A First Report of Molecular Typing, Virulence Traits, and Phenotypic and Genotypic Resistance Patterns of Newly Emerging XDR and MDR Aeromonas veronii in Mugil seheli

Aeromonas veronii is associated with substantial economic losses in the fish industry and with food-borne illness in humans. This study aimed to determine the prevalence, antibiogram profiles, sequence analysis, virulence and antimicrobial resistance genes, and pathogenicity of A. veronii recovered from Mugil seheli. A total of 80 fish were randomly gathered from various private farms in Suez Province, Egypt. Subsequently, samples were subjected to clinical, post-mortem, and bacteriological examinations. The retrieved isolates were tested for sequence analysis, antibiogram profile, pathogenicity, and PCR detection of virulence and resistance genes. The prevalence of A. veronii in the examined M. seheli was 22.5 % (18/80). The phylogenetic analyses revealed that the tested A. veronii strains shared high genetic similarity with other A. veronii strains from India, UK, and China. Using PCR it was revealed that the retrieved A. veronii isolates harbored the aerA, alt, ser, ompAII, act, ahp, and nuc virulence genes with prevalence of 100%, 82.9%, 61.7%, 55.3%, 44.7%, 36.17%, and 29.8%, respectively. Our findings revealed that 29.8% (14/47) of the retrieved A. veronii strains were XDR to nine antimicrobial classes and carried blaTEM, blaCTX-M, blaSHV, tetA, aadA1, and sul1 resistance genes. Likewise, 19.1% (9/47) of the obtained A. veronii strains were MDR to eight classes and possessed blaTEM, blaCTX-M, blaSHV, tetA, aadA1, and sul1 genes. The pathogenicity testing indicated that the mortality rates positively correlated with the prevalence of virulence-determinant genes. To our knowledge, this is the first report to reveal the occurrence of XDR and MDR A. veronii in M. seheli, an emergence that represents a risk to public health. Emerging XDR and MDR A. veronii in M. seheli frequently harbored aerA, alt, ser, ompAII, and act virulence genes, and blaTEM, sul1, tetA, blaCTX-M, blaSHV, and aadA1 resistance genes.


Introduction
The fast-growing demand for seafood presents a significant challenge for the enhancement of fisheries and aquaculture production worldwide. In 2014, the contribution of aquaculture to the human food supply overtook the production of natural water resources Pathogens 2022, 11, 1262 3 of 18 bags to the microbiology laboratory at the National Institute of Oceanography and Fisheries (NIOF), Suez, Egypt, for clinical and bacteriological examination.

Clinical and Postmortem Examinations
The naturally infected M. seheli were screened for detection of any abnormalities. The clinical inspection was carried out as previously described [26]. Necropsy was performed on moribund M. seheli [27].

Isolation and Identification of A. veronii
A loopful of the obtained samples (liver, kidney, and gills) was streaked directly onto tryptic soy agar (TSA) and Aeromonas isolation medium base (supplemented with ampicillin) (Oxoid, Hampshire, UK) and incubated at 28 • C for 18-24 h. [28,29]. The identification of A. veronii was performed using Gram's staining, culture characteristics, hemolysis on blood agar, and biochemical characterization (oxidase, catalase, methyl red, Voges-Proskauer, citrate utilization, gelatin liquefaction, casein, starch liquefaction, sugar fermentation, H 2 S production, urea hydrolysis, bile esculin hydrolysis, and nitrate reduction tests). Additionally, the identification of A. veronii was confirmed by PCR detection of the 16srRNA gene as previously stated [30], and gene sequencing was carried out.

16S rRNA Gene Sequencing and Phylogenetic Analyses
PCR amplification of the 16S rRNA gene was performed for all recovered A. veronii isolates. The retrieved A. veronii strains displayed congruence in their phenotypic features. Consequently, the PCR products of three isolates (chosen at random) were subjected to direct sequencing in both directions following purification using a PureLink PCR-Product purification kit (Thermo-Fisher Scientific, Bremen, Germany). The obtained sequences were placed in the GenBank with accession numbers MW831507, MW836109, and MW599727. Multiple alignments were performed on the obtained sequences. The phylogenetic tree was established according to the neighbor-joining approach with 1000 bootstrap resampling using MEGA X software [31,32].

Determination of Virulence and Antimicrobial Resistance Genes in the Recovered A. veronii Strains
PCR was employed to determine the virulence-determinant genes (aerA, ser, act, alt, ahp, nuc, and ompAII) and antimicrobial-resistant genes (bla TEM , bla SHV , bla CTX-M , aadA1, sul1, and tetA) in the retrieved A. veronii strains. DNA extraction was carried out using the PureLink DNA extraction kit (Thermo-Fisher Scientific, Bremen, Germany/Cat. No. A29790). Negative controls (DNA-free) and positive control strains (provided by the AHRI, Dokki, Egypt) were included in the PCR assay. The obtained PCR products were Pathogens 2022, 11, 1262 4 of 18 separated on agar gel by electrophoresis. Afterwards, the gel was photographed. The primer sequences (Thermo-Fisher Scientific, Karlsruhe, Germany) and cycling conditions are presented in Table 1. Approximately 180 apparently healthy Tilapia zillii (weighting 45 ± 10 g) were collected from private farms in Suez Governorate, Egypt and acclimatized in large fiberglass tanks of 1500 L capacity containing aerated sea water (supplied from the same source as the fish) for 15 days before testing began. T. zillii was selected as a typical model of marine fish due to its simplicity of handling, whereas the handling of M. seheli is difficult. The water parameters were maintained within the permissible limits for T. zillii. The tank was filled with aerated sea water. Dissolved oxygen was monitored at 5 ± 1 mg L −1 using automatic air suppliers (RINA, Genova, Italy), while the water temperature was maintained at 27 ± 0.52 • C. The tank pH was regulated at 7.5, and a cycle of 13 h light/11 h dark was adopted. Ammonia and nitrite levels were measured twice a week and never exceeded 0.05 or 0.25 mg L −1 , respectively. Moreover, six fish were randomly sampled and subjected to parasitological examination (gills and body surface were microscopically examined for the presence of parasites) and bacteriological examination. Only apparently healthy fish with no signs of disease were collected for experimental challenge.

Experimental Setup
Approximately 120 acclimated T. zillii were assigned into 6 groups, each containing 2 subgroups (n = 10). Each subgroup contained 10 fish in 100 L capacity glass tanks. Five groups of fish (G1-G5) were experimentally challenged I/P with 0.2 mL sterile saline cotaining (3 × 10 8 cfu/mL) virulent A. veronii. Each group was challenged with a corresponding A. veronii strain: Strain 1 harbored the aerA gene; Strain 2 harbored aerA and ser genes; Strain 3 harbored aerA, ahp, ser, and nuc genes; Strain 4 harbored aerA, ompAII, alt, ahp, and act genes; Strain 5ed harbored aerA, alt, ahp, act, ser, nuc, and ompAII genes. Another group (C: negative control) was I/P injected with sterile saline solution (0.85% NaCl). Five strains of A. veronii were selected and cultured on tryptic soy broth (Oxoid, UK) for inoculum preparation at 28 • C for 24 h. Then, the bacterial suspension was modified to the final concentration (3 × 10 8 vcfu/mL) using a 0.5 McFarland standard as previously described [44]. The clinical signs, post-mortem findings, and mortality rates were checked for 14 days post-challenge as previously described [45]. To establish Koch's postulates, dead fish were bacteriologically examined for bacterial re-isolation.

Statistical Analyses
The Chi-square test was applied to analyze the data frequencies using SAS software (version 9.4, SAS Institute, Cary, NC, USA); the level of significance was p-value< 0.05. Moreover, the correlations between antimicrobial drugs and antimicrobial resistance genes were determined using R-software (version 4.0.2; https://www.r-project.org/) (accessed on 1 July 2022).

Clinical and Post-Mortem Findings
In the current study, the clinical inspection of naturally infected M. seheli revealed dark skin discoloration with detached scales ( Figure 1A) and distinct hemorrhages at the base of the fins ( Figure 1B). Moreover, the post-mortem findings of naturally infected M. seheli revealed hepatomegaly, friable liver with hemorrhagic patches ( Figure 1C), and congested kidneys ( Figure 1D).

Phenotypic Features and the Prevalence of A. veronii in the Examined M. seheli
All the recovered A. veronii isolates were Gram-negative, motile, straight rods. After 24 h at 28 • C, the bacteria grew effectively on the TSA medium, giving characteristically creamy, round, convex, shiny colonies. Colonies subsequently appeared green with black centers on Aeromonas-selective agar media. Moreover, the recovered colonies were convex, round, and hemolytic on blood agar, turning dark green after prolonged incubation. Biochemically, the obtained A. veronii isolates tested positive for oxidase, catalase, Voges-Proskauer, gelatin liquefaction, methyl red, casein, starch liquefaction, citrate utilization, and fermentation of glucose and sucrose. Moreover, the recovered A. veronii isolates were negative for H 2 S production, urea hydrolysis, bile esculin hydrolysis, nitrate reduction, and mannose fermentation.
The prevalence of A. veronii among the examined M. seheli was 22.5% (18/80). To measure the intensity of A. veronii among various examined organs of M. seheli, three different organs (liver, kidney, and gills) from the same fish were examined, with the highest prevalence noticed in the liver (38.3%), then the kidneys (34.1%), and gills (27.6%), as revealed in Table 2 and

Phenotypic Features and the Prevalence of A. veronii in the Examined M. seheli
All the recovered A. veronii isolates were Gram-negative, motile, straight rods. After 24 h at 28 °C, the bacteria grew effectively on the TSA medium, giving characteristically creamy, round, convex, shiny colonies. Colonies subsequently appeared green with black centers on Aeromonas-selective agar media. Moreover, the recovered colonies were convex, round, and hemolytic on blood agar, turning dark green after prolonged incubation. Biochemically, the obtained A. veronii isolates tested positive for oxidase, catalase, Voges-Proskauer, gelatin liquefaction, methyl red, casein, starch liquefaction, citrate utilization, and fermentation of glucose and sucrose. Moreover, the recovered A. veronii isolates were negative for H2S production, urea hydrolysis, bile esculin hydrolysis, nitrate reduction, and mannose fermentation.
The prevalence of A. veronii among the examined M. seheli was 22.5% (18/80). To measure the intensity of A. veronii among various examined organs of M. seheli, three different organs (liver, kidney, and gills) from the same fish were examined, with the highest prevalence noticed in the liver (38.3%), then the kidneys (34.1%), and gills (27.6%), as revealed in Table 2 and Figure 2. Statistically, there was no significant difference in the distribution of A. veronii among the examined internal organs of naturally infected M. seheli (p> 0.05).     All the isolated A. veronii strains were positive for the 16srRNA gene. The 16srRNA gene sequencing showed that the tested A. veronii strains (accession nos.: MW831507, MW836109, and MW599727) had a common ancestor. Likewise, the tested A. veronii strains exhibited high similarity of genetic identity compared with other A. veronii strains from different sources, such as A. veronii strain zy01 (accession no.: KX768735) from China (94.5-98.9%), A. veronii strain ATCC35624 (accession no.: NR_118947) from UK (94.8-98.8%), A. veronii strain IIGc_SK_CIFE (accession no.: MN809117) isolated from Nile tilapia in India (94.8-98.8%), and A. veronii strain NBH8 (accession no.: MT071583) from China (94.8-98.8%), as illustrated in Figure 3.

Pathogenicity Test
Five A. veronii strains (harboring one, two, four, five, and seven virulence genes, respectively) were selected for the pathogenicity test, as illustrated in Table 6. The clinical signs, pathological lesions, morbidity, and mortality rates in the different groups were monitored for 14 days after challenge. The results showed that fish in the control group had no deaths or pathologic lesions. In contrast, the other groups had substantial mortality rates and septicemic lesions, identical to those reported in naturally infected fish, including dark skin discoloration with detached scales, skin ulcers, and distinct hemorrhages at the base of the fins. Moreover, the mortality rate positively correlated with the virulencedeterminant genes, and the highest mortality rate (100%) was recorded in the group (G5) inoculated with A. veronii Strain 5 which harbored seven virulence genes (as described in Figure 9). Clinically, the majority of infected fish showed detached scales, darkness of the skin, a hemorrhagic vent, slow movement, and hemorrhagic patches, mainly at the base of fins. Post-mortem inspection demonstrated that the tested fish exhibited characteristic septicemia, including enlarged kidneys, a congested liver, and accumulated bloody serous fluid in the abdominal cavity. Furthermore, A. veronii was re-isolated from various internal organs of the diseased and dead fish.

Discussion
Aeromonads are the most common septicemic bacterial fish pathogens, and are considered emerging food-borne pathogens associated with a significant threat to public health [46]. In the present work, the findings of clinical and post-mortem examinations were consistent with the results of [47][48][49], who observed congested gills, scattered hemorrhages on the skin, and detached scales, in addition to congested, friable and enlarged liver, and degenerative changes in the kidneys and spleen of fish naturally infected with Aeromonads. The degree of pathological alterations and the mortality rate are correlated with the severity of infection, fish immunity, and virulence determinants of Aeromonas species [50,51].
During the bacteriological examination, all retrieved isolates were recognized as A. veronii according to their morphological and biochemical features, and the recovered isolates revealed coordination of their phenotypic features. These results were consistent with those recorded by [6], who recovered A. veronii from O. niloticus in Egypt.
In the present study, A. veronii was recovered from moribund M. seheli with a prevalence of 22.5%, and the liver was the most predominant affected organ. The prevalence of A. veronii in this study was higher than that described by [6], who recorded only three isolates from diseased O. niloticus, and nearly similar to that reported by [11], who isolated 87 A. veronii strains from freshwater fish. A. veronii affects a variety of fish species and can live in environments where it may pose harm to the aquaculture industry and threaten food safety [52]. The prevalence of infection is attributed mainly to various predisposing variables, including stress resulting from fish density in intensive systems, poor management, poor hygienic conditions, poor water quality, insufficient oxygen, inappropriate pH, and temperature [14].
Aeromonas species are difficult to differentiate at the species level by conventional methods, due to the lack of a precise biochemical scheme to discriminate between them. Hence, molecular identification is essential for the differential diagnosis of Aeromonas species. The technique of 16S rRNA sequencing is one of the most reliable molecular methods for identifying A. veronii [52]. In this study, all recovered isolates of A. veronii tested positive for 16S rRNA using specific primers. Moreover, the 16SrRNA phylogenetic analysis highlighted that the tested A. veronii strains originated from a common ancestor (accession nos: MW831507, MW836109, and MW599727). Furthermore, the tested A. veronii strains revealed a remarkable genetic similarity with other A. veronii strains from different geographical regions, such as A. veronii strain zy01 from China, A. veronii strain IIGc_SK_CIFE from India, A. veronii strain NBH8 from China, and A. veronii strain ATCC35624 from UK [53]. These results emphasize the epidemiological map and underline the public health significance of A. veronii.
Regarding the antibiogram profiling, ciprofloxacin showed an optimistic antimicrobial activity against the retrieved A. veronii strains from M. seheli. Areomonads are generally susceptible to fluoroquinolones [12]. In contrast, the retrieved A. veronii strains were highly resistant to sulfonamides, penicillin, tetracycline, cephalosporin, β-Lactam-β-lactamaseinhibitor combination, polymyxin, aminoglycosides, and macrolides. Our findings were similar to those recorded by [9,47]. The resistance of A. veronii to various antibiotics affects the health of animals and humans. Inappropriate application of antibiotics in the aquaculture system and the capability of A. veronii to obtain resistance genes from other MDR pathogens are the key predisposing causes contributing to the emergence of multiple drug-resistant superbugs. Therefore, regular use of antimicrobial sensitivity tests and screening for the existence of MDR strains are essential for selecting suitable antibiotics. The emergence of multidrug resistance in bacterial pathogens is attributed mainly to the propagation of antimicrobial resistance genes by horizontal transfer mediated by plasmids [54,55].
The detection of virulence-determinant genes is vital for understanding their potential pathogenicity and the prevention of probable infectious disease [56]. In this study, PCR revealed that the tested A. veronii strains frequently carried the aerA gene, followed by alt, ser, ompAII, act, ahp, and nuc virulence genes. Our findings are consistent with the results of [7,14,56]. Screening of virulence-determinant genes is a vital tool for identifying the possible pathogenicity of Aeromonads [57]. The pathogenicity of A. veronii is related to the expression of certain virulence determinants. Its pathogenicity is attributed mainly to the aerolysin toxin, cytotonic enterotoxins, serine proteases, outer membrane protein, and nuclease enzymes that are encoded by aerA, alt, act, ser, ahp, ompAII, and nuc genes, respectively [6,58]. The aer gene encodes for aerolysin toxin, which plays a significant role in the occurrence of infection. Aerolysin toxin is the primary virulence-determinant factor in Aeromonads, contributing to disease pathogenesis [7]. Moreover, cytotoxic enterotoxins (encoded by alt and act genes) and aerolysin toxin are essential virulence determinants for Aeromonads, and are categorized as potent foodborne pathogens. Both of these virulence determinants exert a substantial effect on the pathogenesis of disease [58]. Protease enzymes (encoded by ser and ahp genes) are common in Areomonads; they play a significant role in the proliferation of bacteria. Furthermore, they endorse the destruction of the mucosa and discoloration of the scales in fish, facilitating the invasion of bacterial pathogens. Serine proteases are characterized by potent caseinolytic activity [59]. The outer membrane proteins (encoded by the ompA gene) are responsible for mucosal adhesion in A. veronii. They exert a significant role in the attachment of A. veronii to the intestinal mucosa of the host [60].
Concerning the multi-drug resistance patterns in the retrieved A. veronii strains, a high percentage of the recovered A. veronii was XDR to nine different classes and carried bla TEM , bla CTX-M , bla SHV, tetA, aadA1, and sul1 resistance genes. Furthermore, most of the isolated A. veronii were MDR to seven or eight different classes and possessed bla TEM , bla CTX-M , bla SHV, tetA, sul1, and aadA1 resistance genes. Multi-drug resistance is thought to be one of the major hazards to public health across the world. It occurs due to the misuse of antibiotics in the aquaculture sector and in medical practice, and may include acquisition of antimicrobial resistance genes via mobile genetic elements [55,[61][62][63]. The bla TEM and bla SHV resistance genes mainly mediate resistance to penicillin. Interestingly, the bla TEM gene is the most predominant β-lactamase gene, commonly found in Aeromonads [14,64]. The resistance to sulfonamides and tetracycline is attributed mainly to the sul1and tetA resistance genes, respectively, which were the most predominant resistance genes found in this study. This was similar to the results of [64], who stated that tetracycline-and trimethoprim-resistance genes were demonstrated in all A. veronii genomes, an observation attributed mainly to the wide use of tetracycline and trimethoprim/sulfamethoxazole in the health sector and in veterinary settings. Moreover, the bla CTX-M gene is responsible for cephalosporin resistance as well as resistance to β-Lactam-β-lactamase-inhibitor combinations. Furthermore, aadA1 is one of the most common aminoglycoside-resistance genes. The development of genes encoding antibiotic resistance on either the bacterial chromosome or plasmid is commonly attributed to the widespread unregulated use of antibiotics. The remarkable increase in antimicrobial resistance represents a rising obstacle in the treatment of diseases caused by MDR pathogens in humans and fish, and is considered a public health threat [40,55,65].
In the results of the pathogenicity tests, fish challenged with A. veronii showed different mortality rates that positively correlated with the prevalence of virulence genes in the inoculated strain. They exhibited typical clinical signs observed in naturally infected fish. These findings are similar to the results reported by [66]. The pathogenicity testing highlighted the virulence and pathogenicity of the A. veronii strains recovered from M. seheli. The pathogenicity tests revealed that the more virulence genes carried by a strain, the higher was the mortality rate.

Conclusions
In summary, to the best of our knowledge, this is the first study to have revealed the occurrence of XDR and MDR A. veronii strains in M. seheli. The recovered A. veronii strains commonly harbored the aerA, alt, ser, ompAII, and act virulence genes. The emerging A. veronii strains were XDR or MDR to several antimicrobial classes (for example, sulfonamides, penicillin, tetracycline, cephalosporin, β-Lactam-β-lactamase-inhibitor combination, polymyxin, aminoglycosides, and macrolides) and frequently carried bla TEM , sul1, tetA, bla CTX-M , bla SHV , and aadA1 resistance genes. Ciprofloxacin revealed optimistic antimicrobial activity against the XDR and MDR A. veronii strains retrieved from M. seheli. Conventional isolation methods and molecular assays are reliable epidemiological tools for identifying A. veronii in fish. Distressingly, the occurrence of XDR and MDR A. veronii strains is currently recognized as a public health threat, which moreover adversely affects the fish industry. Accordingly, regular practice of antimicrobial sensitivity tests and the proper use of antibiotics are called for in the aquaculture and health sectors.