Colistin Susceptibility Testing Research Articles

Genomic Characterization of ESBL- and Carbapenemase-Positive Enterobacteriaceae Co-harboring mcr-9 in Japan.

Worldwide spread of Enterobacteriaceae resistant to colistin, a polypeptide antibacterial drug for last-resort treatment of carbapenemase-producing Enterobacteriaceae (CPE) infections, is concerning. This study aimed to elucidate colistin MICs and molecular characteristics of mcr-1 to mcr-9 of ESBL-producing Escherichia coli (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring mcr genes (especially mcr-9). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting mcr-1 to mcr-9 were performed for all strains with S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridization, and whole-genome sequencing (WGS) with hybrid assembly performed for mcr gene-carrying strains. Two CPE strains showed a MIC ≥ 4 μg/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three mcr-9-carrying strains showing colistin susceptibility. The blaCTX–M–62-positive E. coli THUN648 strain simultaneously carried blaCTX–M–62 and mcr-9 on a 275-kbp plasmid. Besides, blaIMP–6 + blaCTX–M–2-positive Klebsiella pneumoniae THUN262 and blaGES–24-positive Enterobacter kobei THUN627 had mcr-9 encoded on the chromosome. Only THUN627 encoded qseB/C, which is suggested to be a regulatory gene for mcr-9, downstream of mcr-9. However, this strain showed no increased expression of these genes in mRNA quantitative analysis under colistin exposure. Colistin MICs of ESBL-Ec and CPE in Japan were all below 2 μg/ml, which is below the epidemiological cutoff (ECOFF) value (https://eucast.org/) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither “microbiological” nor “clinical” resistance. Several colistin-susceptible Enterobacteriaceae carrying silent mcr-9 encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by mcr-9 remains unclear.

Comparing Quantitative and Qualitative Methods for Detecting the In Vitro Activity of Colistin against Different Gram-Negative Bacilli

The surge in the prevalence of Multidrug-Resistant (MDR) Gram-negative bacterial infections with limited treatment led to colistin reusing to treat MDR infections. This study aimed to determine economical, simple, and reliable colistin susceptibility testing methods as an alternative to the microdilution technique. We compared seven colistin susceptibility testing methods, including quantitative and qualitative, namely: Disk diffusion, E-test, ComASPTM SensiTest Colistin, Colistin broth disk elution, and colistin agar test CHROMagarTM COL-APSE, and BD Phoenix ID/AST automated identification and susceptibility testing system to the gold standard Broth Microdilution (BMD). Whole-genome sequencing was performed on all isolates to determine if the genetic resistant factors affect the phenotypic profile of the colistin resistance. Our results revealed that disk diffusion is still an ineffective method for measuring colistin susceptibility in Gram-negative Bacilli with the highest major error (31.75%), the lowest Kappa 0 (0%), and categorical agreement (68.25%) values. Phoenix, and CompASPTM SensiTest colistin methods have remained superior in reproducibility, sturdiness, and simplicity of use, similar to the currently recommended broth microdilution procedure; with high sensitivity of 95.56%, and 97.73%, specificity of 95.24, and 100%, and Kappa values of 0.89 and 0.95, respectively. This study revealed that Phoenix, and ComASPTM SensiTest colistin methods are recommended for routine microbiology laboratories with a large workload.

Detection of Colistin Resistance in Carbapenem Resistant Enterobacteriaceae by Reference Broth Microdilution and Comparative Evaluation of Three Other Methods.

Objective Challenges in susceptibility testing of colistin along with increase in the prevalence of colistin-resistant carbapenemase-producing Enterobacteriaceae (CRE) pathogens needs addressal. Evaluation of user-friendly methods is necessary as an alternative to broth microdilution (BMD), the reference susceptibility testing method, for routine implementation in diagnostic clinical microbiology laboratories. Genotypic detection of the plasmid-mediated colistin resistance is also needed for infection control purposes. Materials and Methods Colistin susceptibility of 200 nonduplicate clinical CRE isolates from December 2017 to June 2019 was determined by BMD, agar dilution (AD), E test, and rapid polymyxin NP test and interpreted as per the European Committee on Antimicrobial Susceptibility Testing. The results of AD, E test, and NP test were compared with that of BMD, considering minimal inhibitory concentration (MIC) ≤ 2 µg/mL as susceptible and > 2 µg/mL as resistant. Presence of any plasmid-mediated colistin resistance (mcr-1 and 2) was evaluated in 27 colistin-resistant CRE isolates by polymerase chain reaction. Statistical Analysis Performance of different phenotypic methods was analyzed by comparing MIC results of AD and E test with that of reference BMD method. Agreement between BMD and the other two methods was expressed in terms of categorical agreement and essential agreement. Errors were expressed as very major error (VME: false-susceptible) and major error (ME: false-resistance) by AD/E test. VME and ME of 3% disagreement were considered unacceptable. Results Colistin resistance was found in 27 (13.5%) isolates by BMD method. The VME rates of both AD (11%) and E test (37%) could not meet the Clinical and Laboratory Standards Institute recommendation (< 3% VME rate is acceptable) as alternative tests to the reference BMD. Colistin NP test showed sensitivity and specificity of 85% and 98%, respectively. The percentage discordant result in NP test was highest in Enterobacter spp. (17%). None of the 27 colistin resistant isolates showed presence of mcr-1 and mcr-2 genes. Conclusion High VME rate in AD and E tests precludes their use as alternatives to BMD for colistin susceptibility testing. NP test with moderate sensitivity but excellent specificity can be a good alternative for testing colistin susceptibility in CRE isolates, except in Enterobacter spp. Absence of mcr-1 and mcr-2 gene necessitates the exploration of other mechanisms of colistin resistance.

A comparative evaluation of mikrolatest kit broth microdilution and VITEK2 in assessing colistin susceptibility to Gram-negative bacterial isolates

Introduction and Aim: The increasing resistance in colistin is a major concern. The aim of the study was to compare the methods to identify the prevalence of colistin resistance and antibiotic susceptibility patterns of colistin resistant strains isolates from blood, urine and sputum samples at all ICUs including NICU and PICU and wards.&#x0D; &#x0D; Materials and Methods: A total of 1458 consecutive Gram-negative isolates were tested for colistin susceptibility by standard broth microdilution method, Mikrolatest method and VITEK 2 automation system. Intrinsically colistin resistant organisms including Proteus sps, Providencia sps, Serratia sps., and Morganella morganii were excluded. Enterobacteriaceae (e.g., Escherichia coli, Klebsiella), Pseudomonas aeruginosa and Acinetobacter baumanii were included. The sensitivity, specificity, positive predictive value, negative predictive value of Vitek-2, BMD and Mikrolatest methods were compared. &#x0D; &#x0D; Results: Sixteen (1.09%) colistin?resistant isolates were reported over 24 months. K. pneumoniae constituted 8(50%), E. coli 6 (37.5%) and Enterobacter cloacae 2 (12.5%) of the 16 resistant isolates. The sensitivity, specificity, positive predictive value, negative predictive value of Mikrolatest compared with that of VITEK2 were 87.5% vs 56.25%, 90.84% vs 82.65%, 9.58% vs 3.47%and 99.8% vs 99.3%, respectively for resistant isolates. Mikrolatest shared good Category agreement of 1.24% with BMD Essential agreement was 1.5%. Comparing MICs of BMD with other tests, essential agreement was the lowest for the VITEK2, while the Mikrolatest MIC showed essential agreement greater than 1.5%. No errors and 100% categorical agreement (for E. coli, K. pneumoniae) were observed while comparing colistin susceptibility test results of the Mikrolatest MIC and BMD tests. The difference between the two methods in assessing colistin resistance were not statistically significant (P=0.89). Blood[37.5%] and pus[37.5%] samples recorded as the common sources of the isolate, followed by Urine [12.5%], and respiratory [12.5%) samples.&#x0D; &#x0D; Conclusion: Automated VITEK, Mikrolatest MIC methods give variable susceptibility results and colistin should be prescribed only after Mikrolatest and BMD. K. pneumoniae and E. coli, the Mikrolatest showed better performance for isolates with ? 0.5 or ? 16 µg/mL MICs. For P. aeruginosa isolates, colistin resistant isolates must be confirmed Colistin resistance among Gram-negative bacteria, especially K. pneumoniae, is emerging in Indian hospitals. Re-evaluation is required of the methods available to address the numerous technical challenges associated with colistin susceptibility testing, and to determine which method yields the most meaningful results. These studies will provide critical information on the appropriate selection of colistin therapy, as well as evaluating novel and upcoming compounds with structure and properties similar to the Polymyxin.

Colistin Susceptibility Testing by Rapid Colistin Disk Elution Test Among Enterobacteriaceae in Low-Resource Setting.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-μg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 μg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-μL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Comparative evaluation of broth microdilution with E-test, Vitek 2, and disk diffusion for susceptibility testing of colistin on Gram-negative bacteria

Objectives: With the increasing threat of multidrug-resistant organisms, colistin has become popular in clinical practice. A better understanding of antimicrobial susceptibility testing methods for colistin is needed for optimal patient management. The aim of the study was to determine the accuracy of E-test, Vitek 2 system for the detection of colistin minimum inhibitory concentrations (MIC) against broth microdilution (BMD). Material and Methods: A total of 100 isolates of Gram-negative bacilli were subjected to susceptibility testing for colistin using the following methods: BMD, E-test, Vitek 2, and disk diffusion. Using BMD as the gold standard, comparative analysis between different methods was carried out. Results: Comparison of MIC values of E-test (GM = 0.488 mg/ml) against BMD (GM = 0.611 mg/ml using unpaired t-test (t = 2.015, P = 0.045) showed that geometric means of MIC values of E-strip were significantly lower than BMD. Similarly, comparison of MIC values of Vitek 2 system (GM = 0.615 mg/ml) against BMD (GM = 0.611 mg/ml) using unpaired t-test (t = −0.050, P = 0.960) showed no statistical significant differences in geometric means of MIC values. Taking reference as BMD method – the EA for E-strip is 57%, CA is 97%, VME is 2%, and no ME. Similarly, for the Vitek method EA is 64%, CA is 98%, VME is 1%, and ME is 1%. Conclusion: Different susceptibility testing methods for colistin show great variation in their results and BMD is the best candidate as gold standard. The Vitek 2 method showed good concordance with BMD.

Revisiting colistin susceptibility testing: will adding calcium to Mueller–Hinton agar improve the detection of colistin resistance?

ObjectiveThe aim was to investigate whether adding calcium to Mueller–Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories. MethodsAn isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca2+ in concentrations from 2.5 to 40 mM was added to the Mueller–Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes. ResultsResults were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca2+ to Mueller–Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca2+ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca2+ did not improve separation between susceptible and resistant populations. DiscussionThe results from this study show that addition of Ca2+ to the Mueller–Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.

Evaluation of Colistin and Tigecycline Susceptibility Testing Methods for Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

Background: The evolution of nosocomial infections by multidrug resisitance (MDR) and extensive drug resistance (XDR) Acinetobacter baumannii and klebsiella pneumoniae are considered a major health problem owing to the relatively limited treatment options. Colistin and tigecycline are increasingly used as a last choice for treatment of these infections. The most accurate antibiotic susceptibility methods for colistin and tigecycline are still challenging. Objectives: The aim of the current study was to detect colistin and tigecycline antibiotic susceptibility of K. pneumoniae and A. baumanii and evaluate disk diffusion (DD), E- test and VITEK 2 automated system compared to broth dilution (BD) test. Methodology: This study was performed on 35 K. pneumoniae and 15 A. baumanii clinical isolates collected from patients admitted to Benha University Hospitals. The isolated strains were identified by the standard laboratory technique with subspecies identification by VITEK 2 automated system. Colistin and tigecycline antibiotic susceptibility for K. pneumoniae and A. baumanii were evaluated by E-test, disk diffusion and VITEK 2 compared to BD as the reference method. Results: Through the study of the studied k. pneumoniae and A. baumanii strains, The essential and categorical agreements of colistin suscebtibility were (82% &amp; 80 %) for E-test, (92% &amp; 98%) for VITEK 2 and categorical agreement for DD was 54%. The essential and categorical agreements of tigecycline suscebtibility were (96% &amp; 98%) for E-test, (88% &amp;78%) for VITEK 2 and categorical agreement for DD was 74%. Conclusion: For colistin, VITEK 2 is considered a reliable method to detect colistin susceptibility while E-test and disk diffusion showed a poor performance. For tigecycline, E-test showed the best performance compared to the gold standard test while shortcomings of automated VITEK 2 and manual DD were observed.

English

Colistin is an important drug of last resort against lethal infections with multidrugresistant gram-negative bacteria, this antibiotic interacts on phosphate groups of lipopolysaccharides present on the outer membrane of gram-negative bacteria. This first mechanism of transferable colistin resistance involves a gene called mcr-1. Here, we described different tools such as Colistin susceptibility test, Minimal Inhibitory Concentration to find the colistin profiles of Mannheimia haemolytica and Pasteurella multocida serogroup A Moroccan strain isolated from nasal swabs and lung taken from sheep, goat and cattle with respiratory diseases during January 2015 to December 2017 in six different regions, in addition, we investigated either real time PCR to detect mcr-1 gene. Antimicrobial sensitivity test, was achieved for Fortyone isolates, the resistance rates of isolates from Pasteurellaceae species were between 59 and 71%, which respectively correspond to Mannheimia haemolytica and Pasteurella multocida segroup A. The sensitive ratios were between 29 and 41% which respectively correspond to Pasteurella multocida segroup A and Mannheimia haemolytica. Also, the MIC test was done against colistin, the results showed a resistant profile with a MIC &gt;=64 μg/ml except for 3 strains from ruminants which have a value &lt;2 μg/ml. The real time PCR screening test was detected with ct values ranging from 23 to 31 and confirmed the results obtained by MIC test. To our knowledge the present study is the first study which reports the resistance to colistin in Pasteurellaceae species strains isolated from ruminants in Morocco, our results suggest the necessity and the urgency for establishing a national program for monitoring antibacterial resistance against colistin

Susceptibility Testing of Colistin for Acinetobacter baumannii: How Far Are We from the Truth?

Acinetobacter baumannii is involved in life-threatening nosocomial infections, mainly in the intensive care units (ICUs), and often colistin may represent the last therapeutic opportunity. The susceptibility to colistin of 51 epidemiologically typed A. baumannii strains isolated in 2017 from clinical samples of patients hospitalized in the ICU of a tertiary care academic hospital was investigated. All isolates were carbapenem-resistant due to the presence of the blaOXA-23 gene in sequence group 1 (international clonal lineage II) and sequence group 4 (related to international clonal lineage II) isolates, and to the blaOXA-24/40 gene in sequence group 2 (international clonal lineage I) isolates. Vitek®2, agar diffusion, and broth microdilution tests showed major discordancy (≥2 dilution factors) in the minimum inhibitory concentration (MIC) values for colistin in 24 out of 51 isolates, resulting in erroneous reporting of qualitative susceptibility data for eight isolates. In growth kinetics experiments in the presence of colistin, five isolates grew with drug concentrations above the susceptibility breakpoint when incubated for >12 h, and three isolates showed the presence of heteroresistant subpopulations. This study highlights that the high frequency of isolation of carbapenem-resistant A. baumannii strains in high-risk infectious wards requires an accurate application of methods for detecting susceptibility to antibiotics, in particular to colistin, so as to ensure a correct therapeutic approach.

Evaluation of a Commercial Broth Microdilution Panel for Colistin Susceptibility Testing of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae.

Colistin is among the last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Antimicrobial susceptibility testing of colistin is challenging due to its physicochemical properties. Broth microdilution (BMD) is the recommended method for colistin susceptibility testing. However BMD is not practical for clinical microbiology laboratories as manual preparation of BMD plates is time-consuming and labor intensive. Recently, some more user-friendly BMD products with commercial panels have become available. Our objective was to evaluate the performance of a commercial broth microdilution (BMD) product [Sensititre (Thermo Fisher Scientific)] for colistin MIC determination by comparison with reference BMD method using a collection of E. coli and K. pneumoniae isolates. A total of 323 unique patient isolates (102 E. coli, 221 K. pneumoniae) were included in the study. Isolates were stored at -70°C and subcultured twice on sheep blood agar before testing. Colistin MICs of the isolates were determined using Sensititre (a premade BMD product with dried antibiotics) and an 'in-house prepared BMD panel prepared in accordance with CLSI guidelines' (reference method). MIC determination with Sensititre was performed according to manufacturer's instructions. The reference method was performed using untreated 96-well sterile polystyrene plates. Colistin MIC results were interpreted according to EUCAST breakpoints (susceptible, ≤ 2 mg/L; resistant, > 2 mg/L). Overall susceptibility rate of isolates to colistin by reference BMD was 75.9%. Overall categorical agreement (CA), essential agreement (EA), very major error (VME), and major error (ME) rates for Sensititre were 98.5%, 72.5%, 3.8%, and 0.8%, respectively. The CA and EA between Sensititre and reference BMD for the isolates with reference colistin MICs close to the susceptibility breakpoint (2 - 8 mg/L) was 94.2% and 48.1%, respectively. Sensititre yielded a VME rate of 15% and ME rate of 0%, respectively, for this subset of isolates. In conclusion, Sensititre showed high CA but low EA with reference BMD for entire collection of isolates. The VME rate was just slightly above 3% and ME rate was acceptable. The rates of CA and EA were decreased and the rate of VME was increased when a subset consisting of more challenging isolates was used.

Comparative Assessment of Disk Diffusion, E-test and Broth Dilution Methods for Determining Colistin Susceptibility in Acinetobacter Species

Introduction: Colistin is the ultimate reserve drug for gram- negative bacilli especially non fermenting bacilli like Acinetobacter species and only therapeutic option available against carbapenem resistant bacteria. Clinical and Laboratory Standards Institute (CLSI) recommended broth dilution method for colistin susceptibility testing but this method require expertise and very time consuming and is also not feasible to routinely perform in clinical laboratories. In the background of ever expanding problems of drug resistance coupled with increasing use of colistin, it becomes essential to find a testing method which is faster, accurate and can be used as an alternative to broth dilution method. Aim: To assess disk diffusion, E-test and broth dilution method to determine colistin susceptibility in Acinetobacter species. Materials and Methods: This cross-sectional observational study was done in the Department of Microbiology, ABVIMS and Dr RML Hospital, New Delhi, India, from November 2016 to March 2018. Various clinical specimens were received from wards and Intensive Care Unit (ICU) and Acinetobacter species were isolated and identified following standard protocol. All strains were screened by disc diffusion test for various antibacterial drugs and among them 50 MDR strains were selected. Colistin susceptibility by disc diffusion and Epsilometer test (E-test) were done and compared against the reference broth dilution method following. Minimum Inhibitory Concentration (MIC) of colistin for the isolates were determined using macro broth dilution method using colistin sulphate. Chi-square test was used to investigate distributions of categorical variables. Results: All 50 strains were tested for colistin susceptibility by disc diffusion method. Among the 50 isolates, 10 isolates (20%) showed resistance to colistin by disc diffusion method while none of the isolate was found resistant by E-test or broth dilution method. High error rate and poor concordance of disc diffusion with reference to broth dilution method rules out the utility of disk diffusion method in clinical laboratories. Conclusion: A complete agreement between the results of E-test and broth dilution method makes E-test a suitable alternative to broth dilution test in clinical laboratories.

Simple Phenotypic Tests To Improve Accuracy in Screening Chromosomal and Plasmid-Mediated Colistin Resistance in Gram-Negative Bacilli.

CLSI and EUCAST recommend that only broth microdilution (BMD) should be used for routine colistin susceptibility testing; however, this technique can be difficult to perform in resource-poor settings. The purpose of this study was to evaluate the accuracy of a colistin agar spot test (COL-AS) and a colistin drop test (COL-DT) compared to BMD. COL-AS and COL-DT were assessed with a collection of 271 Gram-negative bacilli clinical isolates: 195 Enterobacterales (including 63 mcr-1 positive strains), 37 Acinetobacter spp., and 39 Pseudomonas aeruginosa For COL-AS, 3.0 μg/ml (final concentration) of colistin was added to a Mueller-Hinton agar plate and subsequently swabbed with a 0.5 McFarland standard suspension of the tested strain within a 1 cm2 spot. For COL-DT, 10 μl of a 16 μg/ml colistin solution was dripped on the surface of a Mueller-Hinton agar plate, previously inoculated with a lawn of the tested strain (0.5 McFarland standard). Colistin solution was made either by dissolving powder or by disk elution in cation-adjusted Mueller-Hinton broth (CA-MHB). Overall, 141/271 (52%) isolates were categorized as colistin resistant by reference BMD. COL-AS yielded a categorical agreement (CA) of 95.5% compared to BMD, with 0.7% very major errors and 3.8% major errors. COL-DT yielded a CA of 96.2% compared to BMD, with 0.7% and 0% very major errors and 3.1% and 3.8% major errors, for colistin powder and disk elution solutions, respectively. Most major errors occurred for mcr-1 strains with MICs that fluctuated from 2 to 4 μg/ml according to the method used. In conclusion, we developed and validated methods suited to the systematic screening of resistance to colistin in Gram-negative bacilli.

Comprehensive Statistical Evaluation of Etest®, UMIC®, MicroScan and Disc Diffusion versus Standard Broth Microdilution: Workflow for an Accurate Detection of Colistin-Resistant and Mcr-Positive E. coli.

Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 mcr-positive E. coli, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest®, 0.7% for UMIC®), except for the disc diffusion assay (breakpoint ≤ 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of mcr-positive isolates (17.3%) categorized as susceptible (≤2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 mcr-positive E. coli were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many mcr-positive E. coli remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all mcr-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the mcr screening.

In-house pre-prepared broth microdilution plates: A simple, cheap and pragmatic approach for susceptibility testing of colistin

Background: With the upsurge in multi-drug resistant Gram-negative bacilli, it is imperative to optimize and standardize the antibiotic susceptibility testing (AST) methods for polymyxins (colistin & polymyxin B). The long overdue confusion regarding the susceptibility testing of colistin was put to rest in 2016 when the CLSI-EUCAST joint Polymyxin Breakpoint Working Group recommended broth microdilution (BMD) in polystyrene plates as the reference method for AST. Though several commercially-available BMD plates have been assessed by EUCAST, the high cost of such commercial plates during contemporary times hinder their use in routine microbiological laboratories especially in low and middle income countries. Thus, a suitable alternative to commercial BMD plates should be sought for to ensure a cost-effective, rapid and reliable reporting of AST of colistin. Methods and materials: With the aim of reducing this redundancy in the workflow, we designed an experiment to charge several microtiter plates with the drug dilution and the media on day 0, storing them frozen at −200C and then retrieving them sequentially for AST (henceforth called pre-prepared frozen BMD plates (PFBP). The PFBP were charged with same representative organisms on every 2–4 days for two months to check for the reliability of AST reporting. Essential agreement (EA), categorical agreement (CA), very major error (VME) and major errors (ME) were computed in accordance with ISO standard 20776-2. For validation, the criteria of EA3 90%, CA 3 90%, VME £1.5% AND ME£3% was used. Pearson correlation and Cohen's kappa (k) score was calculated using GraphPad (San Diego, CA, USA). Results: For colistin, EA and CA of PFBP was 97.5% and 98.3%, respectively. For polymyxin B, EA and CA of PFBP was 99.1% and 90%. Further, the performance of PFBP for colistin susceptibility testing of A. baumannii in the present study (EA=97.5%, CA=98.3%) was superior to five commercial plates available and evaluated by EUCAST. Conclusion: Based on these observations, we propose that pre-prepared frozen BMD plates can be incorporated in routine workflow of microbiological laboratory as reliable, cheap and rapid method for susceptibility testing of colistin.

‘Breakpoint broth microdilution plate’ for susceptibility testing of Gram negative bacilli against colistin sulfate

The MIC method applicable to Gram negative bacilli including Acinetobacter spp. is broth microdilution (BMD). Cost and/or availability issues limit the use of commercial MIC panels in resource limited settings. ObjectivesTo design and implement an in-house breakpoint BMD panel (BBMD) for colistin against Gram negative bacilli. DesignBBMD panel was prepared in 96-well plate. MIC concentrations of 1, 2, & 4 ​μg/mL for test, and 0.25, 0.5, 1, 2 & 4 ​μg/mL for control strains were selected to accommodate 19 test and 3 quality control strains per plate. Plates were frozen at −80 ​°C until testing. Validation was performed using strains from a previously published study and compared with freshly prepared MIC panel of 16–0.03 ​μg/mL. ResultsValidation showed 100% agreement with the reference method and BBMD was introduced into routine laboratory practice for colistin susceptibility of carbapenem resistant Enterobacterales (CRE), Acinetobacter baumannii complex and Pseudomonas aeruginosa. From 2nd July-16th September 2018, a total of 1294 (mean 16.8 ​± ​5.5 isolates/day) clinical isolates were tested; 1157/1294 were reported (MIC ≤2 ​μg/mL) within 24-h, whereas 133 required resistance confirmation by full-range BMD. Resistance was confirmed for all but 24 isolates. These discrepancies were mostly due to contamination with bacterial genera inherently resistant to colistin. ConclusionThis BBMD plate is a high through-put and practical method that could reliably be utilized in a routine microbiology laboratory for colistin susceptibility testing of CRE, A. bauamanii complex and P. aeruginosa.

Evaluation of Vitek®2 performance for colistin susceptibility testing for Gram-negative isolates.

BackgroundThe emerging resistance to the last-resort antimicrobial colistin is being reported globally. Underestimation of the burden of colistin resistance and misinterpretation of colistin susceptibility test results, using suboptimal testing methods, may be causing unexplained treatment failures and even mortality among critically ill patients. Thus, this study was conducted at an apex trauma centre to assess the performance of Vitek®2 for colistin susceptibility testing.MethodsA total of 910 clinical isolates of Gram-negative bacteria (GNB), including Enterobacterales, Acinetobacter baumannii and Pseudomonas aeruginosa, were tested and analysed for colistin resistance using Vitek®2. Broth microdilution (BMD) was taken as the reference method. The essential (EA) and categorical (CA) agreements and very major error (VME) and major error (ME) rates were calculated. An MIC correlation was taken to be positive with EA ≥ 90%, CA ≥ 90%, VME ≤ 1.5% and ME ≤ 3.0% rates. Spearman’s coefficient was calculated and P < 0.05 was considered statistically significant.ResultsA total of 64% of isolates were MDR. Overall, 196 (21.5%) and 110 (12%) of isolates were resistant to colistin by BMD and Vitek®2, respectively. The automated Vitek®2 method failed to detect the resistance in up to 48.5% of GNB tested. When comparing Vitek®2 colistin interpretive results with reference BMD for all 910 isolates, the CA was 88% (798/910) with 10% (95/910) VMEs and 1% (9/910) MEs.ConclusionsThe Vitek®2 method for colistin susceptibility testing, still in use in some settings; is a suboptimal and unreliable method.

Clinical and Microbiological Analysis of Risk Factors for Mortality in Patients With Carbapenem-Resistant Acinetobacter baumannii Bacteremia

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) infection is associated with significant mortality, causing worldwide concern, yet there are limited data on contributing microbiological factors. This study aimed to identify the clinical and microbiologic risk factors for mortality in CRAB bacteremia. MethodsAdult patients with monomicrobial CRAB bacteremia in a 2700-bed tertiary hospital between December 2012 and December 2018 were retrospectively enrolled. Risk factors for 30-day mortality were evaluated. All isolates collected on the first day of bacteremia were subjected to colistin susceptibility testing by broth microdilution and to genotyping by multilocus sequence typing. ResultsA total of 164 patients were enrolled, and 90 (55%) died within 30 days. The most common genotype among the isolates was ST191 (49%), and 12 isolates (7%) were resistant to colistin. Genotype, colistin minimum inhibitory concentration, and colistin resistance were not significantly associated with mortality, in contrast to several clinical factors. In multivariable analysis, ineradicable or not-eradicated focus (adjusted odds ratio [aOR], 4.92; 95% CI, 1.95–12.42; P = .001), septic shock (aOR, 4.72; 95% CI, 2.12–10.49; P < .001), and inappropriate antimicrobial therapy (aOR, 2.54; 95% CI, 1.05–6.16; P = .04) were independent risk factors for mortality. Among antibiotic strategies, colistin combined with tigecycline or other antibiotics were significantly associated with lower mortality after adjustment for confounding factors. ConclusionsClinical factors such as the nature of the infection source and source control, severity of bacteremia, and appropriateness of antibiotics, rather than microbiological factors, contribute to mortality in CRAB bacteremia. A specific antibiotic combination may help improve outcomes.

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii

ObjectivesBoth EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. MethodsOne hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. ResultsOverall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. ConclusionsFASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

Detecting In-Vitro Colistin Resistance- A Comparative Study Between Broth Microdilution Versus Vitek-2 For Colistin Susceptibility Testing

Background: Susceptibility testing for polymyxins is a great challenge for a Clinical Microbiology laboratory. There are several methodological issues associated with MIC (Minimum Inhibitory Concentration) determination of colistin.&#x0D; Methods: In our study, we have compared the results of colistin susceptibility testing by Automated system (Vitek-2, Biomerieux, France) with the reference Broth Microdilution method (BMD) to identify the type of discrepancies by Vitek-2 method and thus develop a practical and accurate approach for colistin susceptibility testing in a Clinical Microbiology laboratory. A total of 730 strains of Gram negative bacteria [Escherichia coli (325), Klebsiella sp.(346), Acinetobacter baumanii complex (37) and Pseudomonas aeruginosa (22)] from 485 patents were tested simultaneously by BMD and Vitek-2 method for colistin susceptibility testing.&#x0D; Results: The Essential agreement (EA), Categorical agreement (CA), Very major error (VME) and Major error (ME) rates for Klebsiella sp. were 87.3%, 89.3%, 8% and 2.3% respectively, for Escherichia coli were 88.3%, 89.5%, 9.2% and 1.2% respectively, for Acinetobacter baumannii complex were 89.1%, 91.8%, 8.1% and 0% respectively, for Pseudomonas aeruginosa were 68.1%, 72.7%, 0% and 27.2% respectively.&#x0D; Conclusions: Colistin susceptibility testing by Vitek-2 method is an easily adoptable method and the results of Vitek-2 with reference to BMD are acceptable to a great extent in Klebsiella sp., Escherichia coli and Acinetobacter baumanii complex. So, we believe that Vitek-2 method may be used for colistin susceptibility testing in low risk patients. However, BMD should be used in high risk immunosupressed and immunocompromised patients who are admitted in critical care units. For Pseudomonas aeruginosa, BMD should be routinely used.