Fourteen presumptive Aeromonas isolates were identified at the genus level based on biochemical tests (positive oxidase, nitrate reductase reactions, catalase; fermentation of D-glucose and trehalose; utilization of dulcitol, D-arabitol, erythritol and xylose; and resistance to vibriostatic agent O/129), as described by Abbott et al. [12] and Nagar et al. [10]. Based on the additional biochemical characteristics: aesculin hydrolysis, and acid production from L-arabinose, urocanic acid, salicin, sucrose, D-cellobiose and L-fucose [13, 14], they were further identified up to the species level (Table 1).

Relying solely on morphological and biochemical characteristics for Aeromonas identification is often contentious and unreliable, leading to potential misidentifications [5]. Therefore, the gyrB gene, which has higher discriminatory power for phylogenetic analysis [16], was employed for species-level identification of the biochemically characterized Aeromonas strains. Partial gyrB gene sequences were submitted to GenBank at NCBI (Accession numbers: OQ743456-64, OQ789562-65, and OQ920270). Based on biochemical tests and gyrB gene sequences, these Aeromonas strains were identified and confirmed as A. dhakensis (50%), A. hydrophila (28.6%), and A. jandaei (21.4%). Initially misidentified as A. hydrophila in 2002, A. dhakensis was later recognized as a distinct species in 2013 [8]. Since then, A. dhakensis has been isolated from diverse sources across different countries with varying frequencies [6, 7, 9].

Pairwise and mean distances of aligned gyrB sequences from these strains were estimated for a continuous stretch of 920 bases (positions 852,606–853,528 according to E. coli ATCC 25922 numbering, NZ_CP009072) using ClustalW. The sequence homology among all Aeromonas strains for the gyrB gene ranged from 89.91 to 99.67%, representing 3 to 93 nucleotide variations. The mean sequence similarity, serving as a measure of discriminatory capability, was determined to be 95.0%, comparable to the 92.2% for the gyrB gene of Aeromonas strains described by Soler et al. [16]. The alignment revealed a total of 173 variable positions (18.8% of the sequenced fragment), along with a single triplet (ACA) insertion in A. salmonicida (CECT 894T) and A. bestiarum (CECT 4227T). Intra-species nucleotide substitution rates, determined by calculating mean distance within each species, ranged from 1.18% in A. hydrophila to 1.93% in A. jandaei. A phylogenetic tree constructed using these genetic matrices showed significant divergence among all Aeromonas species, with consistent clustering patterns between the investigated strains and their type or reference strains (Fig. 1).

Unrooted phylogenetic tree (UPGMA) of Aeromonas isolates and other known Aeromonas species based on the gyrB gene sequences. CECT numbers indicate the Spanish Type culture collection numbers of the Aeromonas reference strains. Numbers in the parenthesis represent the GenBank accession numbers. Numbers shown next to each node indicate bootstrap values (percentage of 1,000 replicates). The bar indicates a sequence divergence

This study reinforces the importance of a combined phenotypic and genotypic approach for reliable taxonomic classification of Aeromonas from diverse samples, as previously reported [2, 9]. A. dhakensis, A. hydrophila, and A. jandaei have been reported from fish and aquatic environments [11], as well as clinical and outbreak samples [6, 8]. The rising prevalence of Aeromonas species, known pathogens for both humans and aquatic animals, in aquatic environments raises concerns about increased infections in both populations, especially given the growing demand for seafood and the potential for fish-to-human transmission.

Virulence factors in Aeromonas strains contribute to their pathogenesis and transmission. All aquatic Aeromonas strains in this study carried the genes act (232 bp), ser (350 bp), gcat (237 bp), ahyB (540 bp), and lip (390 bp) (Table 2). The aer (252 bp) and hlyA (597 bp) genes were present in all A. dhakensis and A. hydrophila strains, but only 33.3% and none of the A. jandaei strains, respectively (Table 2). Cytotonic enterotoxins, alt and ast, were found in 78.6% and 64.3% of the strains, respectively (Table 2). Both genes were highly prevalent in A. dhakensis and A. hydrophila strains (> 70%), but only 33.3% and none of the A. jandaei strains, respectively. Structural genes for polar flagella (fla) and lateral flagella (lafA) were present in 100% and 28.6% of these Aeromonas strains, respectively (Table 2). All A. dhakensis and A. hydrophila strains harbored act, aer, hlyA, and fla genes, while ast, hlyA, and lafA genes were absent in all A. jandaei strains. Recently, Aeromonas strains with various virulence genes have been reported from water [9], aquaculture farms [2], and clinical samples [6]. In this study, all strains exhibited distinct β-hemolysis zones on blood agar plates, indicating potential pathogenicity (Table 2). Hoel et al. [26] documented β-hemolysis in 91% of food strains.

Biofilm formation enhances bacterial survival and virulence. Aeromonas can adhere to and colonize both biotic and abiotic surfaces [1]. Biofilm formation was measured using the SBF index, which incorporates bacterial growth rate (OD600nm) for consistent categorization, as described by Naves et al. [20]. In this study, biofilm formation by 14 Aeromonas strains in TSB ranged from 0.39 to 1.38. The strains were categorized as weak (n = 3, 21.4%), moderate (n = 5, 35.7%), and strong (n = 6, 42.9%) biofilm producers (Fig. 2). All A. jandaei strains were strong biofilm producers, while A. dhakensis and A. hydrophila showed no clear pattern. Aeromonas strains from fish produced moderate to strong biofilms, whereas those from water samples were predominantly weak to moderate producers. This aligns with research by Chenia and Duma [27], showing that most Aeromonas strains from freshwater fish and seafood produce moderate to strong biofilms, while strains from estuarine and river waters typically produce weak biofilms [28]. Chen et al. [8] reported that clinical A. dhakensis strains form stronger biofilms compared to A. hydrophila strains in Taiwan.

Biofilm formation by Aeromonas strains in TSB 30 °C. Bars represent average SBF values and standard errors

The antimicrobial susceptibility of 14 confirmed Aeromonas strains was evaluated against 18 antibiotics to create an antibiogram profile. All strains were resistant to ampicillin, cephalothin, imipenem, ampicillin/sulbactam, aztreonam, piperacillin-tazobactam, and clindamycin, but were sensitive to gentamicin, co-trimoxazole, amikacin, chloramphenicol, nitrofuran, and ciprofloxacin (Table 3). Notably, strains from water samples in Ernakulam and Thrissur showed resistance to ceftriaxone and nalidixic acid, whereas strains from fish samples were sensitive to these antibiotics. This antimicrobial resistance (AMR) pattern aligns with findings by Dubey et al. [2].

Aeromonas, a ubiquitous water-borne organism, easily acquires and exchanges AMR genes, making it a recognized reservoir of antibiotic resistance genes (ARGs) [3]. Water environments host diverse microbial communities and ARG reservoirs. Overuse of antibiotics and inadequate sanitation expose these communities to external ARGs, accelerating their acquisition and dissemination. In this study, 43% of strains were resistant to tetracycline, although this was lower than in recent studies by Jacobs and Chenia [29].

Multidrug resistance, defined as resistance to at least one antimicrobial agent from three or more different classes [24], was observed in all Aeromonas strains. Six strains (A4, A5, A6, A7, A8, and A9) resisted six antibiotic classes: penicillin and its derivatives, tetracycline, first-generation cephalosporins, penems, monobactams, and fluoroquinolones. Eight strains (A1, A2, A3, M14, M22, P31, N14, and N46) resisted four classes: penicillin and its derivatives, first-generation cephalosporins, penems, and monobactams. The prevalence of AMR microorganisms is rising and is expected to become a major public health concern.

Our findings corroborate earlier research on the emergence of multidrug-resistant Aeromonas strains in river water, fish, food, and clinical settings [5, 29, 30]. The Multiple Antibiotic Resistance (MAR) index, a valuable risk assessment tool, indicated MAR values between 0.39 and 0.56 (Suppl Table 2) for Aeromonas strains in this study, with all values exceeding 0.2. This suggests contamination from sources with frequent antimicrobial use, indicating a high-risk environment. The MAR index range observed is consistent with previous research [28, 29], further implying potential antimicrobial contamination in the surveyed aquatic environments. To control antimicrobial-resistant Aeromonas in these environments, farmers and veterinary teams should prioritize strong biosecurity measures, such as maintaining water quality and reducing animal stress, while incorporating alternatives like phage therapy, probiotics, vaccines, plant-based antimicrobials, and silver nanoparticles. Phage therapy, in particular, offers a targeted approach to combat resistance, while responsible antibiotic use, effective water management, and staff education are essential for the long-term health and sustainability of aquaculture systems.

Class 1 integrons were identified in 71.4% of the Aeromonas aquatic strains, with variable region sizes ranging from 500 to 1100 bp; the remaining 4 strains showed no amplification (Table 1). These results are consistent with previous research showing 21.7% of Aeromonas strains from ornamental freshwater fish farms carrying class 1 integrons [9]. Additionally, 42.9% of strains exhibited class 2 integrons (250–700 bp) (Table 1), aligning with findings by Jacobs and Chenia [29] and Nagar et al. [19], who reported class 2 integrons in 27% of aquaculture and 50% of food strains, respectively.

Integrons, mobile genetic elements, facilitate the integration and expression of diverse gene cassettes, including antibiotic resistance genes. They play a significant role in promoting antibiotic resistance within environmental bacterial populations via horizontal gene transfer [3]. In Aeromonas strains, integrons could potentially enhance the transfer of multiple antibiotic resistance genes among environmental microorganisms.