Phenotypic antimicrobial resistance traits of Aeromonas isolated from freshwater-farmed Asian seabass, Lates calcarifer

Introduction

Aeromonadaceae is family of Gram-negative, rod-shaped bacteria that have been isolated from various sources such as aquatic environment, soil, fish, higher vertebrates, agricultural products and several other food products. They are ubiquitous in nature and are known to cause diseases particularly when the host is under stress. Aeromonads are recognized as emerging human pathogens, as they are associated with a wide range of food-and waterborne infections. These include gastroenteritis, diarrhoea, urinary tract infections, necrotizing fasciitis, septicaemia and respiratory tract infections (Fernández-Bravo and Figueras, 2020) [18] . For several decades, antibiotics have been used to treat bacterial infections. While antibiotics play a crucial role in disease management, the growing threat of antimicrobial resistance (AMR) has emerged as a major global concern (Dodds, 2017) [16] . Alarmingly, various species of Aeromonas, including both clinical and environmental isolates have been found to exhibit multidrug resistance (Odeyemi and Ahmad, 2017) [34] . The emergence and spread of these resistant bacteria can make the treatment difficult and less effective. It is estimated that drug-resistant diseases could cause 10 million deaths each year by 2050 (Alara and Alara, 2024) [2] . Aeromonas species are among the most important disease-causing bacteria in freshwater aquaculture systems. Several Aeromonas species have been associated with fish diseases, including A. hydrophila (Junior et al., 2019) [21] , A. veronii (Li et al., 2020) [27] , A. caviae (Sharma et al., 2022) [38] , A. dhakensis (Carriero et al., 2016) [7] , A. schubertii (Liu and Li, 2012), A. salmonicida (Cipriano and Bullock, 2001) [9] , A. jandaei (Assane et al., 2021) [4] and A. sobria (Wahli et al., 2005). Frequent disease outbreaks in aquaculture compel farmers to rely heavily on antibiotics for disease management (Preena et al., 2020) [36] . Fish farms and aquaculture systems have increasingly been identified as hotspots for antibiotic resistant pathogens (Watts et al., 2017) [41] . There are numerous studies reporting the presence of multi-antibiotic resistant Aeromonas strains isolated from widely cultured fish species such as catfish, tilapia and carps with presence of various AMR genes among them (Nguyen et al., 2014; Suresh and Pillai, 2023) [32,39] . This is alarming as resistant genes can spread among bacteria and it has direct implications for human health, as resistant bacteria can enter the food chain and are extremely difficult to manage once established (Čížek et al., 2010) [10] . Despite the growing concern, there is a lack of data on the antibiotic resistance patterns of bacterial pathogens from freshwater fish culture systems. Further, the adoption of Asian seabass culture in freshwater is new and therefore, if we record the resistance profile of the bacterial pathogens at this stage of the culture, this data would help us to understand as well as to formulate culture policies that can be followed to obtain a sustainable production of Asian seabass from freshwater culture system without increasing the current resistance pattern of the bacterial pathogens associated with freshwater culture system of Asian seabass. Therefore, the objective of this study was to investigate antibiotics resistance patterns and phenotyping of Aeromonas species isolated from freshwater farmed Asian seabass. The findings are expected to provide baseline data on antimicrobial resistance bacteria in freshwater aquaculture systems and contribute to ongoing efforts in controlling antimicrobial resistance.

Materials and Methods

Ethical statement: The study complies with the current animal welfare laws in India. The care of sampled animals used in this study were following the guidelines of the CPCSEA [(Committee for the purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of India] on care and use of animals in scientific research. The study was approved by the board of Studies and authorities of the ICAR-Central Institute if Fisheries Education, Mumbai, India. As the sampled animal L. calcarifer is not an endangered fish, the provisions of the govt. of India's Wildlife Protection Act of 1972 are not applicable for research on this fish.

Sample collection and isolation of bacteria

Freshwater farmed Asian seabass samples (n = 60) were collected from 12 different freshwater farms located in Andhra Pradesh, Gujarat, Karnataka and Maharashtra, February, 2024 to July, 2024. Moribund fish exhibiting clinical symptoms were transported in live condition to the laboratory for microbiological analysis. Swab samples were collected from kidney, liver and eyes in aseptic condition and inoculated into 5ml Brain Heart Infusion broth (HiMedia Ltd, Mumbai). After 24hrs incubation at 28±2 °C, they were streaked onto BHI agar (HiMedia Ltd, Mumbai). Following an 18-24 hr incubation at 28±2 °C, distinct colonies from the culture plates were selected and subcultured to obtain pure isolates for further examination.

Biochemical and molecular characterization of bacterial isolates

Isolates were subjected to presumptive identification based on colony morphology, gram staining, catalase and oxidase (HiMedia, India) tests. Biochemical tests such as citrate utilization (MacWilliams, 2009a), indole production (MacWilliams, 2009b) and carbohydrate fermentation (Lehman, 2005) were performed to aid in bacterial species identification. Genomic DNA extraction from all bacterial isolates were done using the CTAB (Cetyltrimethylammonium bromide) method (Chachaty and Saulnier, 2000) [8] . PCR was performed to amplify 16S rRNA gene for the bacterial isolates using specific primers (Table 1). Annealing temperature for PCR was determined by performing gradient PCR. Cycling conditions under which PCR was performed are summarised in Table 1. 1.5% (w/v) agarose gel electrophoresis with ethidium bromide dye was used to check the quality of amplified product. The PCR product was extracted from gel using the Purelink Quick Gel Extraction Kit (Invitrogen, Thermo Fisher Scientific, USA) in accordance with the manufacturer's protocol, The PCR amplified product was then sent for Sanger's dideoxy sequencing (Eurofins Genomics India Private Limited, Karnataka, India). The obtained sequences were trimmed by using BioEdit software and were compared to the NCBI nucleotide database using BLASTn. The trimmed sequences were submitted to the GenBank database of the National Centre for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). [37] 16S rRNA 1492R GGTTACCTTGTTACGACTT

Antimicrobial susceptibility tests

Antimicrobial sensitivity testing of bacterial isolates was carried out using the Kirby-Bauer disk diffusion method (Hudzicki, 2009) on Mueller-Hinton agar (HiMedia, India) prepared following the manufacturer's instructions. Each bacterial isolate was inoculated onto the surface using the spread plate method, then antibiotic discs (Table 2) were placed onto the inoculated agar plates. After 24 hours incubation at 28±2 °C, the diameters of the zones of inhibition around each antibiotic disk were measured in millimetres. According to CLSI standards and EUCAST breakpoints, isolates were classified as Intermediate (I), Resistant (R), or Sensitive (S) (CSLI, 2016; CLSI, 2020; CLSI, 2022; EUCAST, 2025) [11,12,13,17] . A high-risk source of antibiotic exposure is indicated by a MAR index score greater than 0.2.

Phenotypic typing of isolates

Isolates were phenotypically typed based on their antimicrobial susceptibility profiles. A numerical code was assigned to each isolate, with 3 points for resistance, 2 points for intermediate susceptibility, and 1 point for susceptibility (Odeyemi and Ahmad, 2017) [34] .

Results

A total of 21 Aeromonas species were identified based on biochemical and molecular characterization (Table 3). The Aeromonas isolates were tested against 28 antibiotics. AST analysis is provided in Fig. 1. Among isolates, the resistance to different antibiotics varied significantly. All the isolates showed resistance to oxacillin (100%) and methicillin (100%). High resistance rates were observed for ampicillin (90%), cefazolin (86%), ticarcillin (57%) and nalidixic acid (57%) while meropenem (38%), tigecycline (29%), norfloxacin (29%), trimethoprim (24%), piperacillin (19%), ceftazidime (14%), rifampicin (14%), cefoxitin (10%), aztreonam (10%), tetracycline (10%), ofloxacin (10%), nitrofurantoin (10%), cefuroxime (5%) and chloramphenicol (5%) showed low antibiotic resistance rates (Table 4).

Isolates were found susceptible to cefotaxime, cefepime, doripenem, tobramycin, gentamicin, levofloxacin, ciprofloxacin and erythromycin. The multiple antibiotic resistance (MAR) index for Aeromonas isolates ranged from 0.11 to 0.50 (Table 5). There were 21 distinct phenotypes of the Aeromonas isolates identified by antibiotic phenotyping utilising number codes.

Discussion

Aeromonads are widely distributed bacterial species in aquatic environments, posing a significant threat to both environmental and human health due to their high adaptability and growing antibiotic resistance (Pinto et al., 2025) [35] . The present study investigated the antibiotic resistance patterns of Aeromonas species isolated from freshwater-farmed Asian seabass. Aeromonads have been reported to cause a disease known as aeromoniasis in Asian seabass, characterized by hemorrhagic lesions, fin erosion and significant mortality (Kathirkaman et al., 2018) [22] . Antibiotic sensitivity profiling of all Aeromonas isolates revealed complete resistance to oxacillin and methicillin (100%), along with varying degrees of resistance to ampicillin (90%), cefazolin (86%), ticarcillin (57%), meropenem (38%), piperacillin (19%), ceftazidime (14%), cefoxitin (10%), aztreonam (10%) and cefuroxime (5%). Similar resistance patterns of Aeromonas spp. isolated from freshwater cultured fish species i.e., walking catfish, tilapia, striped catfish, African catfish, rohu, catla, etc., against β- [5,30,26,24,1,33] . High resistance rates to β-lactam antibiotics (First generation) are attributed to presence of genes that encode β-lactamases and metallo-βlactamases (MBLs) (Bello-López et al., 2019; Nasser et al., 2022) [6,31] . However, the second and third generation antibiotics such as cefoxitin, cefuroxime and ceftazidime showed lower resistance in Aeromonas spp. in the study.

Though the usage of these antibiotics are rare in the commercial aquaculture systems, >5% resistance may be due to the transfer of AMR genes/integrons via the existing barter system among the microbial communities present in the aquatic environment. Furthermore, all Aeromonas isolates were sensitive to the third-and fourth-generation cephalosporins, such as cefotaxime and cefepime. These cephalosporins are reported to resist hydrolysis of the pathogen by plasmid or chromosomally mediated betalactamases and they are weak inducers of group 1 betalactamases (Hardefeldt and Prescott, 2024) [19] . [15,14,33] . This suggests that resistant bacterial populations are still persisting in the aquaculture environment against this quinolone. It is also reported that resistance genes can be maintained even without active antibiotic use, especially through horizontal gene transfer. Norfloxacin (29%) and ofloxacin (10%) i.e., second generation quinolones also showed moderate resistance against Aeromonas isolates. In general, Aeromonas species are found to be resistant to these quinolones due to the presence of plasmid-mediated quinolone resistance genes and chromosomal mutations in drug target genes (Kosikowska et al., 2022) [23] . Furthermore, resistance to tetracycline class antibiotics, including tigecycline (29%) and tetracycline (10%), was also evident from the current study. Resistance against this antibiotic class mainly occurs due to the acquisition of tetracycline resistance genes which encode for efflux pump or ribosomal protection (Hossain et al., 2019) [20] . In this study, some isolates displayed resistance to nitrofurantoin (10%) and chloramphenicol (10%), which are banned in veterinary and aquaculture. Although low, the presence of resistant strains to banned antimicrobials suggests possible environmental persistence of resistance genes, crossresistance mechanisms or historical selective pressure of these antibiotics (Nguyen et al., 2014) [32] . In the study, all Aeromonas isolates were found to be susceptible to [5,29,3,24,1] . These findings suggest that these antibiotics can still be effective for treating Aeromonas infections in commercial aquaculture practices. Multiple Antibiotic Resistance index analysis provides a quantitative value that estimates the extent to which a bacterial isolate is resistant to multiple antibiotics. In general, MAR values >0.2 typically suggests that the bacteria originated from high-risk sources where antibiotics are often used heavily (e.g., intensive aquaculture, hospitals, wastewater) while low MAR index (<0.2) indicates bacteria from environment with less antibiotic exposure. In the current study, the MAR index of Aeromonas ranged from 0.11 to 0.50, which is comparatively lower than values reported in other freshwater culture systems. For instance, study in freshwater farms of Haryana recorded MAR indices between 0.07 and 0.73, with 29.9% of Aeromonas isolates showing MAR index values above 0.2 (Nokhwal et al., 2025) [33] . Similarly, studies from Andhra Pradesh reported MAR index values consistently above 0.2, ranging from 0.33 to 0.78 (Lavanya et al., 2021, 2023) [25,26] , while isolates from Uttar Pradesh showed values between 0.39 and 0.56 (Kumar and Rathore, 2024) [24] . A high proportion of Aeromonas isolates (71%) exhibited MAR index values >0.2, indicating exposure to high-risk environments with frequent antibiotic application (Dhanapala et al., 2021) [15] , and highlighting the potential emergence of antimicrobial resistance in freshwater-farmed Asian seabass culture systems.

Conclusion

This study provides critical insights into the antibiotic resistance profiles of Aeromonas species isolated from freshwater-farmed Asian seabass. Antibiotic resistance can arise from both intrinsic and acquired resistance mechanisms resulting from antibiotic application or contamination of water source with AMR pathogens at the farm-level. The widespread resistance poses potential health risks not only to aquatic animals but also to humans through possible transmission and environmental dissemination of resistance genes. By cataloguing the resistance profiles of these pathogens, it provides a baseline data for predicting and understanding the patterns of antimicrobial resistance in freshwater aquaculture systems. This study also highlights the need for continued surveillance, targeted monitoring and gene-level investigations for better management of antibiotic resistance in aquatic environment.

Acknowledgement

AcknowledgementsThe present study was financially supported by Department of Biotechnology, Government of India (Project No. BT/PR47267/AAQ/3/1060/2022). The authors acknowledge the Director/Vice chancellor of ICAR-Central Institute of Fisheries Education, Mumbai for providing facilities for this research.

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