Colistin and Polymyxin B sensitivity by broth microdilution among carbapenem-resistant clinical isolates of Gram-negative bacilli

Introduction and Aim: The increasing incidence of multidrug-resistant gram-negative bacilli (MDR-GNB) in recent times has led to the revival of older neglected antibiotics such as Polymyxins. Hence, there is a need to determine the minimum inhibitory concentration (MIC) of these antibiotics by conventional and automated antibiotic sensitivity testing methods to assess their clinical utility. This study aimed to assess the in vitro sensitivity of clinical isolates grown with carbapenem-resistant GNB (CR-GNB) to Colistin and Polymyxin B by broth micro-dilution method (BMD). Materials and Methods: Thirty isolates of CR-GNB obtained from various clinical samples received in the microbiology laboratory of Yenepoya Medical College Hospital between August 2018 to November 2019 were included in the study. In vitro assessment of Colistin and Polymyxin B sensitivity among CR-GNB was performed by BMD as per CLSI guidelines. Results: Twelve isolates (40%) were carbapenemase producers. Among the 30 isolates of CR-GNB, 56.6% and 63.3 % of them were sensitive to Colistin and Polymyxin, respectively. Colistin MICs of 12 isolates obtained by BMD assay were concordant with the MICs obtained by BD Phoenix. Conclusion: The results of this study indicate that Colistin and Polymyxin B can be considered as reserve therapeutic agents for treating infections caused by CR-GNB after obtaining the in vitro sensitivity results.


INTRODUCTION
he last two decades have witnessed a remarkable increase in antibiotic resistance among gram-negative bacilli.Many of these multidrug-resistant gram-negative bacilli (MDR-GNB) are associated with hospital-acquired infections, leading to increased morbidity, mortality, and healthcare costs (1).MDR-GNB are ubiquitous in hospitals and can cause infections of the urinary tract, lower respiratory tract, biliary tract, bloodstream, etc.One of the main factors driving the development of antimicrobial resistance (AMR) among GNBs is the selective pressure due to the irrational use of antibiotics (2).These bacteria acquire genes encoding multiple antibiotic resistance mechanisms, including extended-spectrum β-lactamases (ESBLs), AmpCs, and carbapenemases.Carbapenems are considered the best therapeutic option available against MDR-GNB.However, the emergence and spread of carbapenemresistant Enterobacteriaceae (CRE) is a significant clinical and public health concern.CRE are often resistant to all β-lactam drugs and frequently carry mechanisms conferring resistance to other antimicrobial classes, further limiting treatment options.Infections caused by CRE are associated with higher mortality rates than those infections caused by carbapenems-susceptible bacteria due to which World Health Organization (WHO) has labelled CRE, extensively drug-resistant (XDR) Pseudomonas aeruginosa (P.aeruginosa) and XDR Acinetobacter baumannii (A.baumanii)as a "critical" threat (3,4).(5).Therefore, with rising rates of infections caused by CR-GNB, there is a need for a well-established and reliable Colistin susceptibility testing method that routine clinical laboratories can perform to guide appropriate therapeutic decision-making.

MATERIALS AND METHODS
This prospective descriptive study was conducted on 30 consecutive clinical isolates of CR-GNB in a tertiary care medical college hospital in South India.Clinical samples for bacterial culture were collected and processed following standard operating procedures (6).Bacterial identification and antibiotic sensitivity testing were performed by an automated BD Phoenix system based on which CR-GNB was identified.Detection of carbapenemase and metallo beta-lactamase production among the CR-GNB isolates were by Modified Hodge test and Meropenem-EDTA double-disc synergy test, respectively, as recommended by CLSI (7).

MIC testing of Colistin and Polymyxin B by BMD assay
Antibiotic stock solutions were prepared from pure powders of Colistin Sulfate and Polymyxin B (Himedia), using sterile distilled water (DW) as the solvent and diluent.The quantity of antibiotic powder needed for a particular dilution was calculated using the formula: Weight (mg) = [Volume (mL) × concentration (mg/mL)] / Potency (µg/mg) Colistin and Polymyxin B dilutions from 0.25µg/mL to 256 µg/mL were prepared by serial two-fold dilutions.Well-isolated 3-5 bacterial colonies of the same morphological type were picked from an 18-24 hour-old bacterial culture, inoculated into a test tube containing 4-5ml of tryptic soy broth, and incubated at 35ºC for 3-4 hours.The inoculum was then adjusted to 0.5 McFarland standard using sterile normal saline to give approximately 1-2×10 8 CFU/ml.The final inoculum for the BMD was prepared by diluting the above suspension (1:20) using sterile normal saline to yield 5×10 6 CFU/ml.When 0.01mL of this bacterial suspension was inoculated into 0.1mL of MHB in the microtiter well, the final bacterial concentration was approximately 5×10 5 CFU/ml.Microdilution was prepared by taking 90 µL of cationadjusted MHB in each well to which 10 µL of the bacterial suspension and 100 µL of antibiotic solution were added.Sterility control and growth control wells were included for each microtiter plate, which was covered and incubated at 35±2 0 C for 18-20 hours.The lowest concentration of the antimicrobial agent that completely inhibited the growth of bacteria as detected by the unaided eye was considered as the MIC.EUCAST and CLSI guidelines were used for interpreting the MIC values (8).
Among 30 CR-GNB, 12 (40%) were carbapenemase producers by MHT, and 18 (60%) were MBL producers by meropenem-EDTA double disc synergy test.Table 2 shows the MIC of Colistin and Polymyxin B of CR-GNB isolated by MBD method and comparison of Colistin MIC with BD Phoenix automated method.This comparison showed the concordance of Colistin MICs of 12 isolates by MBD assay and BD Phoenix (Table 3).Kappa agreement measure= -0.297 with p-value =0.062 (p>0.05)Ho: Assumption of concordance.Kappa test was used to test the concordance of MIC of Colistin by MBD assay and BD Phoenix.Null hypothesis of the Kappa test states that there is no agreement between the MIC results obtained by the two testing methods as per the values of Kappa & significance.This proves that there is no significant agreement of the results for Colistin MIC between MBD and BD Phoenix method.

Table 2 :
MIC of colistin and polymyxin B by MBD method and its comparison with BD Phoenix

Table 3 :
Colistin sensitivity by BD phoenix