Non-fermenting Gram-negative bacilli (NFGNB) are a heterogeneous group of aerobic bacteria increasingly recognized as important causes of healthcare-associated and opportunistic infections. Pseudomonas aeruginosa and Acinetobacter baumannii are prominent pathogens, while Stenotrophomonas maltophilia, Burkholderia cepacia complex and other species also contribute to clinically significant infections, particularly among hospitalized and critically ill patients [1–3]. Management of NFGNB infections is challenging because these organisms possess intrinsic and acquired antimicrobial resistance mechanisms that may limit therapeutic options. Resistance patterns vary across species and geographical settings, emphasizing the importance of local surveillance and routine antimicrobial susceptibility testing [2]. Biofilm formation can facilitate bacterial persistence on surfaces and medical devices and may contribute to reduced antimicrobial effectiveness and persistent infections [4–6]. Congo red agar (CRA) provides a simple phenotypic screening approach, although its performance varies and results should be interpreted in light of the limitations of qualitative methods [7,8]. This study aims to determine the species distribution of NFGNB isolated from clinical specimens, assess biofilm-associated characteristics and evaluate antimicrobial susceptibility patterns in a tertiary care hospital.

Study design and setting A descriptive observational study was designed in the Department of Microbiology at a tertiary care teaching hospital over a period of nine months i.e from Jan 2026 to Sept 2026. A total of 100 NFGNB were processed during the study period. Inclusion Criteria 1. Clinical specimens received in the Department of Microbiology during the study period and processed for bacterial culture. 2. Non-fermenting Gram-negative bacilli (NFGNB) isolated from clinical specimens and identified using standard microbiological methods. 3. Isolates identified as Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia cepacia complex or other NFGNB. 4. Isolates with sufficient growth and viability for biofilm assessment and antimicrobial susceptibility testing, as applicable. Exclusion Criteria 1. Duplicate isolates of the same species from the same patient during the same episode of infection, if the study protocol includes only one isolate per patient episode. 2. Isolates with insufficient growth or loss of viability that prevents reliable identification or further testing. 3. Isolates with incomplete identification or inadequate laboratory data for the planned analysis. Specimen processing and identification Specimens included pus/wound swabs, urine, respiratory specimens (sputum, endotracheal tube secretions and bronchoalveolar lavage), blood and other body fluids. Specimens should be processed using the standard procedures actually followed in the laboratory. Identification was based on colony morphology, Gram staining, glucose non-fermentation and relevant biochemical. Biofilm detection Biofilm-associated characteristics were assessed using CRA. The medium was prepared using brain–heart infusion broth supplemented with sucrose, agar and Congo red dye. Following inoculation and incubation according to the laboratory protocol, colony colour and morphology were assessed. Black colonies were considered positive and red/pink colonies negative according to the adopted protocol. Biofilm production was not graded. Positivity was calculated as the proportion of positive isolates among the isolates of each species. Antimicrobial susceptibility testing Susceptibility testing was represented as Kirby–Bauer disc diffusion on Mueller–Hinton agar, interpreted using the CLSI guidance. The antibiotic discs were imipenem, meropenem, piperacillin–tazobactam, ceftazidime, cefepime, amikacin, ciprofloxacin and cotrimoxazole.

11%), B. cepacia complex (3; 3%) and other NFGNB (8; 8%). Respiratory specimens contributed 33% of isolates, followed by pus/wound specimens (30%), urine (17%), blood (13%) and body fluids/other specimens (7%). Table 1. Sample-wise distribution of NFGNB species Sample type P. aeruginosa A. baumannii S. maltophilia B. cepacia complex Others Total % Pus/wound swab 15 9 3 1 2 30 30% Urine 10 5 1 0 1 17 17% Respiratory (sputum/ET secretion/BAL) 12 13 4 1 3 33 33% Blood 4 6 2 0 1 13 13% Body fluids/others 2 2 1 1 1 7 7% Total 43 35 11 3 8 100 100% Others includes Alcaligenes/Achromobacter spp., Chryseobacterium spp., Elizabethkingia spp. and unspeciated NFGNB. Biofilm production In the present study, 78/100 isolates (78%) were CRA-positive. Species-specific positivity was highest for A. baumannii (30/35; 85.7%), followed by P. aeruginosa (33/43; 76.7%), S. maltophilia (8/11; 72.7%), B. cepacia complex (2/3; 66.7%) and other NFGNB (5/8; 62.5%). Table 2. Biofilm production by Congo red agar method Species Positive (n) Non-producer (n) Total (n) % positive P. aeruginosa 33 10 43 76.7% A. baumannii 30 5 35 85.7% S. maltophilia 8 3 11 72.7% B. cepacia complex 2 1 3 66.7% Others 5 3 8 62.5% Total 78 22 100 78.0% Antimicrobial resistance In our study, antibiogram showed higher reported resistance percentages for A. baumannii than P. aeruginosa for several listed agents. Percentages require confirmation against the actual number tested and applicable species-specific interpretive criteria. Table 3. Antimicrobial resistance among NFGNB isolates Species N tested Imipenem Meropenem Pip-Tazo Ceftazidime Cefepime Amikacin Ciprofloxacin Cotrimoxazole P. aeruginosa 43 38% 36% 41% 48% 45% 32% 46% — A. baumannii 35 58% 56% 68% 72% 70% 54% 64% 40% S. maltophilia 11 — — — — — — 35% 12% B. cepacia complex 3 — — 38% 34% — 30% 22% — Others 8 — — — — — — — — Total isolates 100 — — — — — — — —

Non-fermenting Gram-negative bacilli (NFGNB) are important opportunistic pathogens associated with healthcare-associated infections because of their persistence in hospital environments and their intrinsic and acquired antimicrobial resistance. The present study evaluated the distribution of NFGNB, biofilm production using Congo red agar (CRA), and antimicrobial susceptibility patterns. Pseudomonas aeruginosa was the predominant isolate (43%), followed by Acinetobacter baumannii (35%), Stenotrophomonas maltophilia (11%), Burkholderia cepacia complex (3%), and other NFGNB (8%). Respiratory specimens accounted for the largest proportion of isolates (33%), followed by pus and wound specimens (30%). Similar predominance of P. aeruginosa was reported in Kakinada, Andhra Pradesh, where it constituted 49% of NFGNB isolates [8]. Grewal et al. also identified P. aeruginosa as the predominant species (87.96%) in a tertiary-care hospital in Patiala [9]. In contrast, Sarkar et al. reported A. baumannii as the predominant isolate (51.34%) in eastern India [10]. A five-year study from eastern India further identified Acinetobacter species as the predominant NFGNB group, with respiratory specimens representing an important source of isolates [13]. These variations may reflect differences in patient populations, specimen distribution, referral patterns, and local hospital epidemiology. The distribution of isolates by specimen type should not be interpreted as specimen-specific prevalence without the total number of specimens processed. Biofilm production was detected in 78% of isolates in our study. The highest proportion was observed among A. baumannii (85.7%), followed by P. aeruginosa (76.7%) and S. maltophilia (72.7%). Biofilm formation facilitates microbial adherence and persistence on surfaces and medical devices and may contribute to prolonged infections. CRA is a relatively simple and economical screening method for detecting phenotypic characteristics associated with biofilm production. However, its results depend on the organism, culture conditions, and interpretation criteria. Research involving clinical Gram-negative bacilli has demonstrated the use of CRA for biofilm screening [11], while studies of multidrug-resistant A. baumannii have shown that biofilm detection varies with the method employed [12]. Thus, CRA positivity should be interpreted as a qualitative screening result rather than a quantitative measurement of biofilm biomass. Our study demonstrated higher resistance percentages among A. baumannii than P. aeruginosa for several agents. Resistance among A. baumannii was 72% to ceftazidime, 70% to cefepime, 68% to piperacillin–tazobactam, and 64% to ciprofloxacin. In comparison, the corresponding percentages among P. aeruginosa were 48%, 45%, 41%, and 46%, respectively. Reddi and Israel reported high resistance to cefepime and ceftazidime among NFGNB in Kakinada, although susceptibility to imipenem was high [8]. Grewal et al. also reported substantial susceptibility to imipenem, while multidrug-resistant A. baumannii was identified in their study [9]. Sarkar et al. documented differences in susceptibility between Acinetobacter, Pseudomonas, and other non-fermenters [10]. A central Indian study similarly highlighted the clinical importance of multidrug-resistant NFGNB [14]. These findings emphasize the need for local antibiograms and periodic surveillance rather than extrapolating susceptibility results between institutions. The findings for S. maltophilia and B. cepacia complex require particular caution because of their small sample sizes. Furthermore, species-level resistance percentages cannot establish a direct association between biofilm production and antimicrobial resistance. Such an association requires isolate-level comparison between CRA-positive and CRA-negative groups. The study also has limitations associated with the qualitative nature of CRA, the small numbers of less frequently isolated species, and its single-centre setting. Overall, combined surveillance of species distribution, biofilm-associated phenotypes, and antimicrobial susceptibility may provide useful information for infection prevention and antimicrobial stewardship.

NFGNB represent important clinical pathogens with varied species distribution, biofilm-forming potential, and antimicrobial susceptibility patterns. Pseudomonas aeruginosa and Acinetobacter baumannii were the predominant isolates in the provisional dataset, with CRA-detected biofilm production observed across species. The comparatively higher resistance percentages among A. baumannii highlight the importance of species-specific surveillance. CRA provides an economical screening method, although its qualitative nature limits assessment of biofilm strength. Periodic antimicrobial susceptibility monitoring and appropriate infection-control measures are essential.

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