Resistance of Aeromonas hydrophila isolates to antimicrobials and sanitizers

ABSTRACT: In recent decades, Aeromonas hydrophila has emerged as a foodborne bacterial pathogen of public health concern, especially as it exhibits resistance to the major chemical sanitizers commonly used in the food industry. Meanwhile, this pathogen may be spread from diseased fish flesh through the contamination of equipment contact surfaces during food processing, thus posing a food safety risk. Thise determined the susceptibility profiles of retail fish-borne A. hydrophila isolates to 24 common antibiotics and five major sanitizers used in the food industry. The polymerase chain reaction technique was used to confirm all A. hydrophila isolates to the species level, and the agar diffusion method was applied to determine the antimicrobial susceptibility profiles. All isolates were confirmed to be A. hydrophila species. This bacterium was observed to have resistance to multiple antibiotics, with the highest resistance index being for those of the beta-lactam class. Additionally, the isolates showed high resistance to four of the five chemical sanitizers tested, with the highest resistance rate being toward sodium hypochlorite. The results suggested that A. hydrophila isolates with multiple resistance to the antimicrobials and main sanitizers used in the food industry can be found in retail fish sold in the Cuiabá region of Mato Grosso, Brazil.


INTRODUCTION
Fish constitutes an important food in the human diet.However, they may contain Aeromonas hydrophila, a bacterial species found ubiquitously in aquatic environments (CHATTOPADHYAY & ADHIKARI, 2014).This human pathogen represents a serious health risk owing to its production of enterotoxins, hemolysins, and cytotoxins as well as other metabolites related to its pathogenicity and virulence (DASKALOV, 2006;IGBINOSA et al., 2012;STRATEV &;ODEYEMI, 2016;PESSOA et al., 2019;FERNÁNDEZ-BRAVO & FIGUERAS, 2020).More importantly, it has shown resistance to different antimicrobials (PRAVEEN et al., 2016).Although several studies have reported the presence of Aeromonas in various food sources, including retail fish (ABD-EL-MALEK, 2017;PASTRO et al., 2019;SANTOS et al., 2019), there is no specific legislation that sets standards for this microorganism in Brazil, resulting in a potential health risk to consumers.
Ciência Rural, v.53, n.12, 2023.Martins et al.Antimicrobial resistance in emerging pathogens is a potential threat to human health and has become one of the biggest challenges to public health systems worldwide (MCEWEN & COLLIGNON, 2018).Most antimicrobials are common to veterinary and human use, which increases the potential of resistance development in pathogens that infect humans and animals and intensifies the risk of spread through food contamination (EMA, 2014;STRATEV & ODEYEMI, 2016).
The infective dose of A. hydrophila is not yet fully understood (PARK et al., 2021).According to GONZÁLEZ-SERRANO et al. (2002), an efficient cooking process can inactivate this pathogen, but insufficient cooking and cross-contamination will pose a potential health risk, primarily in immunosuppressed individuals, children, and the elderly.
Pathogens that exhibit resistance to antimicrobials also exhibit resistance to sanitizers (ROZMAN et al., 2021).The effectiveness of sanitizers, especially against gram-negative bacteria, is crucial in food manufacturing and processing units (ODEYEMI et al., 2022), and their ineffectiveness can represent a threat to food safety and consumer health (GONÇALVES, 2011;SHENG & WANG, 2021).
In Brazil, only two antimicrobials for treating infections in aquatic animals are regulated (SINDAN, 2021).According to the normative instruction number 26 declared by the Ministry of Agriculture, Livestock and Supply (MAPA) on July 9, 2009, the use of betalactams, tetracyclines, sulfonamides, quinolones, and amphenicols as additives for enhancing animal performance or as animal food preservatives is prohibited (BRASIL, 2009).The indiscriminate use of antibiotics in fish farming has favored the development of resistance genes through selective pressure and is one of the main factors responsible for the development and spread of multidrug-resistant bacteria (MUZIASARI et al., 2016).
To reduce the microbial load that can contaminate food, the industry uses sanitizers, which are defined as substances that contain one or more active ingredients with biocidal activity against harmful microorganisms (ROZMAN et al., 2021) at safe levels, are not harmful to health, and are intended for use on surfaces, objects, and environments (KUAYE, 2017).Chlorinated compounds, iodinated compounds, quaternary ammonium compounds, and peracetic acid are among the main groups of sanitizers used in the food industry (ANDRADE, 2008;KUAYE, 2017).
The resistance of A. hydrophila to antimicrobials and the main sanitizers used in the food industry makes this microorganism a target of study and has stimulated the search for better performing sanitizers.It is likely that fish sold in the retail environment in Brazil are contaminated with A. hydrophila, considering the non-application of good handling and sanitation practices in an adequate manner.In view of this, the study determined the resistance of retail fish-borne A. hydrophila isolates to 24 common antibiotics and five main sanitizers used in the food industry.

Samples
In this study, 19 A. hydrophila isolates obtained from the Food Microbiology Laboratory of the Federal Institute of Education, Science and Technology of Mato Grosso (IFMT) Campus Cuiabá Bela Vista were used.The bacteria were from a previous study by SILVA (2023), having being isolated from samples of the Amazonian Pintado (Pseudoplatystoma fasciatum × Leiarius marmoratus), tambacu (Colossoma macropomum × Piaractus mesopotamicus), and tambatinga (Colossoma macropomum × Piaractus brachypomus) acquired from a supermarket, fair, and fish market in the city of Cuiabá, Mato Grosso, Brazil.The bacterial isolates cultured on tilted trypticase soy agar (TSA) in test tubes were stored at 4 °C refrigeration until the time of this study.The cultures were recultured daily for maintenance of the strains according to the method described by SILVA et al. (2017).

Molecular characterization of the A. hydrophila isolates
Young colonies of A. hydrophila on TSA were suspended in sterile water and boiled for 10 min in a dry bath (thermoblock) according to the methods described by PARK et al. (2021) andLAU et al. (2020).Then, each suspension was centrifuged at 24,104 × g for 5 min, following which the supernatant containing total genomic DNA was collected and quantified using a NanoDrop spectrophotometer.An aliquot of the extracted DNA was used for molecular identification of the isolate and the remaining was stored at -20 °C.
According to the method described by EL-GHAREEB et al. ( 2019), the following PCR program was used: denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 s, hybridization of primers at 50 °C for 40 s, and extension at 72 °C for 1.5 min; and a final extension at 72 °C for 5 min.After electrophoresis of the amplified products on a 1.5% agarose gel (100 V, 30 min), the bands were visualized using a UV LTB-HE transilluminator (Loccus Biotechnology).For the standard positive control bands, a commercial 100 bp DNA ladder (Sinapse Inc.) with fragments of known molecular weights was used.
For the antimicrobial susceptibility test, each bacterial suspension was first prepared to a turbidity of 0.5 (McFarland scale) in sterile 0.85% saline solution.Then, the suspension was inoculated into Mueller-Hinton agar medium, and the mixture was overlaid atop each antibiotic-impregnated disc.Incubation was carried out in a bacteriological oven at 35 °C (±2 °C) for 18 -24 h.
After 18-24 h growth of the isolate, the diameter (in millimeters) of the inhibition halo was measured using a meter stick and interpreted using the standard table for classification of the level of strain sensitivity to the antimicrobial tested.As no halo was formed for some of the antibiotics tested (ampicillin, cephalothin, erythromycin, penicillin G, and polymyxin B), they could only be classified as resistant.

Sanitizer susceptibility testing
The susceptibility profile of A. hydrophila to sanitizers was determined using the method described by WANJA et al. ( 2020), with modifications.
The following commercial sanitizers commonly applied in the food industry were tested: 300 mg•L -1 peracetic acid, 70% alcohol, 700 mg•L -1 quaternary ammonium, 200 mg•L -1 sodium hypochlorite, and 0.3% hydrogen peroxide (YUAN et al., 2020).These were diluted according to the manufacturers' instructions for use and the concentrations cited in the literature.The same steps and conditions applied to the agar diffusion test for antibiotics was applied to that for the sanitizers, starting with the prior preparation of the sanitizer dilutions and of the bacterial suspensions to a turbidity of 0.5 in sterile 0.85% saline solution.After inoculation of the bacterial suspension into Mueller-Hinton agar, the mixture was overlaid atop sterile monodiscs (Laborclin) soaked in the respective sanitizers.Following 18-24 h incubation, the inhibition halos were measured.Results were interpreted according to the criteria described by KUAYE (2017), and the isolates with no halo formation were considered resistant to the sanitizer applied.

Statistical analysis
The data of this study were tabulated, typed, and saved on an electronic spreadsheet.Statistical analysis was conducted using R Core Team software (FOX & WEISBERG, 2020;LENTH, 2020;R CORE 15 TEAM, 2020;JAMOVI PROJECT, 2021), where Ciência Rural, v.53, n.12, 2023.Martins et al. descriptive analysis was performed.Because of the non-normality of the data (Shapiro-Wilk test), the Wilcoxon non-parametric test was used.
Multidrug resistance, characterized as resistance to at least one antibiotic in three or more classes (JEONG et al., 2007), was evidenced in this study, as the A. hydrophila isolates recovered from retail fish showed resistance to at least one antibiotic in all the classes investigated.This phenomenon may be attributed to the indiscriminate use of antimicrobials in aquaculture, genetic mutations, or the horizontal dissemination of resistance genes (LAU et al., 2020).
The high resistance to beta-lactams (ampicillin, cephalothin, ampicillin sulbactam, penicillin G, and cefuroxime) is in accordance with the known intrinsic resistance of Aeromonas species to this class of antibiotics.Production of the chromosomal enzyme beta-lactamase is recognized as a common feature among species of genus Aeromonas and contributes to their resistance to betalactams (ROSSOLINI et al., 1996).This intrinsic resistance is related to the chemical instability of the beta-lactam ring in the antibiotic structure, making the drugs susceptible to hydrolysis through bacterial beta-lactamase activity (ZDANOWICZ et al., 2020).
In the isolation of Aeromonas, the use of ampicillin is recommended (MCMAHON & WILSON, 2001) owing to the intrinsic resistance of the species to this beta-lactam antibiotic, which facilitates selection of the bacterium among other bacteria present in the medium that are inhibited by the antimicrobial agent.The findings of this study confirm this common characteristic of the Aeromonas species and is in agreement with the findings of other studies (DAHDOUH et al., 2016;RAMADAN et al., 2018;WU et al., 2019).
Results obtained regarding A. hydrophila resistance to the cephalosporins were similar to those of other studies (RAMADAN et al., 2018;SANTOS et al., 2019;ZAHER et al., 2021).This drug resistance is mainly due to the degradation activities of extendedspectrum beta-lactamases and ampC (a chromosomal cephalosporinase), the main beta-lactamases involved in bacterial resistance to cephalosporins, which may be inherent to the bacteria or even acquired, resulting in the inability of the antibiotics to reach their site of action (MACHADO et al., 2019).
The resistance and multidrug resistance profiles of Aeromonas species from freshwater fish are characterized mainly by the residues from the aquaculture practice itself, where resistance genes are spread throughout the environment and resistance determinants are transferred from terrestrial animals to bacteria and human pathogens (CABELO, 2006;FAUZI et al., 2021;NHINH et al., 2021).The indiscriminate use of antimicrobials in aquatic animals and the consequent contamination of the environment are primarily responsible for the spread of resistant strains in fish (KIMERA et al., 2020).The high level of antibiotic resistance in A. hydrophila reported in this study suggested that the indiscriminate use of antimicrobials in humans and animals has contributed to the development and dissemination of resistance genes along the food chain.It also indicated that unregulated antimicrobials are being misused and overused in fish farming, given that only oxytetracycline and florfenicol are approved for use in Brazil (CARVALHO & SANTOS, 2016;BUENO et al., 2017;SIDAN, 2021).
It is worth noting that some authors have suggested that the beta-lactamase gene is present in the diverse microbiota in the aquatic environments where these fish are raised, representing a serious problem in view of the high enzymatic potential to hydrolyze betalactam antibiotics (ZDANOWICZ et al., 2020).This poses a threat to human health and potentiates the risk of occurrence of antimicrobial resistance (EMA, 2014;STRATEV & ODEYEMI, 2016).
With regard to the resistance of the A. hydrophila isolates to the main industrial sanitizers at the concentrations tested in this study (Table 2), the bacteria showed the lowest percentage (15.78%) of resistance to 3% hydrogen peroxide, which was the most effective of the tested sanitizers.However, it is important to note that the classification of sanitizer sensitivity is complex.The resistance rate obtained in this study can be justified by the fact that A. hydrophila produces catalase, an important cell-detoxifying enzyme that is responsible for converting hydrogen peroxide (a toxic metabolite) into water and oxygen molecules (KUAYE, 2017).As a strong oxidant, hydrogen peroxide is commonly used as a bactericide and sporicide, with low toxicity and residual effect (GERMANO & GERMANO, 2015).Its sanitizing effectiveness against A. hydrophila was evidenced in this study for more than 80% of the isolates.
In contrast, a high rate of resistance to 200 mg•L -1 sodium hypochlorite was observed in more than 90% of the isolates.Chlorine compounds, including sodium hypochlorite, are generally among the most commonly used sanitizers in the food industry.These chemicals have good sanitizing efficacy at low concentrations, are not affected by water hardness, and have high effectiveness against a wide spectrum of bacteria, notwithstanding the fact that they are the cheapest among most sanitizers (ANDRADE, 2008;KUAYE, 2017).However, at the recommended concentration, this active ingredient was not sufficient for inhibiting the growth of the A. hydrophila isolates in this study.
The chemical sanitizers based on peracetic acid and quaternary ammonium, as well as alcohol, Ciência Rural, v.53, n.12, 2023.Martins et al. were ineffective against the A. hydrophila isolates at the concentrations tested.More than 80% of the isolates were resistant to 300 mg•L -1 peracetic acid and 70% alcohol, whereas more than 60% were resistant to 700 mg•L -1 quaternary ammonium.The resistance to disinfectants is related to several cellular mechanisms -internal and external to the bacterial cell -that had resulted from phenotypic and genotypic adaptations (ROZMAN et al, 2021).
Although, there are several aspects related to the mechanism of bacterial resistance to sanitizers, the most common ones are the restricted permeability of the bacterial cell wall, enzymatic degradation, expression of efflux systems, biofilm formation, and changes in the target sites (LAMBERT, 2002;CHAPMAN, 2003).Efflux pumps comprise the main pathways of resistance to sanitizers, and the exposure of microorganisms to inhibitory and subinhibitory concentrations of antimicrobials contributes to the development of resistance (WEBBER & PIDDOCK, 2003;GNANADHAS et al., 2013).
In nature, gram-negative bacteria tend to be more resistant than gram-positive bacteria to antimicrobials owing to the complexity of their cell wall.According to MAILLARD (2002), the biocidal activity of sanitizers can vary significantly among different types of microorganisms and even among different strains of the same species.Despite its importance, bacterial resistance to sanitizing agents has not been treated with interest and due attention by the academic community.The indiscriminate use of sanitizing agents can significantly decrease their effectiveness against clinically important microorganisms ( ROZMAN et al., 2021).This outcome poses a serious threat to food safety, especially coupled with the fact that foodborne outbreaks can become highly recurrent if sanitizer resistance becomes established, especially in the food industry (CARLIE et al., 2020).Therefore, results of this study suggest the need for further research on the factors that affect the performance of sanitizing agents in the fish processing environment, aiming the ultimate goals of selecting the most efficient sanitizer to be applied in the sanitization process and corroborating the safety of this food type.The sanitizers tested followed the usual concentrations cited in the literature and were prepared according to the manufacturers' instructions.

Table 2 -
Radius (in millimeters) of the zone of inhibition of various sanitizers tested against Aeromonas hydrophila isolates.