Colistin Susceptibility Testing Research Articles

Comparative evaluation of broth microdilution, E-strip and Vitek-2 for colistin susceptibility among carbapenem resistant Acinetobacter Spp and Klebsiella Spp

Gram-negative bacterial infections that are resistant to carbapenems are a serious clinical problem. The only effective drugs against them are still tigecycline and colistin. In fact, polymyxin E, also known as colistin, has been regarded as a "last resort" antibiotic. But in recent years, colistin resistance has increased globally, significantly reducing treatment options and highlighting the significance of accurate colistin testing methods to support appropriate therapeutic decision-making. There is no requirement to cite reference in the abstract. So it is suggested to rephrase the sentence. The primary goal of the study is to compare the effectiveness of E-test and Vitek 2 for colistin sensitivity testing to the conventional microbroth dilution method using 120 clinical isolates of and that are resistant to carbapenem over the course of a year, isolated from January to December 2019. In comparison to Vitek-2, the results indicated that brothmicro dilution and E-test produced the narrowest range of colistin MICs. After comparing the E-test's errors and overall agreement with BMD, 100% of the Essential Agreement, 94.27% of the Categorical Agreement, and 5.72% of the Very Major Errors with no Major Errors were found. Comparing Vitek-2 with BMD, similar results were found: 1.66% MEs, 98.32% CAs, 80.99% EAs, and no VMEs. As a result, it was determined that, in contrast to Vitek-2, MBD and E-test had distinct MICs, making them appropriate for determining colistin MIC. It will take more research on automated systems to standardize colistin susceptibility testing procedures, particularly for and .

Evaluation of the Colistin HiMIC Plate Kit for Colistin Susceptibility Testing of Klebsiella pneumoniae.

Colistin HiMIC Plate Kit (HiMedia Laboratories), a new commercial broth microdilution (BMD) test for colistin susceptibility testing was evaluated. BMD according to ISO standard 20776-1 (2019) with two-fold dilutions from 128 to 0.125 mg/L was used as a reference method. The colistin reference MICs (minimal inhibitory concentration) ranged from 0,25 to 128 mg/L with 15 (20.5%; 15/73) isolates having colistin reference MICs close to the current EUCAST breakpoint (MICs of 2, 4, and 8 mg/L). The study assessed the compliance of a commercial kit with the CLSI criteria, including categorical agreement (CA) and essential agreement (EA ≥90%), very major error (VME rate) <3%, and major error (ME) rate <3%. On 73 carbapenemase-producing Klebsiella pneumoniae isolates Colistin HiMICTM Plate Kit showed CA and EA of 100% (73/73; 95% CI: 0.97-1.00) and 82.2% (60/73; 95% CI: 0.72-0.90), respectively. No ME (false-resistant results) and VME (false-susceptible results) were detected. Kit showed acceptable CA, ME, and VME error parameters, whereas the EA did not meet the ≥90% threshold. Laboratories must check for possible limitations of commercial kits before they can be used for colistin susceptibility testing.

LABORATORY DETECTION OF COLISTIN-RESISTANT &lt;i&gt;ENTEROBACTERALES&lt;/i&gt; IN TANDEM WITH ROUTINE ANTIBIOGRAM

Since the emergence and spread of carbapenemase-producing Enterobacterales, particularly those carrying metallo-β-lactamases with 16S rRNA methyltransferases for which newly introduced antibiotics are inactive, colistin is a last resort therapy for life-threatening extensively drug-resistant infections. This requires that laboratories use an accurate and reliable method for routine colistin susceptibility testing. The aim of this study was to evaluate the performance and applicability of a colistin screening medium for detection of colistin-resistant isolates simultaneously with routine susceptibility testing. It was evaluated with fifty colistin-resistant and the same number of colistin-susceptible comparator isolates. Our results showed that all colistin-resistant isolates grew on DiaPlate™ EMB Agar + Colistin medium within the standard antibiogram period. In contrast, no growth was observed among the colistin susceptible comparators. DiaPlate™ EMB Agar + Colistin is a screening medium that can detect colistin- resistant Enterobacterales isolates when pure bacterial cultures are tested. As this method for detecting colistin-resistant isolates uses the same inoculum as the Kirby-Bauer method, the screening test for colistin resistance can be conveniently performed on clinical isolates along with routine antimicrobial susceptibility testing.

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.

Detection of Colistin Resistant Enterobacteriaceae among Backyard Farm Swine and Swine Raisers in Tanay, Rizal Swine Farm

Addressing difficulties in susceptibility testing of colistin and the emergence of colistin-resistant Enterobacteriaceae became a huge burden to medical practitioners due to the complexity of the method and interpretation of the minimum inhibitory concentration of antibiotics. These circumstances are not only rampant among human infection but also in the animal farm industry – particularly, the swine farms, which have the greatest number of cases of Enterobacteriaceae infection. This study aims to determine the prevalence of colistin-resistant Enterobacteriaceae from 40 fecal samples of backyard farm swine (30) and swine raisers (10) in Tanay, Rizal, the Philippines via culture, biochemical testing, and antimicrobial susceptibility testing for colistin. Enterobacteriaceae from fecal samples were isolated, singly picked, and subjected to biochemical tests. Susceptibility testing was performed to determine the minimum inhibitory concentration. The isolates recovered from fecal samples of backyard swine were Escherichia coli (77%), Salmonella species (16%), Citrobacter freundii (7%), and two non-Enterobacteriaceae Pseudomonas species, whereas all fecal samples from swine raisers were positive for E. coli (100%). Moreover, 13.04% of E. coli isolates from backyard farm swine exhibited resistance to colistin with a minimum inhibitory concentration of 4ug/mL, following the international guidelines for interpretation of colistin resistance, whereas the rest of the isolates were susceptible to colistin. E. coli, Salmonella, and C. freundii were present in fecal samples of backyard swine and three E. coli were resistant to colistin. There is a 13.04% prevalence of colistin-resistant E. coli isolated from pooled fecal samples of a backyard swine farm in Tanay, Rizal, the Philippines. The increasing cases of colistin resistance should be strictly monitored in animal farms to come up with a robust solution to fight against antimicrobial resistance.

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.