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Year : 2019  |  Volume : 37  |  Issue : 4  |  Page : 488--495

Performance of three commercial assays for colistin susceptibility in clinical isolates and Mcr-1 carrying reference strain

Chand Wattal, Neeraj Goel, Jaswinder Kaur Oberoi, Sanghamitra Datta, Reena Raveendran 
 Department of Clinical Microbiology and Immunology, Sir Ganga Ram Hospital, New Delhi, India

Correspondence Address:
Dr. Chand Wattal
Department of Clinical, Microbiology and Immunology, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi - 110 060
India

Abstract

Objective: Commercially available antibiotic susceptibility tests (cAST) for colistin are reported to shows variable performance. The current controversy on the colistin susceptibility testing and scarce data from India has left the clinical laboratories in a dilemma on the appropriate and practical approach to tackle the colistin antimicrobial susceptibility testing (AST) issue. This study was aimed to evaluate the performance of commonly used cAST for colistin against broth microdilution (BMD) as the reference method in the clinical isolates. Materials and Methods: Colistin AST was performed on 225 nonduplicate isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii by BMD as the reference method and compared with Vitek-2, Micronaut-S and E-test. The accuracy of the various cASTs was analysed by assessing categorical and essential agreement (EA). Results: We observed an overall categorical agreement of 98.2%, 99.6% and 96.4% and EA of 92%, 92.4% and 72% for Vitek-2, Micronaut-S and E-test, respectively. Unacceptable rates of major error (10.5%) and very major error (21%) were observed for P. aeruginosa with Vitek-2 and E-test, respectively. All the categorical errors (CEs) (7.7%) with Vitek-2 were seen for minimum inhibitory concentrations ranging within two-fold dilution breakpoint of 2 mg/L. Conclusion: Micronaut-S was found to be an acceptable method for colistin AST. In contrast, E-test was unreliable in terms of EA. Vitek-2 was found to be reliable for colistin AST, although it was more prone to CE near the colistin breakpoints.

How to cite this article:
Wattal C, Goel N, Oberoi JK, Datta S, Raveendran R. Performance of three commercial assays for colistin susceptibility in clinical isolates and Mcr-1 carrying reference strain.Indian J Med Microbiol 2019;37:488-495

How to cite this URL:
Wattal C, Goel N, Oberoi JK, Datta S, Raveendran R. Performance of three commercial assays for colistin susceptibility in clinical isolates and Mcr-1 carrying reference strain. Indian J Med Microbiol [serial online] 2019 [cited 2020 Nov 29 ];37:488-495
Available from: https://www.ijmm.org/text.asp?2019/37/4/488/284534

Full Text

 Introduction



The increasing prevalence of carbapenem-resistant Gram-negative bacteria (GNBs) in the last decade has resulted in the widespread use of colistin as the last therapeutic option.[1],[2] Expectedly, colistin resistance, which was infrequent only a few years back, is now commonly reported.[1] The accurate performance of colistin antimicrobial susceptibility testing (AST) is fraught with difficulties due to two main reasons. First, it being a large-sized molecule diffuses poorly in agar media and second polycationic nature of drug results in its binding to polystyrene plates used for broth microdilution (BMD), causing considerable variations in colistin AST results.[2],[3] To address this concern, joint EUCAST and CLSI working group evaluated various commercially available antibiotic susceptibility testing (cAST) for colistin and recommended BMD as the reference method for performing colistin AST.[4] Disc diffusion (DD) methods are not advised for colistin susceptibility testing due to the reasons mentioned above. Similarly, gradient test methods like E-test are also prone to very major errors (VMEs),[4],[5],[6] due to their tendency to underestimate the colistin minimum inhibitory concentration (MIC).[4] Commercially BMD systems like Micronaut-S have shown acceptable performance (categorical agreement [CA] of 86%–94% and essential agreement [EA] of 91%–100%) but are considered expensive for its routine use.[5] BMD, as per the ISO standard 20766-1, is considered as the reference method for colistin AST but is laborious and requires highly trained staff, which discourages its routine use in the laboratories.[7] Joint EUCAST and CLSI working groups, could not systematically evaluate the widely used automated methods (like Vitek-2, Phoenix) and therefore, advised rigorous quality controls for their conditional use in the respective laboratories. Although there have been few recent reports of Vitek-2 accuracy in assessing colistin AST, many of these studies were performed on a small number of resistant isolates, and besides, this also gave varied results.[6],[8],[9] Only one study has been published from India on the performance of Vitek-2 for colistin AST on 42 isolates of Acinetobacter baumannii. This study showed a high CA of 92.8% but poor EA of 61.9%.[3] The recent emergence of colistin resistance due to mcr-1 plasmid has made colistin AST even more problematic.[5] Mcr-1 plasmid imparts colistin resistance around a MIC of 4 mg/l which is very close to its clinical breakpoint in a fermenters as well as the epidemiological cut-off for Enterobacteriaceae;[10] therefore, MIC variation of even normally acceptable ±1 log dilution by a test method can result in VMEs. The current controversy on the colistin testing and scarce data from India has left the clinical laboratories in a dilemma on the appropriate and practical approach to tackle the colistin AST issue. This study aimed to evaluate the performance of commonly used cAST for colistin against BMD as the reference method in the clinical isolates and also using mcr-1 as a control in every run.

 Materials and Methods



The study was conducted in the Department of Clinical Microbiology and Immunology at a 675 bedded tertiary care centre and teaching institute, Sir Ganga Ram Hospital, New Delhi.

Bacterial isolates

This prospective study was carried out from January 2018 to December 2018, in which 225 nonduplicate isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and A. baumannii were included with the aim of recruiting at least 52% colistin-resistant isolates of each category of the bacteria as per the recommendations for the evaluation of cAST.[11] The breakup of organisms from various sites is given in [Table 1]. All the isolates were characterized phenotypically up to the species level using MALDI-TOF MS (bioMérieux, Marcy-l-Etiole, France). AST of these isolates was previously characterized phenotypically by Vitek-2 (bioMérieux, Marcy-l-Etiole, France) as a routine standard of care at our hospital. All the isolates were stored in 20% glycerol broth at − 80°C (Thermofisher, USA) and sub-cultured twice and checked for purity before use.{Table 1}

Susceptibility testing

Colistin AST for all isolates was performed by BMD and compared with Vitek-2 (bioMérieux, Marcy-l-Etiole, France), Micronaut-S (MERLIN DiagnostikaGmbh, Bornheim, Germany) and E-test (bioMérieux, Marcy-l-Etiole, France). Colistin ASTs were performed in parallel using the same inoculum, to ensure that the same population and count of bacteria were tested simultaneously.

Colistin AST by BMD was taken as the reference method for comparing the performance of the other methods. Briefly, colistinsulphate salt (15,000 U/mg) (Sigma, St. Louis, MO, USA), cation-adjusted Muller Hinton Broth (CAMBH) (Sigma, St. Louis, MO) and round bottom polystyrene 96 well trays (Corning Costar, NY, USA) were used for performing BMD as per the ISO standard 20766-1.[7] Colistin salt was dissolved in the distilled water to achieve a working drug concentration of 256 mg/l and stored in glass vials at-80°C until further use. Each vial was thawed only once before use on the day of testing andtwo-fold MIC dilutions ranging from 0.0625 mg/l to 64 mg/l were prepared and tested with a final inoculum of 5 × 105 cfu/ml of each isolate in CAMBH broth. MIC viewing device (Dade Behring, USA) was used for reading the formation of a button or matt in the wells. All the readings were recorded by two blinded observers.

Colistin MICs by Vitek-2 compact cards were assessed using N-280 cards (for K. pneumoniae and E. coli) and N-281cards (for P. aeruginosa and A. baumannii) as per the manufacturer's recommendations with MICs ranging from ≤0.5 mg/l to ≥16 mg/l. Micronaut-S for colistin AST is a frozen MIC panel containing eight strips for colistin testing with MICs range from 0.0625 mg/l to 64 mg/l. Colistin testing by the Etest gradient MICs was performed as per the manufacturer's recommended media, MHE agar (bioMérieux, Marcy-l-Etiole, France), with MICs ranging from ≤0.016 mg/l to ≥256 mg/l. Isolates were considered as susceptible when MIC was ≤2 mg/l and resistant when MIC was ≥4 mg/l for both P. aeruginosa and A. baumannii as per the CLSI guidelines. Since Enterobacteriaceae breakpoints for colistin are not defined, epidemiological cut-off value of ≤2 mg/l was considered as sensitive, and ≥4 mg/l was considered as resistant.[10]

Analysis of the data

Categorical agreement

Categorical agreement was calculated as the percentage agreement of the interpretative results (sensitive/intermediate/resistant) between the test method and BMD.

Essential agreement

Essential agreement was calculated as the percentage agreement within ±1 two-fold dilution of the test method compared and BMD.

Very major error

Percentage discrepancy (falsely susceptible) of the test method with BMD out of total resistant isolates by BMD.

Major error

Percentage discrepancy (falsely resistant) of the test method with the BMD out of total sensitive isolates by BMD.

Minor errors

Percentage discrepancy (falsely intermediate) when BMD result is either sensitive or resistant out of total tested isolates.

Since Vitek-2 colistin upper MIC range is ≥16 mg/l as compared to Micronaut-S (64 mg/l), BMD (64 mg/l) and E-test (≥256 mg/l), Vitek-2 MICs of ≥16 mg/l were normalised to match MICs of other tests for comparability of EA. Of course, CA was not affected by this method.

Acceptable criteria

cAST performance was considered as acceptable if both the CA and EA were ≥90%. In addition, minor errors, major error (MEs) and VMEs rates were benchmarked as ≤10%, ≤3% and ≤3%, respectively, for acceptable performance.[10]

Quality control

Prior to the start of the study, ten replicates of QC strains for colistin AST of E. coli ATCC 25922 (colistin MIC, 0.25 mg/l to 2 mg/l) and P. aeruginosa ATCC 27853 (colistin MIC, 0.5 mg/l to 4 mg/l) were analysed by all the methods as per the CLSI guidelines.[10]

During the evaluation of cAST for colistin susceptibility testing, mcr-1-positive QC strain of E. coli NCTC 13846 (colistin MIC, 4 mg/l) was run with each batch of (comprising seven test strains and one E. coli NCTC 13846).

Discrepant testing

All the discordant results (MEs and VMEs) were retested in duplicate along with BMD to reconfirm the results. In the repeat testing, if the error was resolved, the repeat results were kept as final. If repeat MIC values were within ±2 log dilution and categorical error (CE) in the form of MEs and VMEs remained, the CE was accepted.

Ethics approval

This study was approved by the institutional review board before the start of the study (letter no: EC/09/17/1209).

 Results



The quality control of colistin BMD, Vitek-2, Micronaut-S and E-test for the ten replicates of P. aeruginosa ATCC 27853 and E. coli ATCC 25922 were found to be within the assigned ranges of the QC for all the methods.

Out of a total of 225 bacterial isolates tested for the colistin MIC using BMD, 152 (67.5%) were colistin sensitive and 73 (32.4%) were colistin resistant. Comparison of various methods with respect to CA, along with CEs and EA for different bacteria, is presented in [Table 2].{Table 2}

Vitek-2

An overall acceptable CA of 98.2% and EA of 92% was observed for colistin AST using Vitek-2 cards. CA of 100% was seen for E. coli, K. pneumoniae and A. baumannii. However, high rates of ME were seen for P. aeruginosa (n = 4, 10.5%), wherein four isolates with MIC of 1 mg/l by BMD were reported as 4 mg/l by Vitek-2. Furthermore, poor EA (88.3%) was seen for P. aeruginosa, as 11 isolates showed a difference of more than two dilutions (EA = 79%) as compared to BMD [Table 2] and [Figure 1]. Acceptable EA was observed for E. coli (100%), K. pneumoniae (93.6%) and A. baumannii (100%).{Figure 1}

Micronaut-S

Micronaut-S for colistin AST also showed a high level of CA (99.6%) and EA (92.4%). CA of 100% for E. coli, A. baumannii and P. aeruginosa and of 99.1% for K. pneumoniae was observed. One isolate of K. pneumoniae showed ME (1.7%), wherein MIC of 2 mg/l by BMD was observed as 8 mg/l by Micronaut-S [Figure 2]. EA of 95.5%, 93%, 95% and 88% were seen for E. coli, K. pneumoniae, A. baumannii and P. aeruginosa, respectively.{Figure 2}

E-test

Although the colistin E-test showed an overall acceptable CA of 96.4%, a poor EA of 72% was observed. CA of 100%, 96%, 100%, and 92% was observed for E. coli, K. pneumoniae, A. baumannii and P. aeruginosa, respectively [Table 2]. One ME (2.6%) and 3 VMEs (21%) were seen in P. aeruginosa [Figure 1]. Similarly, ME of 7% (n = 4) was seen in K. pneumoniae [Table 2]. Poor EA of 68.2%, 73%, 52% and 87% were seen for E. coli, K. pneumoniae, A. baumannii and P. aeruginosa, respectively.

All the discrepant isolates (10 MEs and 3 VMEs) as depicted in [Figure 1], [Figure 2], [Figure 3], [Figure 4] when retested gave similar results, thereby excluding the possibility of CEs due to errors in the methodology.{Figure 3}{Figure 4}

Mcr-1 testing

The assigned colistin MIC value of mcr-1 E. coli NCTC 13846 is considered as 4 mg/l, just one dilution above the epidemiologic cut-off value.[4] The result of the E. coli NCTC 13846 testing for various test methods is given in [Table 3]. EA of 100% was seen for both BMD and Micronaut-S, whereas it was 88.6% and 65.8% for Vitek-2 and E-test, respectively. False susceptibility rates of 11.43%, 48.6%, 25.7% and 71.4% were seen for BMD, Vitek-2, Micronaut-S and E-test, respectively.{Table 3}

 Discussion



With the emergence of carbapenem-resistant Enterobacteriaceae, colistin is being used as the last resort antibiotic, and therefore, its accurate AST is of paramount importance for the appropriate outcomes. The use of cAST has recently been mired in controversies due to the reporting of MEs and more specifically, VMEs.[5],[6] Although BMD is now considered as the reference method for assessing the colistin AST but in-spite of all the precautions, significant uncontrollable variability exists with the BMD method.[12] BMD susceptibility testing requires dedicated staff with good pipetting skills and accurate digital weighing equipment, which may be lacking in many of the current clinical microbiology laboratories. Therefore, in spite of the joint CLSI-EUCAST recommendation for the use of BMD as the only reliable method for colistin AST, most of the laboratories in India continue to rely on automated methods (e.g., Vitek-2 or Phoenix). Laboratories that do not have automated AST methods often use E-test for colistin AST as there are no cut-offs for the DD test. Commercial BMD methods like Micronaut-S, though expensive, are less laborious and have largely been found to be reliable by other workers as well.[5],[6] Therefore, the purpose of the present study was to ascertain the accuracy of the commonly used colistin cAST methods.

In the present study, we observed an acceptable performance of the Vitek-2 system (280/281 Cards). The excellent CA is in agreement with other authors who have reported a high CA of 100%–91.4% and EA of 93.4%–90.1%.[9],[13],[14] Chew et al. in their study on 76 isolates of Enterobacteriaceae, of which 21 were mcr-1 positive also reported high EA (93.4%) but slightly lower CA (88.2%) for colistin AST by Vitek-2 [Table 4]. Interestingly, that study showed high rates of VMEs (36%) using Vitek-2. High rates of VMEs reported in that study could be due to the low number of colistin-resistant isolates (n = 25, 32.9%), which can exaggerate VME rates even with few false sensitive results. In contrast, our study had a high number of resistant K. pneumoniae isolates (n = 52, 47%). However, Girardello et al.reported lower but unacceptable VMEs of 8% in 151 isolates comprising of 84.5% colistin-resistant isolates of Enterobacteriaceae.[14]{Table 4}

Similar to Enterobacteriaceae, we also reported high CA (100%) and EA (100%) in A. baumanii by Vitek-2. Dafopoulou et al., in their study, demonstrated CA of 90% and EA of 85% by Vitek-2 in A. baumanii.[13] Singhal et al. from India included 42 isolates of carbapenem-resistant A. baumannii, and they also reported CA of 100% but a poor EA of 26.2% with Vitek-2, though the results of this study were constrained by the absence of colistin-resistant isolates.[3] On the other hand, Vourli et al. showed unacceptable CA and EA of 89.7% and 88.9%, respectively, on a larger number of A. baumannii isolates (n = 117), which included 29 (24.8%) colistin-resistant isolates. Moreover, Vitek-2 showed unacceptable rates of VMEs (37.9%) in this study.[8] CE rate has been shown to be directly proportional to the percentage of isolates that hover around the MIC breakpoint,[13] and in this study too the maximum VMEs were observed for isolates having MICs of 2 mg/l. Therefore, the evaluation of any AST method for colistin should clearly document the percentage of the isolates having MICs near the breakpoint as this factor may change the accuracy of the test method.

In contrast to other GNBs, we observed an acceptable CA of 98.2% but poor EA of 79% for P. aeruginosa. Unacceptable levels of MEs (10.5%) were also observed, though no VMEs were noted. Similar to our results, Girardello et al. also reported a high CA of 93.4% (MEs: 3.3%, VMEs: 3.3%) but low EA of 68.4% in P. aeruginosa isolates.[14] The high error rates in P. aeruginosa in our study as compared to other isolates could be due to the high percentage (n = 15, 40.4%) of isolates with MICs in the breakpoint range of 2–4 mg/l, where even one fold difference in the MIC value can produce CEs. In contrast, we had few isolates in the breakpoint range of 2–4 mg/l for K. pneumoniae (n = 14, 12.8%), E. coli (n = 0, 0%) and A. baumannii (n = 3, 7.1%), possibly explaining excellent results for Vitek-2. Therefore, more studies are required to validate our results for P. aeruginosa being more prone to errors for colistin AST by Vitek-2.

Micronaut-S showed acceptable CA and EA in our study. We could find only one published study on the performance of Micronaut-S on colistin AST by Matuschek et al., in which high rates of CA (94%) and EA (97%) was observed in enterobacteriaceae and no VMEs were seen for bacteria except for P. aeruginosa (2 VMEs observed of 21 isolates).[5] As compared to enterobacteriaceae, modest correlation in A. baumannii (CA: 86%; EA: 91%), and P. aeruginosa (CA: 86%; EA: 100%) was seen in this study. Chew et al. studied another commercial BMD system, Sensititre (Thermo Fisher Scientific, US) for colistin in Enterobacteriaceae and found a good correlation with BMD (CA: 90.1%; EA: 89.5%) with 4% VMEs.[6]

E-test was generally found to be unreliable for colistin AST in the present study. Our findings are similar to other authors who have reported high CA (100%) and low EA (11.9%) in A. baumannii,[3] and enterobacteriaceae (CA: 94-92%, EA: 75%).[5],[6] In contrast, Dafopoulou et al. have reported poor CA and EA for E-test in K. pneumoniae (CA: 56.1%, EA: 48.8%) and A. baumannii (CA: 65%, EA: 55%).[13] The main reason for the poor performance of the E-test is due to the colistin molecule's poor and unpredictable diffusion in the agar medium.

Though we noted high false susceptibility of colistin in mcr-1 positive E. coli NCTC 13846 for all the cAST in this study, and it is pertinent to notice that even 1 two-fold lower MIC change of mcr-1 strain alters the interpretation. Therefore, for better interpretation of QC of mcr-1 strain, it has been suggested that >80% of values can be at 4 mg/l, and the rest of the values can be between 2 mg/l and 8 mg/l.[5] Even with these criteria, all the evaluated cAST showed false susceptibility in excess of 20%. On the other hand, the superlative performance of cASTfor the QC strains E. coli ATCC 25922, and P. aeruginosa ATCC 27853 is due to the fact that CLSI/EUCAST allows variation of 4 twofold dilutions for colistin AST which is in variance to the allowed variation of only twofold dilutions for the clinical isolates.[10],[11] Therefore, the inclusion of mcr-1 strain with stricter QC range for colistin AST in the CLSI/EUCAST guidelines would be an important step for better evaluation of cASTs in future studies.

To conclude, Vitek-2 shows variable performance between the different classes of GNBs for colistin AST. Furthermore, in our study CE rate of 7.7% (n = 4) was seen in 52 isolates (23.1%) for MICs ranging between 1-4 mg/l by Vitek-2. Whereas in 173 (76.9%) isolates of the total, no CE was observed for MICs of <1 mg/l and >4 mg/l. Other authors have also reported excellent performance of Vitek-2 for isolates with MICs of ≤0.5 and ≥16 mg/l.[14] Therefore, based on the results of our study, it could be recommended that the isolates having colistin MICs between 1 and 4 mg/l by Vitek-2 should be re-tested with BMD and the rest of the isolates having MICs of <1 mg/l and >4 mg/l can be released without additional testing. By choosing this strategy, we could avoid BMD testing in 77% of cases. Micronaut-S performed well for all the bacteria tested with ME and VMEs in the acceptable range as defined by CLSI. It is also easy to perform, but its cost may be a limiting factor for its routine use. Although, laboratories that currently do not have automated systems may selectively use commercial BMD systems for isolates, which are carbapenem resistant. E-test was found to be unacceptable with poor EA and high rates of MEs and VMEs and therefore, cannot be recommended for colistin testing.

The strength of our study is the inclusion of a large number of K. pneumoniae colistin-resistant isolates and the inclusion of mcr-1 isolate for QC. Our study had a few limitations. The number of colistin-resistant isolates of E. coli and afermenters included were small as they are infrequent in our set-up. Though we had prospectively collected the culture isolates during the study period, they were screened for the colistin resistance by Vitek-2 and thereafter stored before including in the study. This was done to include more number of colistin-resistant isolates of E. coli, P. aeruginosa and A. baumannii, which are infrequent at our centre as compared to K. pneumoniae. This may have resulted in the bias in our sample population, and therefore, extrapolation of results to other centres may not be possible. Furthermore, we did not do the genotypic analysis of the colistin resistance mechanism in our strains to confirm the colistin resistance, but we had included mcr-1 positive and negative strains for the quality control to check for the accuracy and standardization of BMD. Finally, this study was designed and conducted in the year 2018, but recently in the year 2020, epidemiological cut-off value for colistin AST by CLSI has been deleted and now assigned breakpoints of ≤2 mg/l as intermediate and ≥4 mg/l as resistant. However still the results of the study are important as it still demonstrates the variabilities associated with colistin AST with various methods.[15]

Finally, the results of cAST against BMD from one centre are difficult to extrapolate to other centers as the performance of any cAST is dependent on the MICs of the isolates being evaluated. Therefore, future multi-centric studies on a larger collection of colistin-resistant isolates may be required so that the better correlation with BMD can be assessed for various cAST methods.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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