Detection of putative virulence traits in Aeromonas spp. from brazilian food chain through phenotypic tests and polymerase chain reaction

Aeromonads are natural inhabitants of aquatic environments and may be associated with numerous infections in humans and animals. The human disease may range from self-limiting diarrhea to a more severe form. The pathogenesis of infections are multifactorial, because of their wide variety of virulence factors. This study aimed to evaluate virulence markers in Aeromonas isolates and determinate their virulence profiles. There were analyzed 120 strains of A. caviae (n = 57) and A. hydrophila (n = 63) from human, animal and environmental sources between 2008 and 2012. All isolates were examined to detect extracellular virulence enzymes by phenotypic activity and the presence of virulence genes hly A, aer A, lip , gcat , ser , act , alt and exu by PCR. We observed more than 90% of positivity for at least one phenotypic virulence factors and all of them had at least two of the virulence genes measured. Among the virulence enzymes detected, the DNase was present in 93.33% of the isolates and hemolytic activity was detected in 62.5%. Collagenase and elastase were found in 13.33% and 10.83% of the strains, respectively. We found exu and gcat in 100% of the isolates, lip in 40.83%, aer A in 40.83%, hly A in 40%, alt in 19.16%, act in 17.5% and ser in 11.66%. It was possible to observe different combinations of virulence factors between the isolates showing the multifactorial virulence among the isolates. The diversity of virulence profiles found in this study hint heterogeneity in clones circulating in our environment.


Introduction
Aeromonas species are Gram-negative rod-shaped ubiquitous free-living organisms found in aquatic ecosystems, which can quickly colonize an exceptionally wide variety of habitats and hosts. These microorganisms have already been isolated from water, mammals, fish, invertebrates, birds, insects and soil (Chaix et al., 2017;Fernandes-Bravo & Figueras, 2020;Grilo et al., 2021).
Recognized as emerging pathogens associated with gastroenteritis worldwide, Aeromonads are also implicated as an etiologic agent in a variety of extra intestinal human diseases. The spectrum of disease severity is broad, ranging from mild diarrhea to life-threatening necrotizing fasciitis, septicemia, meningitis, cholera-like illness, and hemolytic-uremic syndrome (Fernandes-Bravo & Figueras, 2020;Gonçalves Pessoa et al., 2019). A. caviae and A. hydrophila lie between most common Aeromonads isolates from human source and are especially implicated in cases of diarrhea (Mbuthia et al., 2018). While traditionally regarded as a pathogen of immunocompromised humans; there have been several reported Aeromonas infections of immune competent individuals (Batra, et al., 2016).
Aeromonas can be isolated from several natural sources, particularly aquatic environments. Contact with fish and other aquatic animals develop in a continuous and almost inevitable way (Gonçalves Pessoa et al., 2019). These microorganisms are indigenous to aquatic environments and have been isolated from surface, underground, potable, bottled, residual, seawater, and irrigation waters (Fernandes-Bravo & Figueras, 2020) and have been isolated from chlorinated drinking water in many countries. They occur in distribution systems where biofilm formation protect the strains of disinfection (Skwor et al., 2020;van Bel et al., 2020;Miyagi et al., 2021). Metagenomic studies of wastewater revealed Aeromonas to be one of the prevailing bacteria probably due to its capacity to regrow in the sewerage system (Gonçalves Pessoa et al., 2019).
Aeromonas may express different virulence factors such as extracellular products including cytotoxic and cytotonic enterotoxins, hemolysins and hydrolytic enzymes, which may create disturbances and biochemical changes in the host's body and as with all pathogens, disease is the result of complex molecular interactions between bacterium, environment, and host.
Aeromonas virulence factors are involved in the establishment of infection and can act meaningfully in the development of the disease (Barger et al., 2020, Jin et al., 2020. Aeromonas species may produce different toxins like as aerolysin (aerA), cytolytic enterotoxin (act), cytotonic enterotoxin (ast), heat labile cytotonic enterotoxin (alt), as well as many enzymes that are considered virulence factors, and may even possess a Type III Secretion System and extracellular hydrolytic enzymes: amylases, arilamidases, esterases, elastases, desoxirribonucleases, chitinases, peptidases and lipases (Citterio & Francesca, 2015). Although these virulence factors contribute to pathogenesis of animal and human diseases caused by Aeromonas, none of them alone may be responsible for all symptoms of disease stages. Because of the complexity in pathogenesis of Aeromonas spp, due to its multi-factorial nature, identification of multiple virulence factors of this genus, become important (Awan et al., 2018).
In Brazil, several studies indicate the occurrence of Aeromonads from aquatic ecosystem, seafood (fish, oysters, mussels and crabs), marine and estuarine animals (Castelo-Branco et al., 2015;Silva et al., 2017;Cardoso et al., 2018;Marinho-Neto et al., 2019;Vaz Rodrigues et al., 2019). Since Aeromonas are known as zoonotic agents, those studies are important to public health because of association with foodborne disease and extra intestinal infections).
Knowing this background we purpose investigate the production of extracellular hydrolytic enzymes and eight virulence genes in A. hydrophila and A. caviae isolated from different sources in Brazilian food chain and their relevance in public health.

Strain Selection
The study was performed with 120 strains of Aeromonas caviae (n = 57) and A. hydrophila (n = 63) isolated from human, animal and environmental sources from 2008 to 2012 selected in the database of National Reference Laboratory for Enteric Diseases / Oswaldo Cruz Institute / FIOCRUZ / Brazil (Table 1).

Phenotypic identification of enzymes
Aeromonas spp. were analyzed for the presence of virulence factors represented by the proteolytic enzymes' elastase and collagenase, the hemolytic activity and nuclease activity. The phenotypic activities investigated were described previously (Chacón et al., 20003;Lafisca et al., 2008). Briefly they were collagenase and elastase activities in BHI (Brain Heart Infusion) broth (37 ºC/18-24h) and then streaked onto Agar BHI added with 1% of collagen, Agar BHI added with 1% of elastin considering positive colonies result as a clearing zone (translucent and luminous halo) under and around the spots inoculums.

Results
The 120 Aeromonads isolates were found to be more than 90% positive for at least one phenotypic virulence factor and all of them had at least two of the measured virulence genes (Table 3).
One hundred thirteen (94.2%) of the isolates exhibited the virulence factors examined. Of the 120 isolates, 75 (62.5%) showed hemolytic activity, and DNA nuclease activity was detected in 112 (93.3%) isolates. The proteolytic enzymes collagenase and elastase were positive in 16 (13.3%) and 15 (12.5%) isolates, respectively. Among the isolates of human origin (n=43), 97.7% of the isolates had at least one of the virulence factors. Among the isolates of animal origin (n=72), this was the case for 91.7% of the isolates. All isolates from the environment (n=4) had virulence factors. Nine different combinations of virulence factors were detected among the isolates (Table 4). Both Aeromonas caviae and A. hydrophila exhibited six profiles of virulence factors. Proteolytic activity of the enzymes collagenase and elastase was observed primarily in human sourced Aeromonas hydrophila, with the isolate from a skin wound sample positive for all virulence factors tested.
The presence of nuclease activity (DNAse) was detected in 112 isolates. However, the eight phenotypically negative aeromonads were positive for the presence of the exu gene.
Isolates from human sources were distinguished between fecal and nonfecal origin. For genes associated with Aeromonas enterotoxins and hemolysins, isolates of fecal origin had a higher incidence of the act gene. Isolates of nonfecal origin had a higher incidence of the aerA, alt, and hlyA genes.
Twenty-seven combinations ( Among isolates from marine mammals with migratory habits, the most prevalent virulence profile was exu-gcat-hlyA, which was detected in 13 isolates from Megaptera novaeglangiae, three from Arctocephalus australis, and one from Eubalena australis. The exu-gcat profile was found in 11 isolates of Lagenodelphis hosei (n=6), Arctocephalus gazella (n=4), and Kogia sima (n=1). The virulence profiles act-aerA-exu-gcat-hlyA-lip and act-aerA-exu-gcat-lip were found in nine isolates each. The first was present in two isolates of Pontoporia blainvillei and seven of Stenella coeruleoalba, and the second was present in five isolates of Eubalena australis and six of Megaptera novaeangliae. These migratory animals tend to reproduce in tropical waters. "Grounding" probably caused by navigational error, stressful conditions, weak immunity, and disease incidence. The health status of the "grounded" animal may provide an indication of the status of the population that is at sea.

Discussion
Aeromonas have multifactorial virulence profile. Some authors associate the presence of high number of virulence factors to bacteria pathogenic potential. Knowing that, virulence markers in clinical strains point out a relation with the symptoms and it is possible to investigate which mechanisms are behind the disease. Studies reported Aeromonas as a cause of diarrhea in many countries around the World (Qamar et al., 2016;Wenqing et al., 2016;Mbuthia et al., 2018;del Valle de Toro et al., 2020). Some studies propose a correlation between Aeromonas toxin genes and potential diarrheal diseases (Teunis & Figueras, 2016). Ottavianni et al. (2011) found at least two toxin genes with distinct combinations in Aeromonas strains showing their pathogenic potential. Virulence factors could determine how disease develops (Tavares, et al., 2015).
Aeromonas are reported as a cause of diarrhea in many countries around the World but their incidence in human infections worldwide is unknown. Studies therefore show that the incidence of diseases caused by Aeromonas vary by geographical location and can be related to bad hygiene habits in undeveloped regions The human gastroenteritis disease caused by Aeromonas can appear in the form of a self-limiting liquid diarrhea to a more severe and invasive diarrhea, which specially is a problem for young children and infants (Fernandes-Bravo & Figueras, 2020).
Particularly in Brazil, incidence ranges from 2,6% to 19,5% in cases of diarrhea (Assis et al., 2014). In our study, 29% (n=35) of isolates were from diarrhea cases. Among these 35 isolates, the most frequent combinations of virulence factors were dnase-hem and dnase-ela-hem. In relation to virulence genes, the most frequent associations were exu-gcat and exu-gcat-hlyA. Although it is not possible to predict whether a particular Aeromonas strain can induce diarrheal illness, it is possible that certain virulence factors of Aeromonas species such as proteases, hemolysins and lipases are associated with their pathogenicity in cases of diarrhea (Tomás, 2012).
The production of extracellular enzymes and proteases in Aeromonas spp. allows them to survive in different ecosystems. More than a matter of virulence, it is a mechanism of survival and response to environmental stress (Citterio & Biavasco, 2015;Zeynep & Comhur, 2018). All isolates from fish samples showed collagenase activity, indicating a possible association between the presence of this enzyme and the pathogenicity of Aeromonas to Genidens barbus and Oreochromis niloticus.
Collagenase has a role in pathogenicity of certain microorganisms, including some admittedly pathogenic Aeromonas species and along to the enzyme elastase, are present in infections of skin and tissue. They may compromise cartilage, bone and mucosal structures (Citterio & Biavasco, 2015;Barger et al., 2020;Jin et al., 2020). These two enzymes standing in A.
hydrophila from skin infection, in this study, suggests the association of these enzymes to Aeromonads wound disease.
A. caviae and A. hydrophila regularly produce DNase (Khor et al., 2018). Nucleases contribute to the establishment of infection exceeding the initial host defenses or by providing nutrients to cellular proliferation (Tomás, 2012). Likewise, DNases may be associated with cell damage and tissue destruction besides the evasion of host defenses, playing a crucial role in microbial invasion and colonization (Li et al., 2015;Pang et al., 2015). Our findings, in which all strains exhibited the exu gene, corroborate the importance of this virulence gene for the maintenance of Aeromonads in various hosts and also in the aquatic environment.
The hemolytic activity is correlated with Aeromonas group of aerolysins and hemolysin comprising several genes, among them aerA, hlyA and act (Hoel, et al., 2017). Aerolysins are proteins capable of altering the permeability of blood cells, as well as other eukaryotic cells (and consequently promoting osmotic lysis) due to polymerization of the structure induced by binding to a membrane specific glycoprotein site (Gonçalves Pessoa et al., 2019). The enterotoxin act is the most cytotoxic virulence factor of Aeromonas and induces multiple effects including hemolytic, cytotonic, and cytotoxic activities (Rasmussen-Ivey et al., 2016;Miyagi et al., 2021). Among our results, the research of hemolytic activity in plaque presented the second largest number of positive isolates, present in a special way among A. hydrophila from human source. Of the genes associated with hemolytic activity, aerA, hlyA and act, were found in higher number of isolates of A. hydrophila, suggesting higher virulence in this species.
Aeromonads' extracellular enzymes role in pathogenicity is still to be determined, they represent a big potential to adapt to environmental changes. Extracellular proteases contribute to the metabolic versatility that allows Aeromonas to persist in different habitats and that facilitate ecological interactions with another organism (Tomás, 2012). It has been described that the genus Aeromonas produces a wide range of exotoxins. However, all toxins described are not produced by all strains, although strains may possess their genes. Furthermore, some strains only express toxin genes in certain growth conditions. Cytotonic enterotoxins do not produce degeneration of the epithelium and have mechanisms of action similar to those of the choleric toxin, since they increase the cyclic adenosine monophosphate (cAMP) levels and prostaglandins in intestinal epithelial cells. Aeromonas species produces cytotonic enterotoxins that show differen molecular weights and variable reactivity to the choleric antitoxin (Tomás, 2012;Rasmussen-Ivey et al., 2016;Miyagi et al., 2021).
Cytotonic heat-labile enterotoxin (alt) has a similar mechanism of action to cholera toxin by increasing the levels of cAMP and prostaglandins in intestinal epithelial cells (Tomás, 2012). species. The gene was later found to be highly conserved among the clinical and environmental isolates of Aeromonas species (Li et al., 2015;Pang et al., 2015;Hoel, et al., 2017;Khor et al., 2018;Nwaiwu, 2019). Thus, many researchers have used the gene as a specific Aeromonas genus marker (Mendes-Marques, et al., 2013). The results found in our study confirm this aspect of the gcat gene, considering that all isolates independently of species and source of isolation presented gcat + . -Marques et al. (2012) found no lip gene in clinical strains but found it in all environmental isolates concluding that its main activity is directly connected with the extracellular survival of Aeromonads. However, our results

Mendes
show 27% of clinical isolates and 40% of environmental isolates positive for the lip gene.
The serine protease released by certain strains of Aeromonas, assists the microorganism invasion breaking down the host's barriers and it's able to cleave plasma proteins such as fibrinogen generating potential complications such as septic shock and blood clotting disorder (Senderovich et al., 2012).
It was possible to observe in this study the diversity of virulence markers presented by the strains, through virulence profiles. It should be noted that the strains of human origin presented greater diversity of profiles. A comparison between the virulence profiles found in human and environmental strains shows that they present common profiles, and the environmental profiles contain virulence markers distributed among the human strains. All virulence markers of the environmental isolates were found in the human isolates. This association between the virulence factors found in environmental and human strains and at the same time the absence of common profiles points to the adaptive capacity of Aeromonas spp. associated with the multifactorial virulence profile.
When observing the most frequent virulence profiles among isolates of animal origin it is possible to notice the presence of markers of hemolytic characteristic, lipases and nucleases. These markers denote the pathogenicity and invasiveness of Aeromonas spp. in these animals. Studies (Peixoto et al., 2012;Castelo-Branco et al., 2015;Cardoso et al., 2018;Marinho-Neto et al., 2019;Vaz Rodrigues et al., 2019) found A. hydrophila and A. caviae among the main etiologies in sick fish and mammals. The isolated strains of A. hydrophila were mostly producers of protease, elastase, hemolysis and enterotoxin in phenotypic tests and the strains of A. caviae were producers of protease only.
Certain studies suggest that some species of Aeromonas synthesize more virulence factors and more often, suggesting the clonal origin virulence, thus, few clones were responsible for disease progression (Pang et al., 2015;Gonçalves Pessoa et al., 2019). Systematic studies on the distribution of genes coding for Aeromonads virulence factors allow us to evaluate the virulence potential of these microorganisms circulating in our environment.
More extensive genomic and epidemiological investigations will allow us to ascertain the frequency of core and accessory genes in environmental, animal and clinical Aeromonas isolates. Sometimes we will be able to detect novel virulence factors through genomic surveillance and estimate the virulence potential of clinical isolates through sequence analysis alone.

Conclusion
Among the Aeromonads isolates was possible to observe that more than 90% were positive for at least one phenotypic virulence factors and all of them had at least two of the virulence genes measured. This study shows that pathogenic  (1), 22-26. https://doi.org/10.1016/j.ram.2019.03.003 (Descripción de datos clínico-epidemiológicos de pacientes pediátricos con diarrea por Aeromonas spp. y estudio de la sensibilidad antibiótica de dicho agente.)