RESULTS

Bacterial identification and phylogenetic analysis

A total of 20 ARB isolates were successfully recovered from water samples collected from three giant freshwater prawn (_M. _rosenbergii) aquaculture ponds in Kalasin Province, Thailand. Based on 16S rRNA gene sequencing and sequence alignment using the Basic Local Alignment Search Tool, the isolates were taxonomically classified into eight bacterial species across five genera, with sequence identities ≥99% (Table 1).

Isolate | GenBank accession number | Species | Sequence similarity (%)
YAmp1 | PQ432911 | Bacillus cereus | 99
YAmp2 | PQ432912 | B. cereus | 99
YBA1 | PQ432913 | B. cereus | 100
YBA2 | PQ432914 | Bacillus wiedmannii | 99
HAmp1 | PQ432915 | Klebsiella pneumoniae | 99
HAmp2 | PQ432916 | B. wiedmannii | 99
HAE1 | PQ432917 | Aeromonas sanarellii | 99
HAE2 | PQ432918 | K. pneumoniae | 99
HTCBS1 | PQ432919 | Aeromonas veronii | 99
HBA1 | PQ432920 | A. veronii | 100
HBA2 | PQ432921 | A. veronii | 99
HSS1 | PQ432922 | K. pneumoniae | 99
MAmp2 | PQ432923 | A. veronii | 99
MAmp3 | PQ432924 | A. veronii | 100
MAE1 | PQ432925 | Enterobacter aerogenes | 99
MAE2 | PQ432926 | Aeromonas jandaei | 99
MAE3 | PQ432927 | Bacillus wiedmannii | 99
MSS1 | PQ432928 | A. veronii | 99
MSS2 | PQ432929 | A. veronii | 100
MBA2 | PQ432930 | Aeromonas dhakensis | 99

Identification of bacterial isolates was performed using partial 16S rRNA gene sequencing followed by sequence similarity analysis against the GenBank database of the NCBI using the Basic Local Alignment Search Tool for nucleotide sequences. Sequence similarity values are presented as percentages relative to the closest reference strains. GenBank accession numbers corresponding to representative isolates generated in this study are provided.

Among the isolates, _Aeromonas _veronii was the predominant species (n = 6), followed by Bacillus cereus (n = 3), Klebsiella pneumoniae (n = 3), and _Bacillus _wiedmannii (n = 3). Notably, all identified species comprised environmental or opportunistic human pathogens, suggesting anthropogenic influences on pond microbiota through contaminated feed, water sources, or cross-contamination via equipment and surface runoff [10, 20].

The phylogenetic tree constructed from 16S rRNA gene sequences (Figure 2) showed clear genus-level clustering, reflecting both the taxonomic consistency and environmental diversity of the isolates. Notably, Aeromonas spp. formed a well-supported clade, suggesting their ecological dominance under aquaculture conditions. This finding is consistent with previous reports showing a high prevalence of Aeromonas species in semi-intensive aquaculture systems characterized by nutrient enrichment and exposure to antimicrobial residues [21].

Figure 2: Neighbor-joining phylogenetic tree based on partial 16S rRNA gene sequences of bacterial isolates recovered from giant freshwater prawn (Macrobrachiumrosenbergii) aquaculture ponds in Kalasin Province, Thailand. Bootstrap values (>70%) derived from 1,000 replicates are shown at the branch nodes. The scale bar indicates nucleotide substitutions per site. Escherichia coli ATCC 25922 served as the outgroup.

The occurrence of both environmental species, such as _B. _wiedmannii, and zoonotic species, such as K. pneumoniae, highlights the complexity of microbial communities in aquaculture ecosystems and underscores the importance of molecular surveillance to monitor potential public health threats.

Antimicrobial susceptibility patterns

Antimicrobial susceptibility testing using the agar disk diffusion method demonstrated widespread resistance to multiple antimicrobial classes among the isolates. Fourteen of the 20 isolates (70.0%; 95% CI: 45.7–88.1%) exhibited multidrug resistance (MDR) according to CLSI criteria [17].

Bacterial species and isolate | AMP | VAN | AZM | STR | RIF | CHL
Bacillus cereus YAmp1 | R | R | R | I | R | I
B. cereus YAmp2 | R | I | I | S | R | S
B. cereus YBA1 | R | R | I | I | R | S
Bacilluswiedmannii YBA2 | R | R | S | I | R | R
Klebsiella pneumoniae HAmp1 | R | R | I | S | R | S
B.wiedmannii HAmp2 | R | R | I | I | R | R
Aeromonassanarellii HAE1 | R | R | S | I | S | S
Klebsiella pneumoniae HAE2 | R | R | R | S | R | S
Aeromonasveronii HTCBS1 | R | R | S | R | R | S
A.veronii HBA1 | R | R | S | R | R | S
A.veronii HBA2 | R | R | S | R | R | S
K. pneumoniae HSS1 | R | R | R | S | R | I
A.veronii MAmp2 | S | S | S | R | R | R
A.veronii MAmp3 | R | R | I | R | R | R
Enterobacter aerogenes MAE1 | S | R | R | R | S | S
Aeromonasjandaei MAE2 | R | R | S | R | R | R
B.wiedmannii MAE3 | R | R | I | S | R | R
A.veronii MSS1 | R | R | R | R | R | R
A.veronii MSS2 | S | R | I | I | R | R
Aeromonasdhakensis MBA2 | R | R | S | R | S | S

_A. _veronii exhibited the highest resistance burden, with all six isolates showing resistance to AMP, VAN, RIF, STR, and CHL, together with partial resistance to AZM. Similar MDR profiles were observed in B. cereus, B. wiedmannii, and K. pneumoniae (Table 2).

The observed resistance pattern likely reflects selective pressure arising from routine antibiotic use in aquaculture systems, either for prophylactic purposes or as growth promoters, particularly in open-pond systems where regulatory control is limited [22]. Intermediate susceptibility (16–20 mm inhibition zones) was also observed in several isolates, suggesting ongoing resistance evolution that may involve plasmid-mediated mechanisms or efflux systems [8].

The emergence of antibiotic resistance in B. cereus, traditionally regarded as a low-risk environmental species, further supports the hypothesis that environmental bacteria can acquire clinically important resistance determinants under prolonged antibiotic exposure [23].

No statistically significant differences in MDR prevalence were observed among the three sampling districts (p > 0.05). However, Aeromonas spp. exhibited significantly higher frequencies of resistance to RIF and STR than non-Aeromonas isolates (p < 0.05).

Detection of β-lactamase resistance genes

PCR-based molecular screening of plasmid DNA from selected MDR isolates revealed the presence of clinically important β-lactamase genes, particularly blaSHV and blaKPC-2 (Table 3).

Detection of ARGs was performed using PCR assays targeting clinically relevant β-lactamase genes, including blaTEM_, blaSHV, blaOXA, blaKPC-2, blaNDM-1,_ and blaIMP. Positive amplification was determined by the expected amplicon size on agarose gel electrophoresis. The co-occurrence of blaSHV and blaKPC-2 in _A. _veronii isolate MSS1 may indicate the presence of mobile resistance determinants that contribute to the environmental dissemination of AMR. Positive and negative amplification results are represented by “+” and “−”, respectively.

These genes were detected in multiple isolates of A. veronii, B. wiedmannii, and K. pneumoniae. The detection of blaKPC-2, a carbapenemase gene commonly associated with nosocomial pathogens, is of particular concern because it suggests the potential transfer of resistance determinants from clinical settings to environmental ecosystems [7, 19].

Bacterial species and isolate | blaTEM | blaSHV | blaOXA | blaKPC-2 | blaNDM-1 | blaIMP
Bacillus cereus YAmp1 | − | − | − | − | − | −
B. cereus YAmp2 | − | − | − | − | − | −
B. cereus YBA1 | − | − | − | − | − | −
Bacilluswiedmannii YBA2 | − | + | − | − | − | −
Klebsiella pneumoniae HAmp1 | − | − | − | − | − | −
B.wiedmannii HAmp2 | − | + | − | − | − | −
Aeromonassanarellii HAE1 | − | − | − | − | − | −
K. pneumoniae HAE2 | − | − | − | − | − | −
Aeromonasveronii HTCBS1 | − | − | − | − | − | −
A.veronii HBA1 | − | + | − | − | − | −
A.veronii HBA2 | − | − | − | + | − | −
K. pneumoniae HSS1 | − | + | − | − | − | −
A.veronii MAmp2 | − | − | − | + | − | −
A.veronii MAmp3 | − | − | − | + | − | −
Enterobacter aerogenes MAE1 | − | − | − | + | − | −
Aeromonasjandaei MAE2 | − | − | − | − | − | −
B.wiedmannii MAE3 | − | − | − | − | − | −
A.veronii MSS1 | − | + | − | + | − | −
A.veronii MSS2 | − | − | − | − | − | −
Aeromonasdhakensis MBA2 | − | − | − | − | − | −

Most notably, _A. _veronii isolate MSS1 co-harbored both blaSHV and blaKPC-2, suggesting clustering of MDR determinants. This co-occurrence may indicate the presence of mobile resistance elements and suggests possible dissemination of resistance determinants within aquaculture environments [24]. The environmental occurrence of these genes outside clinical settings further supports the concept that aquaculture systems may function as reservoirs or transmission pathways for antibiotic resistance determinants to human-associated bacterial populations [9].

These findings highlight the importance of incorporating molecular diagnostic approaches into aquaculture AMR surveillance programs and underscore the need for sustainable antibiotic use policies within a One Health framework [11].

Notably, blaKPC-2 was detected in several environmental isolates, including A. veronii, Enterobacter aerogenes, and K. pneumoniae, indicating the occurrence of clinically important carbapenemase-associated resistance determinants in freshwater prawn aquaculture environments. Among these isolates, _A. _veronii isolate MSS1 simultaneously harbored blaSHV and blaKPC-2, suggesting possible clustering of resistance determinants within mobile genetic elements.

To the best of our knowledge, this study represents the first report describing blaKPC-2-positive bacteria isolated from _M. _rosenbergii aquaculture ponds in Thailand. The 16S rRNA gene sequences generated in this study were deposited in the GenBank database under accession numbers PQ432911–PQ432930.