DISCUSSION

Occurrence of blaKPC-2-carrying bacteria in freshwater prawn aquaculture

To the best of our knowledge, this study is the first report describing blaKPC-2-carrying bacteria isolated from _M. _rosenbergii aquaculture ponds in Thailand. The detection of blaKPC-2 in environmental isolates, particularly in _A. _veronii, is of substantial concern because carbapenemase-producing bacteria have historically been associated mainly with hospital-acquired infections. Although reports of carbapenem-resistant Aeromonas spp. in aquatic environments are emerging globally, studies focusing specifically on freshwater prawn aquaculture systems remain limited.

The present findings address an important regional knowledge gap regarding AMR in freshwater aquaculture systems in Northeastern Thailand, where previous AMR investigations have focused predominantly on marine shrimp production systems or fish-associated environments, including studies conducted in Lam Pao Dam [3]. In contrast, the present study specifically investigated earthen pond systems used for _M. _rosenbergii farming in Kalasin Province and demonstrated the predominance of Aeromonas spp., consistent with previous reports from aquaculture-impacted environments. However, a high prevalence of MDR isolates and the occurrence of clinically important carbapenemase-associated genes, particularly blaKPC-2, were also identified in freshwater prawn pond environments. These findings provide important baseline data for future regional AMR surveillance programs and suggest that earthen pond aquaculture systems may represent distinct ecological niches favoring the persistence and dissemination of AMR determinants.

Microbial diversity and resistance characteristics

This study provides a comprehensive overview of the microbial diversity and antibiotic resistance characteristics of bacteria isolated from _M. _rosenbergii aquaculture ponds in Kalasin Province, Thailand. The findings reveal the co-occurrence of pathogenic, opportunistic, and environmentally persistent bacterial species, many of which exhibit MDR and harbor clinically significant resistance genes. These results reflect a broader global concern regarding the environmental spread of AMR and its implications for aquaculture sustainability and public health.

The high prevalence of MDR isolates observed in this study may reflect the use of empirical or unregulated antibiotics, which is common in small-scale rural aquaculture systems, where veterinary oversight and antimicrobial stewardship measures may be limited compared with industrial aquaculture operations. Open-pond systems may further facilitate the environmental dissemination of resistant bacteria and resistance genes through water exchange, runoff, and sediment-associated microbial communities.

Ecological role of ** Aeromonas ** spp.

The predominance of _A. _veronii among the isolated strains is consistent with its ecological adaptability to aquatic environments, especially those affected by nutrient enrichment and subinhibitory antibiotic concentrations. Previous studies have identified Aeromonas spp. as dominant taxa in freshwater ponds due to their metabolic flexibility and tolerance to environmental stressors [1, 21]. This bacterium is also a known pathogen responsible for motile aeromonad septicemia in aquaculture species, contributing to substantial economic losses in Southeast Asia [3]. The consistent detection of B. cereus, _B. _wiedmannii, and K. pneumoniae across multiple samples suggests that these species may persist in aquaculture systems by associating with organic sediments, detritus, or plankton communities.

AMR patterns

The observed resistance profiles are alarming. Nearly all isolates were resistant to at least two antibiotics, and many, particularly _A. _veronii, K. pneumoniae, and B. cereus, were resistant to four or more, including AMP, VAN, RIF, and chloramphenicol. Resistance to VAN and RIF is particularly concerning, as these antibiotics are considered last-resort options in clinical settings [11]. The high levels of resistance observed in B. cereus, traditionally considered a low-pathogenicity species, suggest that even commensal or environmental organisms can serve as reservoirs or vectors of resistance genes under aquaculture conditions [8].

β-lactamase genes and resistance dissemination

Genotypic analysis further supports the phenotypic resistance data. PCR detection of resistance genes revealed the presence of blaSHV and blaKPC-2 in multiple isolates, particularly in _A. _veronii and K. pneumoniae. The blaKPC-2 gene encodes a carbapenemase enzyme that confers resistance to carbapenems, one of the most potent antibiotic classes, and is primarily associated with hospital-acquired infections [7]. Its detection in aquaculture-associated isolates suggests either environmental contamination from anthropogenic sources or the movement of genes facilitated by mobile genetic elements such as plasmids or integrons [19, 24]. Notably, the co-occurrence of blaSHV and blaKPC-2 in _A. _veronii MSS1 reinforces the likelihood of gene clustering, a phenomenon that increases the stability and transmission of resistance determinants across species and environments.

These findings support a growing body of evidence indicating that aquaculture environments, particularly open-pond systems, may serve as important reservoirs and hotspots for the dissemination of ARBs and ARGs [6, 22]. Factors such as poor water quality management, prophylactic antibiotic use, and inadequate treatment regulation may contribute to selective pressures that accelerate AMR development within aquaculture ecosystems. The detection of clinically relevant resistance genes in environmentally adapted species, including _B. _wiedmannii, further suggests that resistance determinants can persist in aquatic microbial communities, sediments, and biofilms, potentially extending beyond harvesting periods and contaminating subsequent production cycles. From a One Health perspective, these findings highlight the interconnected relationships among aquatic environments, animal health, and public health. However, additional investigations involving exposure assessments, food-chain analyses, and gene-transfer studies are necessary to better evaluate the actual public health implications of freshwater prawn aquaculture systems [11, 20].

Detection of blaSHV in B.wiedmannii

Interestingly, blaSHV was also detected in _B. _wiedmannii, an environmental species not commonly associated with clinically important β-lactamase genes. Although Bacillus spp. are frequently detected in aquaculture environments and are often considered part of the environmental microbiota, the occurrence of blaSHV in these isolates may suggest environmental acquisition of resistance determinants under antibiotic selective pressure. Similar observations have occasionally been reported in environmental bacterial communities, although their ecological significance remains poorly understood.

Practical and policy implications

This study highlights several critical implications. First, it supports the call for more stringent antibiotic use regulations in aquaculture, including the adoption of antimicrobial stewardship programs and the enforcement of withdrawal periods before harvest. Second, the integration of molecular diagnostics, including quantitative PCR and metagenomics, into routine monitoring programs would enable early detection of resistance trends and guide targeted interventions. Third, alternative disease management approaches, such as probiotics, vaccination, and improved biosecurity, should be prioritized to reduce reliance on antibiotics [2].

From a policy perspective, these findings support the need for improved antimicrobial stewardship and routine AMR surveillance within Thailand’s freshwater aquaculture sector. The integration of aquaculture-associated AMR monitoring into national One Health action plans may contribute to more sustainable disease management strategies and reduce the environmental dissemination of clinically important resistance determinants.

Limitations of the study

Several limitations should be considered when interpreting the findings of this study. First, bacterial isolation relied on culture-based methods using AMP-supplemented selective media, which may underestimate the diversity of unculturable or non-target bacterial populations present in aquaculture environments. Second, the number of representative isolates included in molecular analyses was relatively limited (n = 20), potentially restricting broader ecological interpretation. Third, detailed farm-level antimicrobial usage records were inconsistently available, limiting direct assessment of associations between antibiotic use practices and resistance profiles. In addition, environmental contamination from external water sources or agricultural runoff could not be completely excluded. The absence of non-aquaculture reference sites or low-antibiotic-use control ponds limited comparative ecological interpretation of resistance prevalence in the present study. Although the co-occurrence of blaSHV and blaKPC-2 may indicate the presence of mobile resistance determinants, no plasmid sequencing, conjugation assays, or whole-genome sequencing was conducted to confirm gene-transfer or mobility. Future investigations integrating metagenomics, quantitative PCR, and resistome analysis are therefore recommended.