Many laboratories are unable to perform colistin susceptibility testing. Diffusion-based antimicrobial susceptibility testing methods are not recommended, and not all laboratories have the capacity to perform broth microdilution (BMD).
KEYWORDS: AST, MicroScan, antimicrobial susceptibility testing, colistin, polymyxin
ABSTRACT
Many laboratories are unable to perform colistin susceptibility testing. Diffusion-based antimicrobial susceptibility testing methods are not recommended, and not all laboratories have the capacity to perform broth microdilution (BMD). Using a multistep tiered approach, we investigated whether the adapted use of the MicroScan colistin well (4 μg/ml) could enhance laboratory capacity for the detection and subsequent molecular characterization of colistin-resistant Enterobacteriaceae. For the MicroScan colistin well, categorical agreement with BMD was 92.7%, and the very major error rate was 10.7%. For gradient diffusion strips, the categorical agreement was 86.4%, and the very major error rate was 53.6%. The MicroScan colistin well detected all isolates carrying mcr-1 or mcr-2 genes (n = 16), but gradient diffusion strips identified an MIC of ≥4 for colistin for only 62.5% of these isolates. A 6-month prospective phenotypic and genotypic study performed at a single clinical microbiology laboratory assessed isolates growing in the MicroScan colistin well for concordance. While 37 of 39 isolates growing in the MicroScan colistin well displayed a colistin MIC of ≥4 by BMD, all were determined to be negative for the mcr-1 and mcr-2 genes by PCR. A retrospective review of all Escherichia coli, Klebsiella spp., and Enterobacter spp. tested by MicroScan at this laboratory in 2016 identified 260 of 7,894 (3.3%) isolates that grew in the MicroScan colistin well. Based on the data presented, clinical and public health laboratories could use the MicroScan colistin well as a first screen for the detection of isolates displaying elevated colistin MICs, which could then undergo further characterization.
INTRODUCTION
Antibiotic resistance is a global public health problem. Antibiotic resistance in Gram-negative pathogens is an especially serious threat given the limited treatment options available for patients with these infections (1, 2). While new drugs, such as ceftazidime-avibactam, meropenem-vaborbactam, and ceftolozane-tazobactam, have provided much-needed additions to the current antibiotic armamentarium, some pathogens display resistance to these new β-lactam combination agents (3–5). The most significant of these resistant pathogens are multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and Enterobacteriaceae with metallo-β-lactamases (MBLs) such as New Delhi MBL (NDM) (6–8). For patients infected with these organisms, the therapeutic use of colistin may still be considered (9, 10).
However, there are some complicating factors to using colistin to treat serious infections. First and foremost, colistin is not a very effective drug (11–13). A recent study demonstrated a 28-day mortality of 43% when using colistin to treat carbapenem-resistant, colistin-susceptible Gram-negative bacterial infections (11). Another study has demonstrated the difficulty in achieving appropriate drug levels with currently recommended doses (13). Lastly, colistin antimicrobial susceptibility testing (AST) is associated with technical and methodological challenges (14). Broth microdilution (BMD) is currently the only recommended method for colistin AST, but few laboratories in the United States have the capacity to perform BMD (15–18). Disk diffusion and gradient diffusion strips are inaccurate but still commonly performed in laboratories (16, 19–21). The automated instruments (e.g., Vitek, MicroScan, BD Phoenix, and Sensititre) are the most common platforms for performing AST in the United States (17). However, these instruments do not have a U.S. Food and Drug Administration (FDA)-cleared test for colistin susceptibility because there are no FDA breakpoints for colistin, although some have research-use-only panels (22). Some of these automated instruments do have colistin susceptibility testing in countries outside the United States.
Since the discovery of mobile colistin resistance (mcr) genes, multiple variants have been found (23–27). The Centers for Disease Control and Prevention (CDC) has developed a real-time PCR assay for the detection of mcr-1 and mcr-2 that has been shared with state and local public health laboratories through the Antibiotic Resistance (AR) Laboratory Network (28). However, surveillance for mcr genes largely relies on the ability to perform AST for colistin. One possible solution to increase laboratory capacity to screen for resistance to colistin is adapted use of the MicroScan colistin well (Beckman Coulter, Brea, CA). On Gram-negative MicroScan panels, there is a well with 4 μg/ml of colistin that is intended to help with bacterial identification. For example, Serratia marcescens, Proteus mirabilis, Providencia spp., and Morganella morganii are intrinsically resistant to colistin, whereas Escherichia coli, Klebsiella pneumoniae, and Salmonella spp. are not. In this study, we compared the MicroScan colistin well to the gold standard (broth microdilution) and a method frequently used by microbiology laboratories (gradient diffusion strips) to investigate whether the MicroScan colistin well could distinguish which isolates were colistin resistant, which in turn would help determine which isolates should be screened by PCR for mcr genes.
MATERIALS AND METHODS
There were three different components to our evaluation of the MicroScan colistin well. We performed a laboratory comparison of AST data generated by MicroScan and gradient diffusion strips compared to reference broth microdilution on a diverse set of Enterobacteriaceae isolates. We also conducted a phenotypic and genotypic characterization of isolates that grew in the MicroScan colistin well of a large academic clinical microbiology laboratory during a 6-month study period. Finally, we completed a retrospective review of isolates tested by MicroScan at Emory University during 2016. This study was conducted under Research Collaborative Agreement D-448-17 between the CDC and Emory University. Portions of the data presented in the manuscript were previously presented at IDWeek 2017, ECCMID 2018, and ASM Microbe 2018 (29–31).
Laboratory comparison of colistin susceptibility testing methods.
Three methods of colistin susceptibility testing (MicroScan well, broth microdilution, and gradient diffusion strips) were evaluated using 111 Enterobacteriaceae isolates from CDC (Table 1 ). The isolates were collected through reference and surveillance activities of the Clinical and Environmental Microbiology Branch (CEMB) or from outside collaborators. Some of these isolates are in the CDC and FDA Antibiotic Resistance Isolate Bank (AR Bank) (Table 1) (32). All challenge set isolates were chosen for their diversity in organism, colistin susceptibilities by reference BMD, and representation of mcr genes. These included 61 Klebsiella pneumoniae, 33 Escherichia coli, 7 Enterobacter cloacae complex, 2 Klebsiella oxytoca, 2 Klebsiella aerogenes (formerly Enterobacter aerogenes), 2 Citrobacter freundii, 1 Escherichia albertii, and 3 Salmonella isolates (Table 1). All but two isolates were of human origin; these two nonhuman isolates were mcr positive and obtained from collaborators.
TABLE 1.
Colistin broth microdilution MIC distribution for 111 Enterobacteriaceae isolates
| Isolate | No. of isolates with the colistin MIC (μg/ml) of: |
|||||||
|---|---|---|---|---|---|---|---|---|
| ≤0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | Skipped wells | |
| mcr-1 positive | ||||||||
| E. coli | 8a | 3b | ||||||
| K. pneumoniae | 1 | |||||||
| Salmonella spp. | 3c | |||||||
| mcr-2 positive | ||||||||
| E. coli | 1d | |||||||
| mcr-1, mcr-2 negative | ||||||||
| C. freundii | 1 | 1 | ||||||
| E. cloacae complex | 6 | 1 | ||||||
| E. albertii | 1 | |||||||
| E. coli | 18e | 2 | 1 | |||||
| K. aerogenes | 1 | 1 | ||||||
| K. oxytoca | 1 | 1 | ||||||
| K. pneumoniae | 42f | 2 | 4 | 2 | 3 | 6 | 1 | |
Includes AR Bank no. 0346 and AR Bank no. 0349.
Includes AR Bank no. 0493 and AR Bank no. 0494.
Includes AR Bank no. 0496.
AR Bank no. 0538.
Includes AR Bank no. 0160.
Includes AR Bank no. 0162.
BMD was performed at CDC using in-house developed frozen reference panels according to Clinical and Laboratory Standards Institute (CLSI) guidelines (33). These panels were prepared using 96-well sterile, U-bottom polystyrene plates (Caplugs, Buffalo, NY) with cation-adjusted Mueller-Hinton broth (BD Difco, Sparks, MD). No surfactant was used in accordance with CLSI guidelines. The colistin MIC range on the plates was ≤0.25 to >8 μg/ml, and colistin was obtained as colistin sulfate salt (Sigma-Aldrich, St. Louis, MO). The 0.5 McFarland solution was obtained using organism and 0.85% saline. The panels were inoculated with a 95-pin sterile inoculator (10 μl/pin pickup; Caplugs). Gradient diffusion strip AST (Etest, bioMérieux, Durham, NC) was performed according to the recommendations in the package insert. The MIC range on the strips was ≤0.016 to >256 μg/ml. The strips were placed on BBL Mueller-Hinton Agar II (Becton Dickinson, Sparks, MD). There were no special reading instructions followed (read with the naked eye), except that all MICs were rounded up to the nearest doubling dilution as recommended by the manufacturer. The MicroScan dried Gram-negative panel (Neg Breakpoint Combo 44; containing the 4 μg/ml colistin well) was inoculated using the MicroScan Prompt method according to the package insert (final well concentration, 3 to 7 × 105 CFU/ml per the package insert). The Prompt inoculation system does contain PLURONIC surfactants (BASF Corporation, Parsippany, NJ). The panels were incubated off-line (not in the MicroScan WalkAway instrument) and read manually. For all three testing methods (BMD, gradient diffusion strips, and the MicroScan colistin well), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used for quality control (American Type Culture Collection, Manassas, VA). Isolates were tested with all three AST methods on the same day from the same bacterial inoculum (same colonies were used to prepare the 0.5 McFarland solution and for the Prompt method). There was no discrepancy resolution or repeat testing performed. For colistin MIC interpretation, a CLSI-established epidemiological cutoff value (ECV) was adapted because not all species tested have an ECV (15); wild-type colistin MIC was defined as ≤2 μg/ml, and non-wild-type colistin MIC was defined as ≥4 μg/ml.
Prospective study to detect elevated colistin MIC values.
From 1 May 2017 to 25 October 2017, all Enterobacteriaceae isolates with growth in the MicroScan colistin well at the Emory University clinical microbiology laboratory were flagged for transfer to CDC. For these isolates, a sterile disposable bacterial loop was dipped into the turbid colistin well and streaked out onto tryptic soy agar with 5% sheep blood (Thermo Scientific Remel Products, Lenexa, KS). These plates were incubated overnight at 35°C in ambient air, held at room temperature, and transferred to the CEMB laboratory. At CEMB, AST was performed using reference BMD, observation of growth in the MicroScan colistin well, and gradient diffusion strips as described above. All isolates displaying a colistin MIC of ≥2 μg/ml by reference BMD were evaluated for the presence of mcr-1 and mcr-2 by real-time PCR (28).
Retrospective review.
The Emory University clinical microbiology laboratory performed bacterial identification and AST on clinical isolates using a MicroScan WalkAway-96 Plus system (Beckman Coulter Diagnostics, Brea, CA) and a Neg Breakpoint Combo Panel Type 41 or 44 (transition from 41 to 44 occurred in March 2016) between 1 January 2016 and 31 December 2016. A retrospective review was performed of the MicroScan AST results recorded for all E. coli, K. pneumoniae, K. oxytoca, E. cloacae, and K. aerogenes isolates with growth in the MicroScan colistin well during this time period.
RESULTS
Laboratory comparison of colistin susceptibility testing methods.
Using reference BMD as the gold standard, 111 isolates with various colistin MICs were evaluated; 16 isolates had an mcr-1 or mcr-2 gene (Table 1). With reference BMD, one isolate MIC could not be interpreted (skipped wells), 82 were wild type (MIC of ≤2 μg/ml), and 28 were non-wild type (MIC of ≥4 μg/ml). Gradient diffusion strips had a categorical agreement of 86.4% and an essential agreement of 77.3%; there were no major errors and 15 very major errors (53.6%) (Table 2). For the MicroScan colistin well, the categorical agreement was 92.7%; there were 5 major errors (6.1%) and 3 very major errors (10.7%) (Table 3). For the 16 isolates with an mcr gene, reference BMD testing yielded an MIC of ≥4 μg/ml in all instances. Gradient diffusion strips detected an MIC of ≥4 μg/ml in 10 (62.5%) mcr-positive isolates and the MicroScan colistin well showed growth for all 16 (100%).
TABLE 2.
Scattergram of colistin MICs for 110 isolates of Enterobacteriaceae measured by reference BMD and gradient diffusion stripsa
| Gradient diffusion strips (μg/ml) | Reference broth microdilution (μg/ml) |
||||||
|---|---|---|---|---|---|---|---|
| ≤0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | |
| ≤0.25 | 62 | 2 | 7 | 3 | 1 | 3 | 0 |
| 0.5 | 6 | 0 | 1 | 0 | 0 | 1 | 1 |
| 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| 2 | 0 | 0 | 0 | 0 | 4 | 1 | 4 |
| 4 | 0 | 0 | 0 | 0 | 5 | 5 | 1 |
| 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| >8 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
Data in the body of the table are the numbers of isolates determined by the specific method to have the indicated MIC.
TABLE 3.
Scattergram of colistin MICs for 110 isolates of Enterobacteriaceae measured by reference BMD and the MicroScan colistin well (4 μg/ml)a
| MicroScan colistin well (4 μg/ml) | Reference broth microdilution (μg/ml) |
||||||
|---|---|---|---|---|---|---|---|
| ≤0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | |
| No growth | 66 | 2 | 7 | 2 | 0 | 2 | 1 |
| Growth | 3 | 0 | 1 | 1 | 10 | 8 | 7 |
Data in the body of the table are the numbers of isolates determined by the specific method to have the indicated MIC.
Prospective study to detect colistin resistance.
Across a 6-month period of isolate collection, there were 39 instances of Enterobacteriaceae growth in the MicroScan colistin well. This included 18 E. cloacae, 12 K. pneumoniae, 8 E. coli, and 1 K. oxytoca isolates. Testing by BMD revealed 37 isolates that were non-wild type (MIC of ≥4 μg/ml) (94.9%). There was one isolate with an MIC of 4 μg/ml, one with an MIC of 0.5 μg/ml, and one with an MIC of ≤0.25 μg/ml. Gradient diffusion strips detected a colistin MIC of ≥4 in 24 of the 39 isolates with growth in the MicroScan well (61.5%) and 24 of the 37 isolates with confirmed resistance by BMD (64.9%). All 37 isolates with a colistin MIC of ≥2 (by BMD) were negative for the mcr-1 and mcr-2 genes by PCR.
Retrospective review.
The Emory University clinical microbiology laboratory identified 7,894 isolates of E. coli, K. pneumoniae, K. oxytoca, E. cloacae, or K. aerogenes in 2016. Of these, 260 (3.3%) had growth in the MicroScan colistin well. Growth in the colistin well differed by species as follows: 1.5% of E. coli (79/5135), 4.2% of Klebsiella spp. (90/2158), and 15.1% of Enterobacter spp. (E. cloacae and K. aerogenes) (91/601).
DISCUSSION
We sought to evaluate the performance of the MicroScan colistin well and gradient diffusion strips compared to reference BMD for detecting Enterobacteriaceae with elevated colistin MICs. We also sought to determine the prevalence in one clinical microbiology laboratory of isolates displaying elevated MICs to colistin using the MicroScan and whether any mcr genes were present in these isolates.
In this study, the MicroScan colistin well performed better than gradient diffusion strips. Compared with reference BMD, categorical agreement for isolates was higher for the MicroScan colistin well (92.7%) than for gradient diffusion strips (86.4%). Of particular importance, there were fewer very major errors with the MicroScan colistin well (10.7%) compared with gradient diffusion strips (53.6%). The MicroScan colistin well also showed growth for all isolates with a known mcr gene, whereas only 62.5% of mcr-positive isolates tested with gradient diffusion strips displayed an MIC of ≥4. Our results reinforce the findings of several others that for colistin, gradient diffusion strips are inaccurate (19–21).
The use of MicroScan for colistin susceptibility testing has been described previously (34, 35). In line with the findings of the present study, these earlier studies reported a categorical agreement of 88.2 and 91.9% with BMD (34, 35). However, these studies were performed outside the United States, where MicroScan panels have two colistin wells (2 and 4 μg/ml). The high rates of very major errors with gradient diffusion strips have been described before (19–21). The very major errors with gradient diffusion strips led a joint CLSI-European Committee on Antimicrobial Susceptibility Testing (EUCAST) polymyxin breakpoint working group to state that BMD without surfactant is the reference standard for colistin susceptibility testing and that diffusion methods should be abandoned (16).
In the present study, an estimate for colistin non-wild-type prevalence in E. coli, K. pneumoniae, K. oxytoca, E. cloacae, and K. aerogenes was established for a single academic medical center using the MicroScan colistin well. Few such colistin resistance prevalence studies have been conducted because few clinical microbiology laboratories perform routine testing for colistin resistance (17, 18). Previous estimates for colistin resistance prevalence have varied. In one study from a single academic medical center over a 6-year period using BMD, 0.45% of all Gram-negative rods (excluding isolates with intrinsic colistin resistance) showed a colistin MIC of ≥4 μg/ml, and 1.5% of all K. pneumoniae had an MIC of ≥4 μg/ml (36). In another study evaluating carbapenem-resistant K. pneumoniae (CRKP) using broth macrodilution, 13% of CRKP had an MIC of ≥4 μg/ml (37). Our study found that the prevalence of colistin MICs of >4 μg/ml varied by species from 1.5% of E. coli to 4.2% of Klebsiella spp. to 15.1% of Enterobacter spp. None of the isolates prospectively collected with an MIC of ≥2 μg/ml by BMD in our study were found to harbor an mcr gene.
There are some limitations to using the MicroScan colistin well in practice. A major drawback of the MicroScan colistin well (4 μg/ml) is that on traditional Gram-negative MicroScan panels in the United States, there is only one well. Thus, it would be presumed that labs can only detect isolates with an MIC of >4, which does not match the current CLSI ECV and that isolates with an MIC of 4 μg/ml should not grow in the MicroScan well. Nevertheless, most of the isolates in our study with an MIC of 4 μg/ml by BMD did grow. Another limitation to using the MicroScan well as a first screen to detect mcr-positive isolates is that there have been several reports of mcr isolates with an MIC of 2 μg/ml (or even less) to colistin or polymyxin B (34, 38). There is not currently a solution to this issue aside from screening all Enterobacteriaceae isolates, which would be very labor-intensive. Using the present study as an example, screening all Enterobacteriaceae would require screening 7,894 isolates, while only screening isolates with growth in the MicroScan well would require screening 260 isolates.
There were also limitations to our study. At the time of the study, our laboratory’s PCR assay covered only mcr-1 and mcr-2. Since this time, several additional mcr variants have been described (25–27). Another limitation is that we tested isolates that do not have an ECV (e.g., Salmonella and Citrobacter spp.) but have been found to harbor mcr genes (39, 40). We did this by adapting the ECV from other similar species.
Colistin susceptibility testing remains a challenge, despite it being a desirable goal. In the United States, microbiology laboratories have few options. Gradient diffusion strips for colistin testing should not be used because of their high rates of very major errors. Reference BMD remains the gold standard, and some laboratories do use this method (36). Others have evaluated Sensititre Research Use Only GNX2F plates (Thermo Fisher, Waltham, MA) (41). A study by Richter et al. examined 106 carbapenem-resistant K. pneumoniae isolates (28 with an MIC of ≥4 μg/ml) and compared Sensititre GNX2F plates to broth macrodilution and found an essential agreement of 94.3% and a categorical agreement of 96.2% (41). Finally, the recently developed colistin broth disk elution test may be another option (42). Nevertheless, colistin testing is difficult for most labs to implement.
The MicroScan colistin well may be an aid to laboratories looking for a way to screen for elevated colistin MICs and select isolates for mcr gene detection. Laboratories could use E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) as quality control strains and isolates from the CDC and FDA Antibiotic Resistance Isolate Bank (especially the “Isolates with New or Novel Antibiotic Resistance Panel”) to help develop an internal quality control program to bring on this testing. The intent would be that if a laboratory wanted to narrow down which isolates should be screened for mcr or elevated colistin MICs, the MicroScan colistin well could be used as a first screen, followed by BMD and/or mcr PCR testing.
ACKNOWLEDGMENTS
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The findings and conclusions in the manuscript are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
We acknowledge Gian Maria Rossolini, Vincenzo Di Pilato, Surbhi Malhotra-Kumar, Basil Britto Xavier, Anette Hammerum, Robert Skov, and Richard Meinersmann for providing mcr-positive isolates. Finally, we thank the medical technologists at Emory University, especially Deborah Hudson, for their assistance in obtaining the data used to generate the manuscript.
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