PurposeMultidrug-resistant (MDR) organisms are being increasingly reported from India. This study aimed to determine the antibiotic susceptibility pattern of non-fermenting Gram-negative bacilli (NF-GNB) isolated from all the clinical samples to estimate the prevalence of MDR MDR NF-GNB and to screen for colistin-resistance genes among all colistin-resistant strains.Materials and methodsThis prospective study conducted from January 2021 to July 2022 at a tertiary care teaching hospital in central India identified MDR NF-GNB from clinical samples using standard procedures and antimicrobial susceptibility testing conducted as per Clinical Laboratory Standards Institute (CLSI) guidelines. Colistin-resistant strains identified by broth microdilution were further subjected to detection of plasmid-mediated colistin-resistant genes (mcr-1,mcr-2,mcr-3) by polymerase chain reaction (PCR).ResultsA total 2,106 NF-GNB were isolated from 21,019 culture positive clinical samples, of which 743 (35%) were MDR. Majority of MDR NF-GNB isolated were from pus (45.50%) followed by blood (20.50%). Out of 743 non-duplicate MDR non-fermenters,the most common werePseudomonas aeruginosa(51.7%), Acinetobacter baumannii(23.4%),and others (24.9%).Around5.2%Pseudomonas aeruginosaand 2.3%Acinetobacter baumanniiwere resistant to colistin, and 88.2% were resistant to ceftazidime.Burkholderia cepaciacomplexwas 100% susceptible to minocycline and least susceptible to ceftazidime (28.6%). Out of 11, 10 (90.9%)Stenotrophomonas maltophiliawere susceptible to colistin and least susceptible to ceftazidime and minocycline (27.3%). All 33 colistin-resistant strains (minimal inhibitory concentration ≥ 4 µg/mL) were found to be negative formcr-1,mcr-2, andmcr-3genes.ConclusionOur study showed a significantly wide variety of NF-GNB, ranging fromPseudomonas aeruginosa(51.7%),Acinetobacter baumannii(23.4%),toAcinetobacter haemolyticus(4.6%),Pseudomonas putida(0.9%),Elizabethkingia meningoseptica(0.7%),Pseudomonas luteola(0.5%), andRalstonia pickettii(0.4%), which have not been commonly reported in literature. Of all the non-fermenters isolated in the present study, 35.28% were MDR, raising the concern for rationalizing antibiotic use and improving infection control measures to avert or slow the emergence of antibiotic resistance.