agreement for all 58 and 36 isolates found to be resistant and susceptible, respectively, by rBMD was found to be 100% with CBDE test. Since < 1 and > 4 µg/mL values could not be determined with the CBDE method, essential agreement (EA) could not be calculated. No major or very major errors were detected. Our results showed that the performance of the CBDE test is good when compared to the rBMD method. According to our data, we believe that the CBDE method can be used in routine laboratories for the detection of colistin resistance.

The Emergence of Colistin- and Imipenem-Associated Multidrug Resistance in Escherichia coli Isolates from Retail Meat.

To determine the prevalence of Escherichia coli and their drug resistance profiles in fresh pork sold at two retail outlets (open-air market and closed retail stores) in Alice, South Africa. Retail meat samples (n = 176) collected from four shops (two from open-air markets and two from closed stores) were analyzed by conventional biochemical and PCR-based molecular confirmatory tests. The confirmed isolates were profiled for antimicrobial susceptibility to a panel of 12 commercial antibiotics: tetracycline, ampicillin, sulphamethoxazole trimethoprim, erythromycin, gentamycin, colistin sulphate, cefotaxime, chloramphenicol, norfloxacin, ciprofloxacin, cefuroxime, and imipenem. Colistin, ampicillin, and erythromycin resistance genes were profiled with the gene-specific primers. Multidrug resistance (MDR) and the association of imipenem and colistin in the MDR profile were determined. A total of 68 (39.08%) E. coli isolates were confirmed by PCR analysis. Resistance was most common to erythromycin (100%), followed by cefotaxime (95.58%), ampicillin (88.23%), cefuroxime (88.23%), trimethoprim-sulphamethoxazole (88.23%), and tetracycline (60.29%). Overall, 27/68 (39.70%) were MDR (≥ 3antibiotics classes). MDR E. coli isolates associated with imipenem resistance (50.00%) and colistin resistance (33.82%) were detected. The resistance genes were detected among the isolates though not in all the phenotypically resistant isolates. The detection of colistin resistance among MDR E. coli isolates from retail meat is troubling as the drug is a last resort antibiotic. Overall, the epidemiological implications of the findings are of public health importance.

Detection of colistin resistance via four methods in multidrug-resistant Gram-negative rods isolated from blood cultures.

The broth microdilution (BMD) method recommended for the detection of colistin resistance is labor-intensive, time-consuming, and difficult to apply in routine laboratories. Thus, various methods, such as disk elution, commercial microdilution, and rapid polymyxin-NP tests have been developed for the detection of colistin resistance. In this study, a total of 102 multi-resistant Gram-negative bacteria isolated from blood cultures were evaluated by four different methods for the detection of colistin resistance, and compared with the reference method. For the detection of the compatibility of these methods with the reference method, categorical and essential agreements, very major, major, and minor error rates were determined. Colistin-tigecycline and colistin-meropenem combinations were investigated in colistin-resistant isolates. Of the isolates, 15 (15%) [K. pneumoniae (n = 12), A. baumannii (n = 2), E. coli (n = 1)] were resistant to colistin with reference BMD method. MIC50 and MIC90 values of all isolates were ≤ 0.25 μg/mL and 16 μg/mL, respectively. The categorical agreement rates were 100% for commercial microdilution, disk elution, and RPNP test. The essential agreement rates of commercial microdilution, disk elution, and broth macrodilution were 78.4%, 86.3%, and 100%, respectively. Although there were no major errors in these methods, the macrodilution (12%) and commercial microdilution (20.6%) methods showed the most minor errors. Colistin-meropenem combination showed a 100% synergistic effect, but the colistin-tigecycline combination showed an 80% synergistic effect and 20% indifference effect. Disk elution and RPNP tests are suitable for routine use because they are the most efficient, easiest, low-cost, and good performance tests in detecting colistin resistance.

Colistin Resistant Gram-Negative Bacteria Isolated from Various Clinical Samples in North Indian Tertiary Care Center

&gt;Background: Antibiotic resistance and their mischaracterization often lead to treatment failure. In the present scenario, resistance to colistin is emerging. Poor testing methods for colistin resistance detection can increase the risk of treatment failures and even mortality. In this study, we analyzed the burden of multidrug-resistant (MDR), Extensively drug-resistant (XDR), pandrug resistant (PDR), and colistin resistant Gram-negative bacteria and also evaluated Vitek-2 compact and Broth Microdilution (BMD) for the detection of colistin resistance at North Indian Tertiary-Care Hospital. Methods: A total of 11237 samples were processed in which 1822 (16.21%) were gram-negative bacilli (GNB). All GNB after identification by Vitek-2 compact, were subjected to antibiotic sensitivity testing by Vitek-2 and characterized as MDR, XDR, and PDR as per guidelines of Centers for Disease Control and Prevention US (CDC) and European Centre for Disease Prevention and Control (ECDC). Identification of colistin resistance in Enterobacteriaceae family, Pseudomonas aeruginosa, Acinetobacter baumannii was made by broth microdilution (BMD) method. BMD was used as a standard gold method for colistin resistance. Results: A total of 43.57% MDR, 9.49% XDR, and 3.5% PDR gram-negative bacteria was isolated from various departments and from various sites. Vitek-2 compact detected 47 (2.75%) and MBD method detected 38 (2.23%) colistin-resistant gram-negative bacilli. Vitek-2 compact failed to detect nine colistin-resistant organisms. The highest resistance towards colistin was seen in A. baumannii (6%), followed by P. aeruginosa (5%), Escherichia coli (1.07%), Klebsiella pneumonia (1%). Conclusions: There is a need for quick and efficient antibiotic resistance management efforts against colistin resistance. Vitek-2 compact gives unsatisfactory and inaccurate results in the detection of colistin susceptibility

Performance evaluation of colistin rapid NP test for detection of colistin resistance in colistin resistant Enterobacterales

BackgroundCarbapenem-resistant Enterobacterales (CRE) have become a global threat, with colistin being the last resort for treatment. CLSI and EUCAST recommend broth microdilution (BMD) as the reference method for colistin susceptibility testing. Nordmann and Poirel have developed a simple and rapid Polymyxin NP test as an alternative to BMD for screening of colistin-resistant Enterobacterale (CoRE) isolates. This study aimed to evaluate the performance of the colistin rapid NP (CRNP) test for the detection of colistin resistance. MethodsCRNP test was performed on thirty-one clinical Enterobacterales isolates [Klebsiella pneumoniae (21), Enterobacter spp, (9) and Escherichia coli (1), which were resistant to Colistin by the reference BMD method. The results of the NP test were interpreted after 4hr by the change in color of NP solution (original orange to yellow as a result of bacterial growth) and compared with that of BMD. ResultsThe CRNP test could accurately detect 26/31 of the colistin resistant Enterobacterale isolates, and thus had an overall categorical agreement of 83.8%. Out of thirty-one CoRE isolates, five Enterobacter spp. 5/31 (16.1%) could not be detected by the CRNP test giving a false-negative result. Excluding those five Enterobacter spp. the categorical agreement (CA) of the CRNP test with BMD was 100%. The overall very major error (VME) of the CRNP test was 16% but decreased to < 3% when Enterobacter spp. (5) were excluded. ConclusionThe CRNP test is technically less demanding, yields faster results compared to the reference BMD method, and has 100% agreement with reference BMD in Enterobacterales except for Enterobacter spp.

ChromagarTM Col-Apse for Detection of Colistin Resistance in Clinical Isolates of Multidrug Resistant Gram Negative Bacilli.

To determine the diagnostic accuracy of CHROMagarTM COL-APSE, for detection of colistin resistance in clinical isolates of multi drug resistant (MDR) gram negative bacilli (GNB). Cross-sectional validation study. Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan from February 2019 to August 2019. Total 96 MDR-GNB in clinical isolates were included. Isolates were identified using gram stain, catalase, oxidase, API 20E and API 20NE. After taking approval from Institutional Ethical Review Committee, colistin susceptibility was determined simultaneously by CHROMagarTM COL-APSE (using 1x105 CFU/ml inoculum) and Broth Micro Dilution (BMD) Minimum Inhibitory Concentration (MIC) method as per CLSI. EUCAST guidelines were followed for interpretation of susceptibility profile. Results were validated with gold standard test, i.e. BMD. Out of 96 MDR clinical isolates, the distribution was K. pneumoniae n=63, E. coli n=18, A. baumannii n=11, C. freundii n=3, and E. cloacae n=1. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of CHROMagarTM COL-APSE for detection of colistin resistance, keeping BMD-MIC method as gold standard, was 97.96%, 97.87%, 97.96%, 97.87% and 97.92%, respectively. CHROMagarTM COL-APSE can be used as screening agar by direct streaking of specimen as well as diagnostic for detection of colistin resistance by the use of standardised inoculum. It can be used as a critical time saving method for colistin resistance detection in absence of expertise or manpower required for BMD as well as the cost required for genetic sequencing to detect MCR genes. Key Words: Multi drug resistant (MDR), Gram negative bacilli (GNB), Colistin resistance, CHROMagarTM COL-APSE.

COLISTIN AGAR; EVALUATION OF A NOVEL DIAGNOSTIC APPROACH TO DETECTION OF COLISTIN RESISTANCE

Objective: To determine the diagnostic accuracy of Colistin agar for detection of Colistin resistance in clinical isolates of MultiDrug Resistant Gram-Negative Bacilli.&#x0D; Study Design: Cross-sectional validation study.&#x0D; Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Feb to Aug 2019.&#x0D; Methodology: A total of 100 Multi-Drug Resistant Gram-Negative Bacilli in clinical isolates were included. Isolates were identified using Gram stain, Catalase, Oxidase, API 20E, and API 20NE. After approval from the institutional ethical review committee, Colistin susceptibility was determined simultaneously by Colistin agar and Broth Micro Dilution Minimum Inhibitory Concentration method as per CLSI. For susceptibility criteria, EUCAST guidelines were followed. Results were validated with the gold standard test, i.e., Broth Micro Dilution.&#x0D; Results: Out of 100 Multi-Drug Resistant clinical isolates, the distribution was K. pneumoniae n=60, E.coli n=16, A. baumannii n=11, C. freundii n=8, and E. cloacae n=5. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of Colistin agar for detection of Colistin resistance, keeping Broth Micro Dilution Minimum Inhibitory Concentration method as the gold standard was 96.67%, 97.14%, 93.55%, 98.55%, and 97%, respectively.&#x0D; Conclusion: Colistin agar has excellent diagnostic accuracy for the detection of colistin resistance with standardized inoculum density. Due to its ease of use, cost-effectiveness, and accurate results, it can be used in lab setups deficient in manpower and advanced equipment for Broth Micro Dilution or genetic sequencing.

Assessment of colistin resistance in Gram negative bacteria from clinical samples in resource-limited settings

Objective: To find a proper method to assess colistin resistance in multidrug resistant Gram negative bacteria (MDR-GNB) on a routine basis in resource limited settings.Methods: Clinical samples were processed. MDR-GNB were identified and were examined for colistin resistance by colistin broth elution method, colistin agar method, and colistin disk elution screening method. Broth microdilution method was used the gold standard.Results: A total of 10 235 clinical samples were processed, in which 857 (8.4%) MDR-GNB were identified. The very significant errors, categorical agreement, major errors, positive predictive values, negative predictive values, specificity and sensitivity of all the phenotypic methods were 5.5%, 0%, 94.4%, 100%, 99.6%, 100% and 94.4%, respectively for the detection of colistin resistance. The colistin elution screening method was cheap and easy to perform with similar results to broth microdilution method.Conclusions: All the evaluation methods for colistin resistance showed similar results. So the laboratories can choose any method for detection of colistin resistance. However, we recommend colistin disk elution screening method because, it is easy and cheap and can be performed in limited resources.

Evaluation of Vitek2 and Colistin broth disk elution test in comparison with Micro broth dilution for Susceptibility testing of Colistin among Carbapenem Resistant Enterobacterales.

Colistin is the last expedient for treating the infections caused by Carbapenem resistant Enterobacter ales (CRE).Expanding usage of colistin as led to development of Colistin resistance. This urges for a need of reliable and accuratetesting method that can be adopted as routine for susceptibility testing of Colistin. The innate cationic property ofcolistin molecules is associated with complexities. Hence evaluation of testing method is needed for detection of colistinresistance. Present study is done to evaluate the results yielding from Vitek2 and CBDE in comparison with rBMD. About200 CRE isolated from clinical specimens were tested, results of Vitek2, CBDE was compared with rBMD. In comparisonwith BMD Categorical agreement (CA) of Vitec 2 was 83% with major error of 17% .CA of CBDE was 99% with 1% of verymajor error. Since the resistance to colistin is increasing accurate reporting of Colistin MIC by a validated method becomesimportant. So, we would recommend CBDE for routine testing of Colistin susceptibility.

DETECTION OF COLISTIN RESISTANCE IN MULTIDRUG-RESISTANT GRAM-NEGATIVE BACILLI: PERFORMANCE OF THE BD PHOENIX AUTOMATED SYSTEM VERSUS THE BROTH MICRODILUTION METHOD

Introduction: The increased dissemination of multidrug-resistant Gram-negative bacilli along with a lag in the development of novel, broad-spectrum antimicrobials determined the reintroduction of colistin into clinical practice. In this context, antibiotic susceptibility testing for colistin using high-performance methods has become a requirement. Objective: We compared the performances of the Becton Dickinson (BD) Phoenix 50 automated system and the Micronaut MIC-Strip, a commercial broth microdilution (BMD) method, for the detection of colistin resistance in clinical multidrug-resistant Gram-negative bacilli, isolated from patients admitted in a tertiary hospital in southern Romania. Methods: 54 clinical isolates with different multidrug-resistant phenotypes (Enterobacterales and Pseudomonas aeruginosa) were tested for colistin susceptibility with the BD Phoenix 50 automated system and the Micronaut MIC-Strip, following the Clinical Laboratory Standard Institute recommendations (CLSI, 2021) and were further classified according to the interpretative categories (resistant and intermediate, respectively). The statistical data was calculated using Microsoft Excel 2007 and OpenEPI software. Results: BD Phoenix system failed to detect 11 colistin resistant isolates (false susceptibility). By contrast, all 38 clinical isolates defined as intermediate by the BMD method were also classified as intermediate by the Phoenix system. Conclusion: BD Phoenix system is a reliable technology for detecting colistin resistance. However, high rates of false susceptibility were observed for the BD Phoenix system, indicating that the intermediate / susceptible results should be confirmed with the standard BMD method. Keywords: colistin susceptibility testing, Gram-negative bacilli, multidrug resistance.

Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS.

BackgroundEven though both EUCAST and CLSI consider broth microdilution (BMD) as the reference method for antimicrobial susceptibility testing (AST) of colistin, the method exhibits potential flaws related to properties of the colistin molecule.ObjectivesTo develop a flow cytometry method (FCM) for colistin AST and to validate it against BMD, a commercial screening test and WGS.MethodsColistin-mediated loss of membrane integrity in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. was detected with the fluorescent probe YoPro-1 by FCM. An international collection of 65 resistant and 109 susceptible isolates were analysed and the colistin concentration required to reach the EC50 was compared with the BMD MIC and the presence of genotypic resistance markers.ResultsThe overall FCM sensitivity and specificity for colistin resistance was 89% and 94%, with E. coli > K. pneumoniae > P. aeruginosa, whereas the performance for Acinetobacter spp. was poor. All tested E. coli were correctly categorized. Three K. pneumoniae isolates with genotypic findings consistent with colistin resistance were detected by FCM but not BMD. Compared with BMD, FCM delivered AST results with a 75% reduction of time.ConclusionsHere, we present a rapid FCM-based AST assay for qualitative and quantitative testing of colistin resistance in E. coli and K. pneumoniae. The assay revealed probable chromosomal colistin resistance in K. pneumoniae that was not detected by BMD. If confirmed, these results question the reliability of BMD for colistin testing.