Colistin Susceptibility Testing Research Articles

Comparative Evaluation of Colistin-Susceptibility Testing in Carbapenem-Resistant Klebsiella pneumoniae Using VITEK, Colistin Broth Disc Elution, and Colistin Broth Microdilution.

The study aimed to compare the results of colistin-susceptibility testing performed using the automated VITEK system, colistin broth microdilution (BMD), and colistin broth disk elution (CBDE) methods. This exploratory study was conducted in a tertiary care center in South India. Carbapenem-resistant Klebsiella pneumoniae (n = 49) isolates collected from a clinical microbiology laboratory over six months (March-September 2023) were used for the study. Among the 49 carbapenem-resistant Klebsiella pneumoniae isolates, 42 were found to be susceptible to carbapenem by all three methods. Seven isolates were found to be resistant to colistin using BMD and CBDE methods. Two isolates were incorrectly detected as colistin-susceptible, and one isolate was wrongly categorized as colistin-resistant using the automated VITEK system. CBDE is a reliable and cost-effective method that can be adopted in the routine microbiology laboratory for colistin-susceptibility testing, as it does not require any specialized equipment or techniques and is 100% consistent with the gold standard BMD method. Although the automated VITEK system is used in most routine microbiological laboratories for antibiotic-susceptibility testing, it cannot be reliably used for colistin-susceptibility testing due to its high error rates (very major error rate of 28.5%; major error rate of 2.4%).

Colistin, the last resort antibiotic: challenges in the implementation of its routine susceptibility testing

Background: colistin has become a critical antibiotic for lifethreatening multidrug resistance Gram-negative infections, particularly carbapenemase-producing bacteria. Detecting colistin resistance in routine microbiology laboratories is crucial for combating these fatal infections poses a challenge. Especially in developing countries, there is a need for a cost-effective, rapid, and user-friendly diagnostic method. Objective: implementing the various available methods for colistin testing is a significant challenge in resource-limited settings due to logistic difficulties and the need for technical expertise. Materials and Methods: this study shares experiences and insights gained while implementing in-vitro colistin susceptibility testing in a high-load bacteriology laboratory of a tertiary care center in Delhi, India. The following test methods for colistin susceptibility testing were incorporated in the routine antimicrobial susceptibility testing of our laboratory: Colistin Agar Test, Colistin Broth Disk Elution Test, Broth Microdilution susceptibility testing. Results: inconsistent growth patterns were observed in the colistin agar dilution Minimum Inhibitory Concentration (MIC) method, which could be resolved only after the preparation of fresh plates containing that specific concentration of colistin. The contamination issue of plates on use over a few days was addressed by pouring agar containing various concentrations of colistin in cottonplugged glass tubes. In the colistin broth disk elution test, due to the non-availability of screw-capped 10 mL glass tubes, MacCornety bottles (30 mL) were used. Subcultures were performed from the turbid wells to rule out the growth of contaminants when encountering discordant MIC values or skipped wells on the colistin broth microdilution test. Conclusions: despite several technical issues in in-vitro colistin susceptibility testing, we have successfully implemented it in our laboratory. Our experiences can offer guidance to laboratories that are still in the process of implementing it.

Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study

BackgroundIn Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. MethodsBetween September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 μg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. ResultsA total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 μg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 μg/ml, 12/15 were mcr-1. ConclusionsWe found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Comparison of Colistin Susceptibility Tests

Introduction: Polymyxins are important antimicrobial agents for the treatment of infections caused by Gram-negative bacteria. The susceptibility testing for polymyxins is a challenge for clinical laboratories due to the difficulty of performance, reproducibility, and accuracy of available methods. Aim: To compare the performance of the colistin susceptibility test of an automated system and a gradient test with the gold standard broth microdilution method (BMD). Materials and Methods: Multidrug-resistant isolates of Acinetobacter baumannii (n=102), Klebsiella pneumoniae (n=40), and Pseudomonas aeruginosa(n=11) were included. The VITEK 2 systems and gradient test were studied according to the manufacturer’s instructions. Broth microdilution tests were performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Commercial susceptibility testing methods were compared to BMD. Results: Rates of essential agreement of colistin test results between BMD, VITEK 2, and gradient test were 96.1% and 79.7%, respectively. The VITEK 2 and gradient test showed 95.4% and 94.8% of categorial agreement. The very major error rate of VITEK 2 was 3.2%, and the gradient test was 5.2%. The major error rate of VITEK 2 was 1.3%, and there was no major error for the gradient test. Conclusion and Suggestions: The very major error rate was higher in the gradient test (5.2%) than VITEK 2 (3.2%). Even if the very major error rate of VITEK 2 was lower, both resistance and susceptility results of VITEK 2 should be confirmed with the BMD test. Further studies for susceptibility testing are needed with a focus on the correlation of MIC’s results of different tests.

Comparative Evaluation of Broth Microdilution With Disc Diffusion and VITEK 2 for Susceptibility Testing of Colistin on Multidrug-Resistant Gram-Negative Bacteria.

Background The rise of antibiotic resistance, particularly in Gram-negative bacteria, poses a significant global health threat. Colistin, a last-resort antibiotic, has witnessed renewed use. However, accurate susceptibility testing for colistin is challenging, with various methods available, leading to potential discrepancies. Ensuring reliable testing is crucial for effective patient treatment and antimicrobial stewardship. This study addresses the need to compare different colistin susceptibility testing methods, providing insights into their accuracy and relevance in clinical settings. Methods In this one-year prospective observational cross-sectional study conducted atIndira Gandhi Institute of Medical Sciences (IGIMS), Bihar, India, atertiary care hospital from July 2021 to June 2022, we aimed to evaluate the concordance between two widely used methods, VITEK 2 and Disc Diffusion, for antibiotic susceptibility testing in clinical multidrug-resistant Gram-negative bacterial isolates. These isolates, including species like Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter freundii, and Escherichia coli, were isolated from various clinical specimens. After rigorous species-level identification and quality control measures, antibiotic susceptibility testing was performed using both methods, and their agreement was assessed through Percentage Agreement analysis. Results In our study, we isolated and identified bacterial isolates from 105 patients, with a mean age of 47.30 years, demonstrating a wide age range. Pus samples were the most common type (25.7%), and K. pneumoniae was the most prevalent organism (45.7%). Antibiotic resistance patterns revealed significant challenges in treating infections caused by K. pneumoniae and A. baumannii, with resistance rates exceeding 70% for certain antibiotics. Among the 48 isolates of K. pneumoniae, the agreement was 93.8%, with 89.6% being sensitive and 6.3% being resistant by Disc Diffusion, while VITEK 2 indicated 0% resistance. E. coli isolates (n=21) had an agreement of 90.5%, with 90.5% sensitivity and 9.5% resistance by Disc Diffusion, and no resistance by VITEK 2. Conclusion The comparative analysis of antibiotic susceptibility testing methods reveals the superior performance of the VITEK 2 system, particularly in sensitivity and negative predictive value, emphasizing its potential as a reliable tool for guiding antibiotic therapy decisions.

Investigation of carbapenem-resistant Enterobacterales isolates at a tertiary laboratory in Pretoria, South Africa.

This study aimed to investigate phenotypic and genotypic characteristics of carbapenem-resistant Enterobacterales isolates (CRE) at a tertiary laboratory in South Africa. A total of 99 CRE isolates were collected between 2019 and 2021. Carbapenemase production was tested using modified carbapenem inhibitory method. Colistin susceptibility testing was performed using the ComASP™ Colistin broth microdilution method. Conventional PCR assays were conducted for detection of mcr-1 gene and common carbapenemase genes (blaVIM, blaNDM, blaIMP, blaKPC, blaOXA-23, blaOXA-51 and blaOXA-48). Rep-PCR assay was conducted to determine the genetic relatedness of the study isolates. Majority of the isolates were Klebsiella pneumoniae (83%). Carbapenem resistant K. pneumoniae cluster was observed from ICU and surgical ward samples. Colistin resistance was observed in 13% (12/93) of the isolates namely, in 11 K. pneumoniae and one Enterobacter cloacae. The blaOXA-48 (65%) was the most prevalent gene detected followed by blaNDM (25%) and blaVIM (22%). Several K. pneumoniae isolates concomitantly carried multiple carbapenemase genes with one isolate carry up to 5 five genes blaVIM, blaNDM, blaOXA-48, blaOXA-23 and blaOXA-51. The mcr-1 gene was not detected in the isolates. Rep-PCR assay showed that most isolates matched cluster A (50%). The high prevalence of blaOXA-48, blaNDM and emerging colistin resistant isolates is of concern for patient management at this institution and needs close monitoring. Rep-PCR is a valuable tool in establishing infection clusters in resource-limited settings.

Assessment of in vitro colistin susceptibility of carbapenem-resistant clinical Gram-negative bacterial isolates using four commercially available systems & Whole-genome sequencing: A diagnostic accuracy study

AimTo analyze the diagnostic utility of commercially available platforms and Whole-genome sequencing (WGS) for accurate determination of colistin susceptibility test results. Material & methodsAn exploratory diagnostic accuracy study was conducted in which sixty carbapenem-resistant Gram-negative bacteria were subjected to identification and AST using MALDI-TOF MS & MicroScan walkaway 96 Plus. Additional AST was performed using the BD Phoenix system and Mikrolatest colistin kit. The test isolates were subjected to Vitek-2 and WGS at CRL, Bengaluru. ResultsThere was no statistically significant agreement between the colistin susceptibility results obtained by WGS, with those of commercial phenotypic platforms. The MicroScan 96 Plus had the highest sensitivity (31 %) & NPV (77 %), and the BD Phoenix system had the highest specificity (97 %) and PPV (50 %), respectively, for determining colistin resistance. ConclusionThe utility of WGS as a tool in AMR surveillance and validation of phenotypic AST methods should be explored further.

Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli.

The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods - colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen's kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2-84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.

Colistin Susceptibility Testing by Colistin Broth Disk Elution MIC Method among Carbapenem-resistant Gram-negative Blood Culture Clinical Isolates in a Tertiary Care Setting, East Delhi, India.

Antibiotic resistance is a growing concern for bloodstream infections (BSIs), especially with the emergence of multidrug-resistant (MDR) gram-negative bacteria. In this study, we aimed to assess the pattern of colistin susceptibility using the colistin broth disc elution (CBDE) method among carbapenem-resistant gram-negative clinical isolates from blood cultures in a high burden tertiary healthcare setting in East Delhi. A total of 106 carbapenem-resistant gram-negative clinical isolates were tested. The most common isolates were Klebsiella pneumoniae, Escherichia coli, Enterobacter species, and Klebsiella oxytoca by CBDE method. All the carbapenem resistant gram-negative bacterial blood culture isolates showed intermediate colistin susceptibility. This was statistically significant by chi-square test (p<0.5). This study highlights the need to monitor colistin resistance trends in the face of increasing antimicrobial resistance. Accurate surveillance of emerging colistin resistance is crucial for effective management of BSIs caused by carbapenem-resistant gram-negative bacteria.

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.

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.

Antimicrobial susceptibility testing and reporting practices of public hospital microbiology laboratories in Greece, 2022: A national observational survey and call for action.

Antimicrobial resistance (AMR) in Greece is among the highest across the European Union/European Economic Area (EU/EEA), with high AMR rates even to last-line antibiotics. To better understand the clinical microbiology laboratory practices and capacities in species identification and antimicrobial susceptibility testing (AST) across public healthcare establishments in Greece, we sent a questionnaire to 98 of 128 public hospital microbiology laboratories between 1 February and 1 April 2022. Of the 73.5% (72/98) that responded, 51.4% (37/72) reported using EUCAST guidelines. Two of three laboratories used an automated instrument for species identification and AST for all laboratory samples. Broth microdilution for colistin susceptibility testing was used by 46 of the laboratories, more frequently in larger (> 400 beds) versus smaller (< 400 beds) hospitals (90.5% (19/21) vs 52.9% (27/51) respectively, p = 0.011). MALDI-TOF mass spectrometry was available in one of 10 laboratories, and more often in larger compared to smaller hospitals (p = 0.035). Although the majority of laboratories had a laboratory information system (LIS) in place, only half had the capacity to extract data directly from the LIS for the purpose of AMR surveillance; 73.6% (53/72) used restrictive antibiograms. Public microbiology laboratory AMR capacities in Greece require improvement, prioritising interventions for EUCAST guidelines implementation.

Comparative Evaluation of Colistin Susceptibility Testing Using Agar Dilution and Broth Microdilution in Multidrug-resistant and Extensively Drug-resistant Gram-Negative Isolates

Comparative Evaluation of Colistin Susceptibility Testing Using Agar Dilution and Broth Microdilution in Multidrug-resistant and Extensively Drug-resistant Gram-Negative Isolates

Antibiotic susceptibility of Pseudomonas aeruginosa isolated at 108 Military Central Hospital from 01/2020 to 06/2022

Objective: To determine antibiotic susceptibility patterns of Pseudomonas aeruginosa isolated at the 108 Military Central Hospital from January 2020 to June 2022. Subject and method: A total of 784 Pseudomonas aeruginosa strains were isolated from patient specimens from different departments. Bacteria identification and antibiotic susceptibility test were performed using the Vitek MS system and the Vitek-2 Compact system, respectively. Multidrug-resistant and/or carbapenem-resistant Pseudomonas aeruginosa (MDR/CPR P. aeruginosa) strains were determined. Colistin susceptibility test was done using cation-modified Muller-Hinton broth diffusion method (CBDE, colistin broth disk elution). Result: Pseudomonas aeruginosa was highly resisting to fluoroquinolones (62.8%), and aminoglycosides (53.4%). The proportion of MDR/CPR P. aeruginosa was 54.3% (426/784), of which the highest proportion was found in the Department of Infectious Diseases (65.52%), ICU (64.39%) and the Internal Respiratory Department (45.69%). Carbapenem-resistant Pseudomonas aeruginosa was highly prevalent in urine specimens (65.38%) and respiratory specimens (56.76%). Of 258 MDR/CPR P. aeruginosa isolates, 11.2% isolates were resistant to colistin, 26.6% and 33.3% of these remained susceptible to amikacin and piperacillin/tazobactam, respectively. Conclusion: The prevalence of antibiotic resistante Pseudomonas aeruginosa remains high, including resistance to carbapenem and colistin. Antibiotic regimen should include amikacin or piperacillin/tazobactam in combination with other antibiotics. The study suggested the routine application of diagnostic tools for the rapid detection of resistant strains.

Genomic characterization of colistin resistance in Klebsiella spp. under the pressure of colistin.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml-1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml-1), and a novel mutation C28R in mgrB was first reported in this study.Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

Evaluation of in-vitro colistin susceptibility and clinical profile of carbapenem resistant Enterobacteriaceae related invasive infections

PurposeTo determine the colistin susceptibility. To compare E-test vs broth-microdilution (BMD) method for invasive carbapenem resistant Enterobacteriaceae (CRE) infections. To study treatment options for the CRE. To analyze the clinical profile and outcome of CRE infections. MethodsAntimicrobial susceptibility testing was performed for 100 invasive CRE isolates. Gradient diffusion and BMD methods were performed to determine colistin MICs. Essential agreement (EA), categorical agreement (CA), very major error (VME), and major error (ME) were worked out between BMD method and E-test. The clinical profile of patients was analyzed. ResultsThe majority of the patients suffered from bacteremia [47(47%)]. Klebsiella pneumoniae was the most common organism isolated overall as well as among bacteremic isolates. 9(9%) CRE isolates were colistin resistant by BMD of which six were Klebsiella pneumoniae. There was 97% CA between E-test and BMD. EA was 68%. VME was found in three out of nine colistin resistant isolates. No ME was found. Among the other antibiotics tested for CRE isolates, the highest susceptibility was seen to tigecycline [43(43%)] followed by amikacin [19 (19%)]. The most common underlying condition was post solid organ transplantation [36(36%)]. A higher survival rate was seen among non-bacteremic CRE infections (58.49%) than bacteremic CRE infections (42.6%). Four out of nine patients with colistin resistant CRE infections survived and had a satisfactory outcome. ConclusionKlebsiella pneumoniae was the most common organism causing invasive infection. Survival rates were higher in non-bacteremic CRE infections than bacteremic infections. Good CA was seen between E-test and BMD for colistin susceptibility, but the EA was poor. VME was more common than ME when E-tests were used for colistin susceptibility testing resulting in false susceptibility. Tigecycline and aminoglycosides are possible adjunct drugs for the treatment of invasive CRE infections.

Evaluating the drop test method in measuring colistin susceptibility of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa.

Introduction. Colistin is an antibiotic used to treat Gram-negative bacterial infections, particularly those caused by multidrug-resistant bacteria.Hypothesis/Gap Statement. The broth microdilution (BMD) reference method is the recommended protocol for detecting colistin susceptibility; however, it is laborious and expensive and cannot be performed in all laboratories.Aim. To evaluate the colistin susceptibility in Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa using an alternative method, which has been referred to as the drop test.Methodology. A 16 µg ml-1 colistin solution was deposited on a Mueller-Hinton agar plate previously swabbed with the strain and incubated overnight, and the presence or absence of an inhibition zone was observed.Results. The categorical agreement (CA) of the drop test with respect to BMD was 100 % when 190 Enterobacterales (19 E. coli and 171 K. pneumoniae) were used, and no major errors (MEs) or very major errors (VMEs) were detected. The CA of the drop test with respect to the BMD was 99.2 % for 119 P. aeruginosa isolates, while no ME was detected and only 1 VME (6.7%) was observed.Conclusion. The drop test is an alternative method for antimicrobial susceptibility testing of colistin against K. pneumoniae and E. coli. It is an adequate method for detecting resistant strains of P. aeruginosa, but susceptible isolates should be confirmed using BMD. The drop test is a simpler alternative to the BMD that does not require additional equipment and allows for the testing of numerous isolates in a short period of time.

1824. Combination therapy of daptomycin/colistin for extreme drug resistant Acinetobacter baumannii bacteremia

Abstract Background There are experimental data demonstrating synergistic in-vitro activity of colistin and daptomycin against extremely drug-resistant (XDR) strains of Acinetobacter baumannii.Aim of this study is the description of a case-series of patients with confirmed XDR A. baumannii bloodstream infection (BSI) receiving an antimicrobial regimen including colistin and daptomycin. Methods Prospective study of patients with BSI caused by XDR A. baumannii from May 2021 to February 2022 in a tertiary level hospital. Demographic, clinical and laboratory data were recorded and evaluated from all patients. BacT Alert system (BiomerieuxR) was used for blood cultures and bacterial identification and susceptibility testing was performed using VITEK® 2. Colistin susceptibility testing (dilution method) were interpreted in accordance to Clinical and Laboratory Standards Institute (CLSI) criteria. Results A total of 16 episodes of XDR A. baumannii were recorded in 16 patients (14 males). Median age was 60 (range 38-85) years. Ten patients (56.2%) were hospitalized in medical wards, and 43.8% in surgery wards. One-third of patients had previously been admitted to the Intensive Care Unit, while all patients had an indwelling central vascular catheter. Fourteen of the 16 XDR strains of A. baumannii were also resistant to colistin. Also, 2 of the 16 strains had a MIC of 2μg/ml for tigecycline, while in the remaining 14 the MIC was &amp;gt; 2μg/ml. All isolates had meropenem MIC &amp;gt;16μg/ml. All patients received a combination of colistin -daptomycin -meropenem -tigecycline. Clinical and laboratory improvement was noted in 15 of the 16 patients within the 14 days of treatment. Fifteen out of the 16 patients showed a microbiological response: the mean duration between the start of treatment and the first negative blood culture was 12 days.Finally, no patient experienced a marked increase in CPK due to daptomycin use. Conclusion The outcome of this case series of patients receiving colistin-daptomycin combination as part of a broader synergistic antimicrobial regimen to treat XDR A. baumannii was satisfactory, with no significant adverse reactions. This potential clinical benefit of the colistin/daptomycin combination should be further evaluated in a larger comparative study. Disclosures All Authors: No reported disclosures.

Comparative evaluation of colistin susceptibility testing by Disk Diffusion and Broth Microdilution methods: An experience from a tertiary care hospital.

Recent times have seen uninhibited use of colistin due to emergence of carbapenem resistant gram-negative bacteria especially in India and the most common method still employed by most of the laboratories for in vitro testing of colistin is disk diffusion method. The study aimed to compare two methods for colistin susceptibility testing Methods: This retrospective observational study was done on a total of 212 non-duplicate carbapenem resistant gram-negative isolates from patients attending our tertiary care hospital from April 2019 to June 2020. Colistin susceptibility testing for these isolates was done by disk diffusion method followed by broth microdilution method. We found out that disk diffusion method showed relatively low sensitivity [34.5%; 95%CI: 19.94-52.65] but high specificity [99.45%; 95% CI: 96.97 - 99.90] in detecting colistin resistant organisms in comparison to broth microdilution method. Disk diffusion method is an unreliable method at detecting colistin resistance. Therefore, we should always rely on susceptibility testing by standard broth microdilution or newly introduced broth disk elution method before dispatching the report even in resource limited settings. The early and accurate reporting of susceptibility results can preserve the therapeutic value of the drug until we have newer treatment options available in the country.