Introduction

Mastitis, an inflammatory disorder of the mammary gland, stands as a primary cause of economic detriment within the dairy sector worldwide1. In bovines, it is triggered primarily by infectious agents and, less frequently, by physical injury2. The disease imposes substantial financial losses, estimated at approximately USD 147 per cow annually, primarily owing to reduced milk yield and quality, and augmented culling frequencies3. While clinical mastitis presents with overt pathological symptoms, subclinical mastitis (SCM) constitutes a more insidious and pervasive form, affecting up to 50% of dairy herds worldwide without visible clinical signs4.

Despite its asymptomatic nature, SCM exerts a significant adverse impact on milk composition and quality, as reflected by elevated somatic cell counts (SCCs) and reduced overall productivity5. S. aureus and E. coli are regarded as the predominant causes of bovine mastitis. However, emerging opportunistic bacteria such as Aeromonas hydrophila are increasingly being implicated in SCM6, raising concern due to their zoonotic potential and multidrug-resistant (MDR) characteristics7. Poor hygienic practices and contact with infected animals facilitate the invasion of Aeromonas species into the mammary gland, leading to bacterial proliferation and excretion in milk8. Moreover, owing to the environmental ubiquity of Aeromonas, post-pasteurization contamination remains a potential risk. Milk provides a highly conducive media for the growth of numerous pathogens, many of which are implicated in the contamination of milk or act as food-borne pathogens that threaten consumer health9. Among these, Aeromonas hydrophila exhibits exceptional adaptability and growth potential in milk, which is supported by its high moisture content, nearly neutral pH, and abundant nutrient composition10.

Members of the genus Aeromonas are Gram-negative, facultative anaerobic, non-spore-forming rods11. The genus Aeromonas is known to induce several human infections. Species within this genus are considered major pathogens commonly associated with intestinal infections12 and are responsible for over 95% of reported cases of Aeromonas-related bacteremia cases13. Although their involvement in bovine mastitis, particularly in SCM, has been sporadically reported, several lines of evidence suggest an emerging epidemiological role, as Aeromonas species have been isolated from milk samples, indicating an underexplored etiological significance14. Annual and strategic antimicrobial resistance (AMR) reports from WOAH focus broadly on resistance trends and global antimicrobial use in animals, but they do not specifically identify multidrug-resistant Aeromonas hydrophila in bovine milk as a priority pathogen. Instead, these reports provide overarching policy and surveillance standards, offering guidelines for monitoring antimicrobial use, harmonizing surveillance systems, and promoting responsible veterinary practices15.

Pathogenicity in Aeromonas spp. is mediated by a diverse repertoire of virulence determinants that confer hemolytic, proteolytic, and adhesive properties. These functions are encoded by key virulence genes, including aerA (aerolysin), hylA (hemolysin), ser (serine protease), and act and ast (cytotoxic enterotoxins), which collectively contribute to host tissue destruction, invasion, and immune evasion16,17. Despite their recognized role in human disease, the expression and regulation of these virulence genes in bovine SCM isolates remain inadequately characterized, underscoring the need for further molecular investigation. The tremendous global increase in antimicrobial resistance poses a foremost health concern18,19,20. Effective control of mastitis in dairy herds relies on rapid pathogen identification and timely antimicrobial intervention. However, the widespread misuse of antimicrobials in veterinary sectors has favored the occurrence of antimicrobial resistance (AMR) among mastitis-associated pathogens. A. hydrophila has shown alarming levels of resistance, driven by excessive antibiotic application in agriculture and intrinsic β-lactamase production21. Recent surveillance studies have revealed high resistance to tetracyclines, along with increasing resistance to fluoroquinolones and aminoglycosides22,23,24. In the context of bovine mastitis, infection with MDR A. hydrophila not only complicates treatment and prolongs infection but also heightens the possibility of zoonotic transmission via milk consumption25,26.

Despite these concerns, comprehensive studies integrating virulence genotyping, phenotypic antibiograms, and MDR profiling of A. hydrophila isolates from bovine SCM remain scarce. This knowledge gap impedes the formulation of evidence-based control and antimicrobial stewardship strategies. Notably, this is the first in-depth, systematic investigation characterizing the existence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. hydrophila strains isolated from subclinically mastitic milk samples. By integrating phenotypic and genotypic analyses, this study enhances the current understanding of A. hydrophila as a bovine milk-borne pathogen and a potential reservoir of antimicrobial resistance, thereby filling a grave gap in the One Health framework.

Materials and methods

Animal ethics

The study was carried out in compliance with the ARRIVE guidelines. All methods were performed according to relevant guidelines and regulations. The handling of cows and all the experiments were approved by the Animal Ethics Review Committee of Suez Canal University (AERC-SCU), Egypt .

Sampling and California mastitis test (CMT)

Approximately 800 quarter milk samples were randomly collected from 200 apparently healthy dairy cows (four quarters per cow) across four commercial farms in Cairo, Egypt, with 200 samples obtained from 50 cows at each farm. Sampling was conducted over the period from June to August 2023. Sampling was performed after disinfecting the udder with ethyl alcohol (70%). The California mastitis test (CMT) was carried out to screen for subclinical mastitis in the examined milk samples follows standard bovine CMT guidelines (AHDB Dairy, UK)27. The test relies on the reaction between the reagent and somatic cell nuclei in milk, which produces a gel proportional to cell concentration. For each udder quarter, 2 mL of milk was placed in a well of a white plastic paddle and mixed with an equal volume of CMT reagent (DeLaval, DeLaval International AB, Tumba, Sweden) using gentle circular motions. The reaction was assessed visually within 20 s and scored according to the degree of gel formation: negative (0), weak positive (1), distinct positive (2), and strong positive (3). Positive CMT samples were subjected to bacteriological analysis.

Isolation and identification of A. hydrophila

CMT-positive samples were incubated at 37 °C for 24 h, followed by centrifugation (3000 rpm for 5 min). The sediments were streaked onto Tryptic Soy Agar (TSA) (Oxoid, UK), and incubated at 37 °C for 24 h. Distinct colonies were sub-cultured on Rimler Shotts (RS) medium and Aeromonas Isolation Media (supplemented with rehydrated ampicillin) (Oxoid, UK) at 37 °C for 24 h28. A. hydrophila isolates were identified using Gram’s staining, assessment of culture features and hemolytic activity, and a series of biochemical tests, including oxidase, catalase, methyl red, Voges-Proskauer, citrate utilization, gelatin liquefaction, casein hydrolysis, starch liquefaction, sugar fermentation, H2S production, urea hydrolysis, esculin hydrolysis, and nitrate reduction tests. Furthermore, the identification of Aeromonas spp was achieved through PCR exploration of 16 S rRNA according to Lee29. In addition, distinguishing A. hydrophila was verified through PCR exploration of the species-specific gyrB gene using a designated primer set (A-hyd F: AGTCTGCCGCCAGTGGC; A-hyd R: CRCCCATCGCCTGTTCG), as described by Persson30.

Antimicrobial susceptibility testing of the retrieved A. hydrophila isolates

It was investigated via a disc diffusion assay on Mueller-Hinton agar (Oxoid, UK). A panel of 9 antimicrobials was employed: amoxicillin (AMX, 30 µg), ceftriaxone (CRO, 30 µg), gentamycin (GM, 10 µg), sulfamethoxazole/trimethoprim (SXT, 25 µg), tetracycline (TE, 10 µg), amoxicillin/clavulanic acid (AMC, 30 µg), chloramphenicol (C, 30 µg), erythromycin (E, 15 µg), and norfloxacin (NOR, 10 µg) (Oxoid, UK). The analysis of the results was consistent with the CLSI procedures31. Besides, all procedures including the use of E. coli-ATCC25922 for quality control were performed. The A. hydrophila isolates were categorized as multidrug-resistant (MDR) or extensively drug-resistant (XDR) following established criteria of Magiorakos32, where MDR is defined as resistance to ≥ 1 agent in ≥ 3 antimicrobial classes, and XDR is defined as resistance to ≥ 1 agent in all but ≤ 2 antimicrobial classes. Besides, the multiple antibiotic resistance index (MAR) was determined33.

Detection of virulence and antimicrobial resistance genes in the isolated A. hydrophila strains

PCR was used to investigate the distribution of the virulence genes (aerA, ser, act, alt, ast, and hlyA) and resistance genes (blaTEM, blaOXA, blaCTX−M−1, aadA1, sul1, and tetA) in the tested A. hydrophila strains. DNA was extracted via a QIAamp DNA Mini Kit (QIAGEN Sciences Inc., Germantown, MD, USA/ Cat. No. ID 51326). Each PCR run included positive controls (strains supplied by AHRI, Egypt), and negative controls (DNA-free). The amplified products were separated on a 1.5% agarose gel and subsequently photographed. The oligonucleotide sequences and their cycling conditions are shown in Table 1.

Table 1 List of primer sequences and PCR-cycling conditions.
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Statistical analyses

The results were analyzed by means of the Chi-square test (SAS software, version 9.4, SAS Institute, Cary, NC, USA) (a p-value < 0.05 donates a significant variation). Moreover, the correlations among the tested antimicrobial agents and antimicrobial resistance genes were determined using the ‘’corrplot’’ package in R-software (version 4.0.2; https://www.r-project.org/).

Results

Phenotypic characteristics and the prevalence of A. hydrophila

Using the CMT, the prevalence of subclinical mastitis in the examined samples was 42.5% (340/800). A. hydrophila was isolated from CMT-positive samples at a percentage of 20.8% (71/340).

A. hydrophila strains were Gram-negative motile rods. The retrieved isolates exhibited significant growth on TSA agar, with creamy, round, convex, and shiny colonies. In addition, the bacteria displayed significant growth on RS medium, producing distinguishing yellow circular colonies. For Aeromonas selective agar, the isolates produced green colonies with a black center. A subset of the isolates demonstrated β-hemolysis on blood agar.

Biochemically, the retrieved strains were positive for oxidase, indole, catalase, Voges-Proskauer, citrate utilization, nitrate reduction, starch liquefaction, esculin hydrolysis, gelatin liquefaction, casein hydrolysis, and fermentation of glucose and sucrose. In contrast, the retrieved isolates tested negative for H2S production, methyl red, and urea hydrolysis. As well, all the retrieved strains were found to carry the 16SrRNA conserved gene and were positive for the species-specific gyrB gene, with an amplicon size of 144 bp.

Antibiogram profile of the isolated A. hydrophila strains

A. hydrophila strains were resistant to amoxicillin (100%), tetracycline (90.1%), amoxicillin/clavulanic acid (85.9%), sulfamethoxazole/trimethoprim (84.5%), ceftriaxone (83.1%), erythromycin (76.1%), gentamycin (53.5%), and chloramphenicol (50.7%). In contrast, all the isolates were sensitive to norfloxacin (100%), which demonstrated remarkable antimicrobial efficiency, as summarized in Table 2; Fig. 1.

Table 2 Antimicrobial resistance profiles of the recovered A. hydrophila strains (n = 71).
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Fig. 1
Fig. 1
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The heat map demonstrates the antimicrobial resistance profiles of the retrieved A. hydrophila isolates (n = 71), with the intensity of the red color directly proportional to the corresponding susceptibility pattern.

The statistical analyses confirmed a significant variance in resistance levels across different antimicrobials (p < 0.05). Moreover, the correlation analyses showed strong positive correlations between CRO and AMC (r = 1); E and GM (r = 1); TE and AMX (r = 1); SXT and TE (r = 1); SXT and AMX (r = 0.99); GM and SXT (r = 0.99); SXT and E (r = 0.99); AMC and AMX (r = 0.99); and GM and TE (r = 0.98), as presented in Fig. 2.

Fig. 2
Fig. 2
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The heat map demonstrates the correlation coefficients (r) between the different tested antimicrobial agents. Color intensity represents the magnitude of the correlation, with blue indicating negative correlations and red indicating positive correlations.

Distribution of virulence and antimicrobial resistance genes in the recovered A. hydrophila strains

PCR revealed that the aerA gene (100%) is the predominant virulence marker associated with A. hydrophila isolates, followed by the alt (53.3%), ast (45.1%), ser (29.6%), act (26.8%), and hylA (22.5%) genes. Similarly, the isolates harbored the blaTEM, tetA, blaOXA−1, sul1, blaCTX−M−1, and aadA1 resistance genes with prevalence of 100%, 90.1%, 85.9%, 84.5%, 83.1%, and 53.5%, respectively, as illustrated in Table 3; Fig. 3. There was significant variation (p < 0.05) in the dissemination of virulence genes in the retrieved A. hydrophila strains. Contrariwise, there was no significant variance (p > 0.05) in the distribution of the antimicrobial resistance genes in the tested A. hydrophila strains.

Table 3 Prevalence of virulence and antimicrobial resistance genes in the retrieved strains (n = 71).
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Fig. 3
Fig. 3
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A radar chart demonstrates the distribution of virulence and antimicrobial resistance genes associated with A. hydrophila strains (n = 71) recovered from subclinically mastitic bovine milk.

Multidrug-resistance profiles of A. hydrophila strains

Herein, 28.2% (20/71) of the A. hydrophila strains were XDR to 8 antimicrobial classes and harbored the blaTEM, blaCTX−M−1, blaOXA−1, tetA, aadA1, and sul1 resistance genes. Moreover, 15.5% (11/71) of the A. hydrophila strains showed MDR to 6 classes and owned the blaTEM, blaCTX−M−1, blaOXA−1, tetA, and sul1 genes. As well, 14.1% (10/71) of the A. hydrophila strains were MDR to 5 classes and had the blaTEM, blaCTX−M−1, blaOXA−1, tetA, and sul1 genes. Also, 8.4% (6/71) of the tested A. hydrophila strains were XDR to 7 classes and had the blaTEM, blaCTX−M−1, blaOXA−1, tetA, aadA1, and sul1 genes (Table 4; Fig. 4). Furthermore, the MAR-index values (0.22–0.89) were > 0.2, signifying that the isolated A. hydrophila strains are yielded from high-risk contamination.

Table 4 Distribution of phenotypic multidrug resistance patterns and antimicrobial resistance genes among the retrieved A. hydrophila isolates.
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Fig. 4
Fig. 4
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Illustrates the distribution of multidrug resistance patterns, including multidrug resistance (MDR) and extensively drug resistance (XDR), among A. hydrophila strains (n = 71) recovered from subclinically mastitic bovine milk.

The results revealed strong positive correlations concerning blaTEM and AMX; blaCTX−M−1 and CRO; tetA and TE; sul1 and SXT; aadA1 and GM; blaCTX−M−1 and AMC (r = 1 for each); blaTEM and AMC (r = 0.99); blaOXA−1 and AMX (r = 0.99); blaOXA−1 and AMC (r = 0.95); and blaOXA−1 and CRO (r = 0.94), as shown in Fig. 5.

Fig. 5
Fig. 5
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The heat map depicts the correlation coefficients (r) between the antibiotic resistance genes present in the recovered A. hydrophila isolates (n = 71) and the various tested antibiotics. Color intensity represents the magnitude of the correlation, with blue indicating negative correlations and red indicating positive correlations.

Discussion

Subclinical mastitis constitutes a condition of profound veterinary and economic significance, exerting substantial adverse effects on milk yield, quality, and overall productivity within the global dairy industry, including in Egypt4. The escalating existence of antimicrobial resistance reflects a critical global health concern, given its capacity for horizontal transmission of resistance determinants, thereby compromising the therapeutic efficacy of conventional antimicrobial agents and threatening both animal and public health security19. The present investigation highlights the notable prevalence and multidrug-resistance prospective of A. hydrophila recovered from subclinical mastitic milk in dairy herds. The detected subclinical mastitis rate (42.5%) corresponds with earlier investigations that underscore the substantial impact of subclinical infections in dairy cattle44,45,46, which frequently pose a greater epidemiological risk than clinical mastitis owing to their insidious, asymptomatic nature and consequent underdiagnosis.

The isolation of A. hydrophila from 20.8% of mastitic milk samples underscores its emerging role as an opportunistic mastitogenic pathogen, corroborating earlier findings by Özavcı6 and Zubairi47, who also documented its increasing implication in bovine and environmental infections. Post-milking contamination of milk with Aeromonas spp. may occur through environmental or fecal sources, given their widespread occurrence in water, animal housing facilities, and bovine feces48,49,50.

Phenotypically, the recovered isolates displayed the defining morphological and biochemical attributes of the genus Aeromonas, in accordance with recognized taxonomic standards51,52. Their biochemical profile was characteristic for A. hydrophila, exhibiting catalase, indole, and gelatinase activities that signify considerable metabolic versatility and environmental adaptability53. The detection of hemolytic activity and diverse hydrolytic enzymes, including gelatinase, caseinase, and amylase, further highlights the organism’s capacity for host tissue invasion and pathogenicity.

The differentiation of Aeromonas species based solely on conventional biochemical assays is thought-provoking due to the absence of a definitive biochemical scheme capable of accurately distinguishing among closely related species. Consequently, molecular identification has become indispensable for precise taxonomic discrimination within the genus. Herein, the isolates harbored the 16 S rRNA and gyrB genes, both of which are conserved inherited genes of A. hydrophila. These findings are consistent with the observations of Algammal17, Persson30, and Tacão54, who similarly reported the utility of these conserved genes in the reliable identification and differentiation of A. hydrophila from other Aeromonas species.

The antimicrobial resistance (AMR) patterns revealed an alarming resistance profile, notably 100% resistance to amoxicillin and substantial resistance to tetracycline, β-lactam/β-lactamase inhibitor combinations, and sulfonamides. Similar resistance trends in A. hydrophila from aquatic, animal products, and environmental sources were reported17,50,55. The intrinsic production of β-lactamases56, efflux pumps, and plasmid-mediated resistance determinants likely underpin these resistance patterns10,56. Interestingly, norfloxacin exhibited complete efficacy against all isolates, suggesting the potential utility of fluoroquinolones in treatment protocols, although their prudent use is advised to prevent the emergence of resistance. Our results are consistent with those recorded by previous investigations17,57. A. hydrophila typically exhibits high susceptibility to quinolone-class antimicrobials58,59, and resistance to these agents remains infrequent. When present, quinolone resistance is predominantly associated with point mutations in the gyrA gene, which induce conformational alterations in DNA gyrase, thereby diminishing the binding affinity of quinolones and conferring reduced susceptibility60.

The development of antimicrobial-resistant A. hydrophila is considered a serious concern for veterinary and public health. Its resistance is driven mostly by the indiscriminate utilization of antimicrobials in livestock systems, together with its capacity to gain resistance genes from other superbugs. Mobile genetic elements enable the horizontal transfer of these genes, promoting multidrug resistance61,62. Therefore, regular antimicrobial susceptibility testing and molecular screening are vital for guiding appropriate treatment and controlling the propagation of resistant strains63,64.

The revealing of virulence genes further elucidates the pathogenicity of these strains. The universal occurrence of the aerA gene, encoding aerolysin, highlights its essential role in cytotoxicity and host tissue damage26,65. Aerolysin is recognized as the principal virulence determinant in Aeromonas species, playing a pivotal role in disease pathogenesis through its cytotoxic and pore-forming activities that disrupt host cell membranes38. Herein, the observed ubiquitous presence of aerA among isolates may reflect either clonal expansion of particular A. hydrophila strains or horizontal gene transfer contributing to virulence gene dissemination, as A. hydrophila exhibits an open pan-genome with dynamic virulence factor distribution across genomes16.

The concurrent detection of the alt, ast, act, ser, and hylA genes suggests a multifactorial virulence mechanism, with synergistic interactions likely enhancing pathogenicity. Comparable gene distributions were reported by previous investigations17,66,67, affirming the global consistency of A. hydrophila virulence profiles. Cytotoxic enterotoxins (encoded by alt and act) and aerolysin are major virulence factors of Aeromonas species, and are central to their pathogenic and foodborne potential. These toxins mediate host cell damage and disruption68, while serine proteases, exhibiting strong caseinolytic activity, enhance tissue invasion and virulence69.

The genotypic antimicrobial resistance findings mirror the phenotypic data, particularly the widespread detection of blaTEM, blaOXA−1, and blaCTX−M−1, genes known to mediate extended-spectrum β-lactamase (ESBL) production21,70. Specifically, the blaCTX−M gene confers resistance to cephalosporins and β-lactam/β-lactamase inhibitor combinations, highlighting its critical role in β-lactam resistance among A. hydrophila isolates21. A key finding in the present study is the high prevalence of ESBL genes, especially blaCTX−M−1. The co-occurrence of blaTEM, blaOXA−1, and blaCTX−M−1 in several isolates is concerning. Although we did not experimentally localize these genes, their typical association with mobile elements in Gram-negative bacteria, and the co-carriage of unrelated resistance genes (e.g., tetA and sul1) within isolates, strongly suggests they are plasmid-borne. If located on conjugative plasmids, they pose a significant risk for horizontal transfer to other bacteria in dairy farm environments and the human gut, underscoring the One Health threat.

The strong correlations observed between specific resistance genes and their corresponding antimicrobial classes confirm the genetic basis of phenotypic resistance. Similar ESBL-mediated resistance was reported in Aeromonas spp. recovered from both milk and environmental settings50,62.

Moreover, resistance to sulfonamides and tetracyclines is predominantly mediated by the sul1 and tetA genes, respectively71,72,73,74, which constitute the most prevalent resistance determinants identified in this study. In addition, aadA1 is frequently detected and is recognized as one of the principal genes conferring resistance to aminoglycosides75.

The high MAR indices (> 0.2) indicate that the isolates were obtained from environments with substantial antibiotic exposure, such as intensive dairy farms where antimicrobial use is frequent. This aligns with the reports of Odeyemi76 and Yucel77, who identified Aeromonas spp. as sources of AMR genes within aquatic and dairy settings. The detection of both MDR and XDR strains underscores the clinical and epidemiological threat posed by A. hydrophila, which is considered a reservoir for resistance gene propagation through the food chain and environment78,79.

In this context, the presence of MDR and XDR A. hydrophila in raw milk presents a potential hazard for consumers. While pasteurization is effective at killing vegetative bacterial cells like A. hydrophila, the risk lies in post-pasteurization contamination from equipment or the farm environment, given the organism’s environmental ubiquity77. Furthermore, the consumption of raw milk or unpasteurized dairy products, which is common in some communities, poses a direct health risk79. These findings highlight the critical need for stringent hygienic practices during milking and milk storage. At a policy level, our results advocate for the inclusion of Aeromonas species, particularly in the context of AMR, in national One Health surveillance programs.

Study limitations

Although species-specific gyrB PCR offers strong evidence for the identification of A. hydrophila, we acknowledge that the lack of sequencing of these amplicons represents a limitation. Future investigations should incorporate multilocus sequence typing (MLST) or whole-genome sequencing (WGS) to better assess the genetic diversity and evolutionary relationships of A. hydrophila strains in dairy environments. Additionally, determination of minimum inhibitory concentrations (MICs) for critical antimicrobials, such as fluoroquinolones, should be considered in subsequent studies to complement phenotypic resistance profiling.

Conclusion

To our knowledge, this is the first investigation to document MDR and XDR A. hydrophila strains isolated from bovine subclinical mastitis in Egypt. The emergence of such superbugs in milk poses a substantial and emerging public health concern. These emerging MDR and XDR A. hydrophila strains commonly harbor the blaTEM, tetA, blaOXA−1, sul1, blaCTX−M, and aadA1 resistance genes. Moreover, the aerA, alt, ast, and ser virulence genes are commonly inherited in A. hydrophila strains of milk origin. A robust genotype-phenotype correlation was confirmed, with a strong association between the existence of definite resistance genes and observed resistance to their consistent antimicrobials. To combat the spread of these MDR pathogens, we recommend the implementation of routine antimicrobial susceptibility testing in milk surveillance programs. Furthermore, accurate identification of MDR and XDR A. hydrophila necessitates a synergistic approach, combining conventional methods and molecular assays.