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  Table of Contents  
CORRESPONDENCE
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 564-565
 

Polymyxin Nordmann/Poirel test for rapid detection of polymyxin resistance in Enterobacteriaceae: Indian experience


1 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Pathology, All Institute of Medical Science, New Delhi, India
3 Department of Hospital Infection Control, Narayana Health, Bengaluru, Karnataka, India

Date of Submission07-Oct-2016
Date of Acceptance18-Oct-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
Vijay Samuel Richard
Department of Hospital Infection Control, Narayana Health, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195382

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How to cite this article:
Bakthavatchalam YD, Veeraraghavan B, Mathur P, Purighalla S, Richard VS. Polymyxin Nordmann/Poirel test for rapid detection of polymyxin resistance in Enterobacteriaceae: Indian experience. Indian J Med Microbiol 2016;34:564-5

How to cite this URL:
Bakthavatchalam YD, Veeraraghavan B, Mathur P, Purighalla S, Richard VS. Polymyxin Nordmann/Poirel test for rapid detection of polymyxin resistance in Enterobacteriaceae: Indian experience. Indian J Med Microbiol [serial online] 2016 [cited 2017 Feb 24];34:564-5. Available from: http://www.ijmm.org/text.asp?2016/34/4/564/195382


Dear Editor,

Increasing multi-drug resistance (MDR) in Gram-negative pathogens has caused considerable therapeutic challenges in clinical practice. This has resulted in an increase in the use of polymyxins in treating MDR infections. However, chromosomal or plasmid-mediated (mcr-1 and mcr-2) polymyxin resistance are being increasingly reported worldwide.[1],[2],[3] Molecular methods are not always effective in detecting chromosomally-encoded polymyxin resistance. For polymyxin susceptibility testing, conventional methods such as disc diffusion and E-test are not reliable, while the standard reference technique of broth microdilution (BMD) is time-consuming (24 h).[4] Moreover, rapid and reliable methods for testing colistin or polymyxin B in the clinical settings are essential for timely and effective patient management. Recently, a rapid polymyxin Nordmann/Poirel (NP) test was developed for detection of polymyxin resistance in Enterobacteriaceae.[4] We sought to evaluate the performance of the rapid polymyxin NP test in detecting polymyxin resistance.

A total of 232 non-duplicate Enterobacteriaceae isolates recovered from bloodstream infection during 2014–2015 were randomly selected and included in this study. This collection included carbapenem-resistant Klebsiella sp., (n = 122) and Enterobacter sp., (n = 50) and intrinsically polymyxin-resistant Proteus mirabilis (n = 30), Morganella morganii (n = 15) and Serratia marcescens (n = 15) as an internal control. Minimum inhibitory concentration (MIC) of colistin and polymyxin B against Enterobacteriaceae isolates (Klebsiella sp., and Enterobacter sp.,) was determined using the BMD method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (M07–A10)[5] and interpreted as per CLSI guidelines (M100–S26). All the isolates were screened for polymyxin resistance using the rapid polymyxin NP test as previously described.[4] This test reagent contains cation-adjusted Mueller-Hinton broth, phenol red (pH indicator) and 10% anhydrous D(+)-glucose and colistin or polymyxin B at a final concentration of 5 µg/ml. This test involved incubation under aerobic conditions to improve glucose metabolism and accuracy of the results.

All the pathogens intrinsically resistant to polymyxin were found to be resistant to polymyxin B and colistin with the rapid polymyxin NP test (100%). Among the tested isolates, 30% (n = 37) of Klebsiella sp. and 18% (n = 9) of Enterobacter sp. were found to be resistant to both colistin and polymyxin B using both BMD and polymyxin NP tests, respectively [Table 1]. All the polymyxin (colistin and polymyxin B) resistant isolates of Klebsiella sp. were multi-drug resistant. However, all the polymyxin-resistant Enterobacter sp. isolates were carbapenem susceptible. Among the tested isolates, 70% (n = 85) of Klebsiella sp. and 18% (n = 41) of Enterobacter sp. were found to be colistin-susceptible (MIC ≤2 µg/ml) and gave negative results in the rapid polymyxin NP test. A high degree of concordance was seen between BMD and the rapid polymyxin NP test. In this study, the rapid polymyxin NP test was found to have a sensitivity, specificity, positive predictive value and negative predictive value of 100%. The performance of the rapid polymyxin NP results was similar with either colistin or polymyxin B. Further analysis of all the 37 polymyxin-resistant Klebsiella isolates, using next generation sequencing, revealed chromosomal mutations (unpublished data).
Table 1: Rapid polymyxin Nordmann/Poirel results, minimum inhibitory concentration (MIC) of colistin and polymyxin B among polymyxin-resistant Klebsiella sp., and Enterobacter sp.

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Interestingly, heteroresistance to colistin and polymyxin B was observed in Klebsiella pneumonaie isolate (n = 1) and this was found to be positive in the rapid polymyxin NP test [Figure 1]. A majority of the tested isolates showed positivity in the rapid polymyxin NP test in <3 h.
Figure 1: Colistin hetero-resistant Klebsiella pneumoniae

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The rapid polymyxin NP test is based on the metabolism of glucose by bacterial growth in the presence of defined concentration of colistin or polymyxin B. The acid metabolites produced are detected by a colour change (orange to yellow) of the phenol red pH indicator. This test was reported to have a sensitivity and specificity of 99.3% and 95.4%, respectively.[4] However, false positive results (30%) were reported while using bacterial colonies recovered from acidifying media.[4] In comparison, this study has shown the sensitivity and specificity of the rapid polymyxin NP test to be 100%; moreover, none of the tested isolates showed false-positive or false-negative results. Similarly, the performance of the rapid polymyxin NP test was reported to be excellent in early identification of polymyxin-resistant Enterobacteriaceae directly from the supernatant of the positive blood cultures (monomicrobial).[6]

It is well known that polymyxin susceptibility testing with Enterobacter sp., may results in multiple skip wells and this may lead to uninterpretable results, and therefore, the results should be performed with extreme caution.[7] However, in the present study, the tested Enterobacter sp. showed multiple skip wells at higher dilutions (MIC >8 µg/ml) rather than at the lower dilutions.

Susceptibility testing of polymyxin is challenging due to the (i) poorly defined relative proportion of polymyxin components, (ii) cationic property of polymyxin and (iii) heteroresistance to colistin/polymyxin B in MDR pathogens. In comparison with BMD (reference method), rapid polymyxin NP showed excellent discrimination between polymyxin susceptible and polymyxin-resistant enterobacterial isolates. It is easy to perform, rapid, reliable and cost-effective. Rapid polymyxin NP is a potential screening method for detecting polymyxin resistance in Enterobacteriaceae.

 
 ~ References Top

1.
Falagas ME, Rafailidis PI, Matthaiou DK. Resistance to polymyxins: Mechanisms, frequency and treatment options. Drug Resist Updat 2010;13:132-8.  Back to cited text no. 1
    
2.
Vasquez AM, Montero N, Laughlin M, Dancy E, Melmed R, Sosa L, et al. Investigation of Escherichia coli Harboring the mcr-1 resistance gene – Connecticut, 2016. MMWR Morb Mortal Wkly Rep 2016;65:979-80.  Back to cited text no. 2
    
3.
Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, et al. Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016. Euro Surveill 2016;21:30280.  Back to cited text no. 3
    
4.
Nordmann P, Jayol A, Poirel L. Rapid detection of polymyxin resistance in Enterobacteriaceae. Emerg Infect Dis 2016;22:1038-43.  Back to cited text no. 4
    
5.
Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow Aerobically; Approved Standard. 10th ed. CLSI Document M07-A10. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.  Back to cited text no. 5
    
6.
Jayol A, Dubois V, Poirel L, Nordmann P. Rapid detection of polymyxin-resistant Enterobacteriaceae from blood cultures. J Clin Microbiol 2016;54:2273-7.  Back to cited text no. 6
    
7.
Landman D, Salamera J, Quale J. Irreproducible and uninterpretable Polymyxin B MICs for Enterobacter cloacae and Enterobacter aerogenes. J Clin Microbiol 2013;51:4106-11.  Back to cited text no. 7
    


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