ESBL-Producing Acinetobacter baumannii from Households near Tertiary Health care settings in Kerala: A One health Surveillance study

Introduction

Antibiotics revolutionized medicine, enabling progress in surgery, organ transplants, cancer treatment, infection treatment and more. The need to survive causes microorganism to become resistant to drugs, which poses a major challenge to modern medicine (Nese et al., 2021) [1] . Although antimicrobial resistance happens naturally, various factors have accelerated its rise. These include improper antibiotic use like self-medication or ceasing medication before completion, inappropriate or frequent antibiotic prescriptions by healthcare providers, widespread use in agriculture and animal husbandry, and the slow pace of developing new antibiotics (Ruiz-Pérez et al., 2024) [2] . Pathogens resistant to antibiotics have spread globally, posing a significant threat to public health as they cause severe and life-threatening infections with rising rates of morbidity and mortality (Ting et al., 2023) [3] . A significant number of antibiotic-resistant infections encountered in clinical settings are caused by a specific group of pathogens known as the 'ESKAPE' organisms, which include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are able to "escape" the effects of antibiotics and resist elimination through standard treatments. The main difficulty in managing infections caused by ESKAPE organisms lies in the prompt identification of their antibiotic resistance. A report from 2019 indicated that antimicrobial resistance was responsible for approximately 3.57 million deaths worldwide and among these, an estimated 0.5 to 1 million deaths were caused by carbapenemresistant A. baumannii (Murray et al., 2022) [4] . Among ESKAPE pathogens, A. baumannii is one of the main organisms causing disease in humans and animals. A. baumannii is an opportunistic Gram-negatives non-flagellated coccobacillus bacterium causing severe nosocomial infections. This opportunistic pathogen is responsible for hospital and community-acquired infections. Its ability to form biofilms and resist desiccation, disinfectants, oxidative stress, and complement mediated killing are characteristics that allow A. baumannii to thrive within healthcare settings (Clémence et al., 2022) [5] . The organism is mainly found as a hospital-associated pathogen, but it is increasingly found in environmental sources like wastewater, sewage, rivers, soil, animal manure, and sometimes on food, especially in areas influenced by human activity. It survives well in moist environments and on dry surfaces, forming biofilms and persisting for long periods, which raises concerns about the environment acting as a reservoir for MDR strains that can spread to humans and animals. (Pulami et al.,2023) [6] Acinetobacter baumannii's ability to resist antibiotics, survive in harsh environments, and lack of known toxin genes indicates that its virulence relies on a "persist and resist" approach. Its resistance to critical last-line antibiotics like colistin, tigecycline, and carbapenems has made it one of the most challenging hospital-acquired ESKAPE pathogens (Clémence et al., 2022) [5] . There has been a global increase in the isolation of multidrug-resistant (MDR) and even pandrug-resistant (PDR) strains of A. baumannii. Key resistance genes associated with β-lactamase production include bla TEM , bla SHV , bla GES , bla PER , bla VEB , bla CTX-M , bla KPC , bla ADC , and bla OXA . Among beta-lactamases, carbapenem resistance is shown more prominently by the A. baumannii. (Kyriakidis et al., 2021) [7] . The use of antibiotics in hospital settings significantly contributes to the growing challenge of antimicrobial resistance (AR). Studying the antibiotic resistance patterns of environmental isolates is important, as it helps uncover potential pathways through which antibiotics may be transferred from hospitals to the environment (Kunhikannan et al., 2023) [8] . Increasing evidence indicated that human waste-related environments, including various municipal wastewater types such as domestic, industrial, hospital, and stormwater serve as reservoirs of resistant bacteria. Among these, hospital wastewater is considered a key source of clinically relevant A. baumannii (Hubeny et al., 2021) [9] . The presence of A. baumannii in non-clinical environments like water and soil further suggests that these habitats may act as reservoirs for antibiotic resistance genes (ARGs), particularly those encoding β-lactamases (Sykes et al., 2024) [10] .

Materials and Methods

Isolation and molecular confirmation of A. baumannii

A total of 180 environmental samples comprising soil (collected from 5-10 cm below the top layer) and water (well or tap water) from houses near the tertiary care veterinary and human hospitals from Thiruvananthapuram, Thrissur and Kalpetta districts of Kerala were collected. From each city, 30 households were selected, ten houses within one Km and five beyond one Km of veterinary and human hospitals. The study was conducted for a period of 10 months (November 2024 to August 2025). All the samples were aseptically collected and transported to the lab under chilled conditions. The samples were subjected to isolation and identification of A. baumannii. The soil samples were enriched at a rate of 1:10 dilution in buffered peptone water and incubated at 37 °C overnight. Enriched soil samples and water samples were streaked onto Leeds Acinetobacter agar. Colonies exhibiting characteristic pink mucoid colonies with pink colour diffused into the medium were selected (Fig 1 .), and species-level identification was carried out using polymerase chai n reaction (PCR) targeting the gyrB gene (Higgins et al., 2007) [11] (Table 1. and Fig 2 .). The DNA was extracted from all the isolates using the phenol-chloroform method and a PCR with the following conditions was carried out: 94 0 C for 2 min, followed by 25 cycles of 94 0 C for 1 min, 60C for 30 s and 72C for 1 min, with a final extension at 72C for 10 min. The amplicons were visualised following electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

Detection of ESBL resistance

The Kirby-Bauer disc diffusion test, employing cefotaxime (CTX 30 μg), ceftazidime (CAZ 30 μg), ceftazidime/ clavulanic acid (CAC 30/10 μg), and cefotaxime/ clavulanic acid (CEC 30/10 μg) antibiotic discs, was used for the phenotypic detection of ESBL as per CLSI (2024) [12] standards. The PCR assay was employed to detect bla CTX-M , bla TEM and bla SHV genes conferring ESBL resistance (Table 1.), following a method outlined in prior research (Bhoomika et al., 2016) [13] .

Results and Discussion

Acinetobacter baumannii exhibits remarkable environmental persistence, enabling it to survive for extended periods often weeks to months on dry surfaces like hospital equipment, bedding, and healthcare worker attire, which facilitates nosocomial transmission. This resilience stems from its ability to tolerate desiccation, form protective biofilms, and resist common disinfectants such as chlorhexidine and phenols, outperforming many other Gram-negative bacteria in harsh conditions. (Gallego, 2016) [14] . Most of the studies in Kerala regarding the organism were done in clinical samples from human hospitals (Sathyanesan et al., 2024 [15] , Rudrapathy et al., 2022 [16] , (Rajan et al., 2023) [17] , Sheeba et al., 2025) [18] etc. Studies conducted in animal samples are rare, with few studies, like that conducted by Hebbal et al. (2025) [19], reporting the presence of A. baumannii. In the current study, out of the 180 samples collected, 50 samples (27.8%) were confirmed to be harbouring A. baumannii. Whereas study by Madalgi et al.

(2025) [20] reported a lower rate of occurrence of the pathogen (5.5%) from water samples from Karnataka. These variations in prevalence may reflect differences in sampling methods, geographic locations, or environmental conditions influencing contamination levels.

Household environmental samples within one Km of selected hospitals had a higher prevalence of A. baumannii isolates than those households that were farther away. Among the households within one Km, the ones near the human hospital in Thrissur showed the highest rate (65.0%), while the lowest (20.0%) were observed near the veterinary hospital in Thiruvananthapuram (Table 2.). Kalasseril et al. (2020) [21] found A. baumannii in 5.3% of the isolates from water bodies near hospitals across three districts of Kerala. These variations may be due to the difference in hospital type, patient load, and surrounding ecology which influenced difference in contamination patterns. Households beyond one km from the selected hospitals showed either no A. baumannii isolates or occurrence rates of only 10-20%. This indicated that environmental dispersion of the organism diminishes with increasing distance from the hospital.

Statistically there was a significant difference in the distance wise occurrence of A. baumannii isolates from households near the two selected hospitals in Thrissur. Zone-wise, the highest A. baumannii occurrence rate came from households near Thrissur's human hospital (50.0%), while the lowest was near Thiruvananthapuram's veterinary hospital (16.67%) (Table 3.). These differences likely stem from factors like urban overcrowding, varying disease prevalence, and inconsistent sanitation practices across the cities. [22] reported a lower rate of occurrence (25%) of ESBL-producing A. baumannii isolates recovered from the waste water from residential areas in Nigeria. A study conducted by Hossain and Dey (2023) [23] reported the resistance rate of A. baumannii isolates recovered from the soil samples from the hospitals in Dhaka towards ceftazidime was 75.0%. The higher prevalence of ESBL-producing A. baumannii near hospitals points to contamination spread via proximity, with effluents and vectors carrying strains to nearby homes. This highlights the pressing need for stronger control strategies and monitoring efforts.

Genotypically, 90% of the A. baumannii isolates were identified as ESBL producers as they harboured any one of the three ESBL-encoding genes viz., bla CTX-M , bla SHV , and bla TEM considered in the study. Most of the isolates harboured bla SHV gene (82.0%). Genotypically highest rate of detection of ESBL-producing A. baumannii was seen in households near human hospital in Thiruvananthapuram and veterinary hospital in Thrissur (Table 5. And Fig 4 .). Rahman (2024) [24] conducted a study in waste water samples in Dhaka city and the most prevalent ESBL-encoding gene in the A. baumannii isolates was bla TEM (55.6%). A study conducted in lagoon waters from Nigeria by Ajoseh et al. (2025) [25] reported that 25.0% of the isolates were characterised as ESBL producers. These genes play a critical role in bacterial drug resistance by producing enzymes that hydrolyze betalactam antibiotics. Mutations in these genes expand the enzyme's substrate range. Often plasmid-mediated, ESBL genes spread rapidly via horizontal transfer and co-carry resistance to other drugs like aminoglycosides, complicating therapy and increasing nosocomial infection risks. This indicates there is an urgent need to combat this issue.

Conclusion

The current study revealed the significant environmental dissemination of A. baumannii from tertiary veterinary and human hospitals in Kerala, where isolation rates were markedly higher from households within one Km of the selected hospitals demonstrating distance-dependent contamination gradients driven by hospital effluents, vectors, and poor sanitation. The ESBL-producing A. baumannii is important because it can inactivate commonly used betalactam antibiotics, survive for long periods in soil and water, and spread resistance genes to other bacteria, making infections difficult to treat. Through contaminated environmental sources such as wastewater, soil, and surface water, it can infect or colonise in humans and animals, facilitating a continuous transmission cycle between hospitals, communities, livestock, and the environment. The ESBLproducing A. baumannii were isolated more from the households in close proximity with the selected hospitals. These results highlighted the fact that households near hospitals can act as sentinel sites for monitoring AMR reservoirs, emphasizing the "persist and resist" strategy of A. baumannii. The findings of the current study call for urgent interventions including enhanced wastewater treatment, antibiotic stewardship, and routine environmental surveillance to curb transmission from clinical to community settings, mitigating the escalating threat of multi/pan-drug resistant strains in Kerala.

Conflict of interest

The authors declare that there are no actual or potential conflicts of interest that could have influenced this work inappropriately.

Acknowledgement

AcknowledgementsThe authors sincerely acknowledge the College of Veterinary and Animal Sciences, Pookode, Wayanad, Kerala, for offering the necessary research facilities that enabled the successful completion of this study.

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