Antimicrobial resistance profile of Aeromonas spp. isolated from tambaqui fish ( Colossoma macropomum )

The current study assesses the resistance profile of Aeromonas spp. individuals isolated from tambaqui fish ( Colossoma macropomum ) grown in fish farms located in São Luís Metropolitan Region - MA. In total, 114 bacterial strains belonging to species Aeromonas spp. were used in the experiment. Antimicrobial resistance profile was defined based on using the disk diffusion method and 10 antimicrobials. Isolates were classified as susceptible, susceptible upon increased exposure and resistant to the assessed principles; phenotypes showing resistance to multiple tested drugs (MDR) were classified. Bacterial strains presented widespread resistance to penicillin; as well as high oxacillin, cefepime-, cefadroxil-and azithromycin-resistance rates. Ampicillin associated with sulbactam, neomycin and ofloxacin were the antimicrobials recording the highest sensitivity rate; high frequency of isolates (34.21%) were categorized as MDR phenotypes. Antimicrobial resistance was observed in the herein assessed Aeromonas spp. isolates, despite the lack of antimicrobials registered in Brazil to be used for C. macropomum fish farming purposes.


INTRODUCTION
Synthetic antimicrobials play an essential role in preventing and treating bacterial infections, in protecting both human and animal health, as well as aquatic organisms produced in intensive systems.However, the use of non-authorized antimicrobials in aquaculture systems has mainly contributed to drug-resistance development in bacterial strains capable of affecting populations' health and safety, as well as the environmental ones.In addition, these substances can leave residues in animal-origin products (AOP), such as fish, and it can pose risks to human health due to their consumption (Gastalho et al., 2014;De Sousa et al., 2019;Pessoa et al., 2019).
The emergence of antimicrobial resistance among food-borne pathogens is widely recorded in the literature; such a phenomenon is attributed to indiscriminate use of antimicrobials in animal production systems (Caniça et al., 2019).Bacteria can undergo selective pressure due to the use of antimicrobials; consequently, they can function as reservoirs of resistance genes and horizontally transfer them to other bacterial species (Hussain et al., 2017).Thus, bacteria presenting multidrug resistance (MDR) phenotypes are a growing concern for One Health, since they can directly transfer resistance genes to humans through AOP intake, and it can make it hard to treat infectious diseases in both humans and animals (Nhung et al., 2017).
Bacterial infection outbreaks are a limiting factor for aquaculture systems, since they affect production by decreasing its yield (Watts et al., 2017); consequently, they lead to significant economic losses (Figueiredo and Leal, 2008;Turker et al., 2009;Zamri-Saad et al., 2014).Bacteria of interest to fish farming systems are classified as opportunistic; they are found in water and in fish microbiomes, as well as can trigger bacterial infections in weakened hosts (Leira et al., 2016).Despite the diversity of bacteria showing potential pathogenicity in fish, Aeromonas spp.stands out for its high frequency rates (Suhet et al., 2011;Sebastião et al., 2015).
Bacteria belonging to genus Aeromonas are quite numerous and acknowledged for their hemolytic and biofilm formation abilities, which can affect strains' virulence (Sebastião et al., 2015;Pessoa et al., 2019).Thirty-six Aeromonas spp.species are listed in the literature; at least 19 of them are considered emerging pathogens (Pessoa et al., 2019;Fernández-Bravo and Figuera, 2020).Aeromonas hydrophila stands out among the aforementioned species as the most isolated one in fish farming systems, at global level, as well as for its ability to infect several hosts (Pessoa et al., 2019).This bacterial species was recorded in tambaqui fish (Colossoma macropomum) grown in Brazil (Leão et al., 2020;Pessoa et al., 2020;Gallani et al., 2020).

Ethical Aspects
All methodological procedures were carried out in compliance with ethical principles established by the Brazilian College of Animal Experimentation, as well as with the Animal Experimentation Ethics Committee (CEEA -Comitê de Ética em Experimentação Animal) of State University of Maranhão (UEMA -Universidade Estadual do Maranhão) (Protocol n. 09/2022).
In order to carry out the resistance test, Aeromonas spp.isolates were inoculated in brain and heart infusion (BHI) broth and incubated at 37°C, for 24 hours, until they showed turbidity compatible to McFarland standard n.0.5.Sowing was carried out with the aid of a sterile cotton swab, on plates filled with Müller-Hinton agar; five minutes later, antimicrobial disks were added to the plates, based on using flamed tweezers.Plates were incubated in a bacteriological oven at 37°C, for 24 hours.After the incubation procedure was over, inhibition halos -formed around the respective active principles -were measured with a millimeter ruler.
Results were compared to the measurement table set by the laboratory accounting for manufacturing the antimicrobial disks.Isolates were classified as susceptible, susceptible upon increased exposure and resistant to the assessed antimicrobial principles (Table 1).The study took into consideration multiple-drug resistance (MDR) phenotypes, as well as isolates showing resistance to three, or more, antimicrobial classes many different at the same time, according to Dos Santos et al. (2014).

RESULTS AND DISCUSSION
Aeromonas spp.isolates, deriving from tambaqui fish (Colossoma macropomum), have shown generalized resistance to penicillin, as well as high resistance to oxacillin, cefepime, cefadroxil and azithromycin (Table 2); this finding suggested environmental contamination and/or likely use of these antimicrobials in fish farms.According to Saavedra et al. (2004), Costa et al. (2008) and Franco et al. (2010), Aeromonas spp.resistance to beta-lactam antibiotics, such as penicillin, oxacillin, cefepime and cefadroxil has been increasing in aquaculture systems (Miranda et al., 2013).According to Harakaeh et al. (2006), the increased number of antimicrobials-resistant microorganisms is explained by the indiscriminate and excessive use of these drugs in intensive animal production systems.
Bacterial infections are one of the main causes of economic losses in intensive production systems like fish farming (Tavares-Dias and Martins, 2017 ;Bandeira Junior et al., 2019;Pessoa et al., 2019).According to Pessoa et al. (2020), Aeromonas hydrophila was the most prevalent bacterial species isolated (41.2%) from tambaqui fish, although other species were also isolated at lower rates, namely: A. dhakensis, A. caviae, A. veronii and A. jandaei.These findings have evidenced that tambaqui fish are not free from epizootic outbreaks caused by bacteria belonging to genus Aeromonas.Therefore, greater attention should be given to this bacterial genus in native fish species.
Only two antimicrobial drugs have their use approved by the Ministry of Agriculture, Livestock and Food Supply (MAPA) to treat bacterial infections in fish, namely: oxytetracycline -used to treat salmonids and catfish; and florfenicol -used to treat trout and tilapia.Both drugs are orally administered through fish feed (Sindan, 2018).Among these drugs, florfenicol is the antimicrobial drug most often used in aquaculture systems to treat several bacterial diseases in fish (Zhang et al., 2020).However, according to Miranda et al. (2013) and Pessoa et al. (2019), antimicrobial drugs are misused as prophylactics and therapeutics in fish farms, a fact that contributes to the selection of resistant bacteria, as well as to the selection and dissemination of their respective antimicrobial resistance-encoding genes.
Ampicillin associated with sulbactam (penicillin), neomycin (aminoglycoside) and ofloxacin (quinolone) were the antibiotics showing the best activity against Aeromonas spp.isolates in the present study, i.e., the ones that presented the highest sensitivity to (Table 2).High sensitivity to antimicrobial "ampicillin" may be associated with the presence of compound "sulbactam", which is an irreversible inhibitor of most beta-lactamases found in penicillinresistant organisms, such as Aeromonas sp., and it indicates synergistic interaction between these components.
Resistance to ampicillin is expected in most Aeromonas spp.species, in association with the production of beta-lactamase enzymes (Miranda et al., 2013); this antimicrobial drug is used as a selective agent in culture media used to grow this bacterium (Jacobs and Chenia, 2007).Costa et al. (2008) observed high ampicillin resistance levels in bacteria isolated from catfish (Rhamdia quelen) grown in Santa Maria County, Rio Grande do Sul State, Brazil.Franco et al. (2010) assessed bacterial isolates extracted from shrimp (Litopenaeus vannamei) and recorded 12.5% isolates' sensitivity to ampicillin.
Quinolones are drugs widely used to treat furunculosis, among other infections caused by Aeromonas spp., in fish (Smith et al., 2010).However, according to Alcaide et al. (2010), there is concern about the indiscriminate use of quinolones, since resistance to this antibiotic emerges from the inhibition of nucleic acid synthesis.Akinbowale et al. (2008) observed sensitivity of bacteria isolated from a wide variety of fish species and aquaculture environments in Australia, in tests conducted with ciprofloxacin, which is a quinolone type.Costa et al. (2008) recorded 84.31% (n= 43) sensitivity to nalidixic acid (another quinolone) in 15 Aeromonas spp.isolates extracted from catfish (Rhamdia quelen).These findings are consistent with those observed for Rev. Ambient.Água vol.18, e2936 -Taubaté 2023 Antibiotic-resistant bacteria transfer to humans through contaminated food is an emerging public health issue.The literature has fewer studies focused on investigating antibiotic resistance in bacteria isolated from aquatic animals than studies focused on investigating this topic in land-based food-producing animals.The present study provides further support to the hypothesis that there is potential risk of transferring resistant bacteria to humans through fish intake.

CONCLUSIONS
Based on findings in the current study, it may be concluded that: • Despite the lack of antimicrobial drugs registered in Brazil to be used for Colossoma macropomum culture purposes, antimicrobial resistance was observed in Aeromonas spp.isolated from tambaqui fish.
• Multiresistance to three or more antimicrobials observed in high frequency in evaluated Aeromonas spp.raises concern about fish farming in São Luís Metropolitan Region -MA, as well as about human health, given the likelihood of human infections caused by resistant bacteria due to fish intake.
• Given the clinical and epidemiological implications of Aeromonas spp.species investigated in the present study, as well as the identification of antimicrobial drug resistance and multidrug resistance profiles, it is worth emphasizing the urgent need of both warning about and clarifying these pathogens' significance for Public Health.This knowledge can help to establish preventive infection control guidelines in intensive fish farming carried out in Maranhão State and countrywide.

Table 1 .
Interpretation pattern of disk diffusion test based on halo measurements, and results observed for sensitivity, sensitivity upon increased exposure and resistance of Aeromonas spp.isolates tested against 10 antimicrobials.