Microbiological analysis of Magellanic Penguins found in coastal regions of Northern Santa Catarina

The Magellanic penguin is the most commonly species of penguin found in temperate regions of South America. During the months of April to September, many of them are found stranded on coastal regions of Brazil, either dead or severely debilitated due to various adverse situations they face during their migratory period. Many of these debilities are due to infections caused by microorganisms present in the microbiota of these penguins, which take advantage of the immunosuppression of these birds to proliferate and generate clinical signs that can ultimately lead to their death. In the present study, several microbiological techniques were performed, including bacterial culture, antimicrobial susceptibility testing (antibiogram), fungal culture


INTRODUCTION
Penguins are considered sentinels of the oceans, as the study of these birds can provide information on how the impacts of human activities affect marine life (CAMPOS et al., 2013;DAMINELLI, 2018).
There are currently 18 species of penguins registered, with the Magellanic penguin (Spheniscus magellanicus) being the most commonly found in temperate regions. They are found in the Falkland Islands and in regions of Argentina and Chile that comprise the Patagonian Desert, mainly during their breeding period between September and March (VANHONI et al., 2018).
The migratory period usually begins in April and goes until mid-September. During this period, penguins leave for the ocean in search of food, traveling thousands of kilometers to the north and converging migration routes following the ocean currents to their wintering locations on the continental shelf, which are generally located in northern Argentina, Uruguay and the southernmost region of Brazil (SKEWGAR; BOERSMA; SIMEONE, 2014). Young penguins seem to start their migratory period later than adults, usually beginning to be found stranded of Brazilian coasts, mainly in the coastal regions of the states of Rio Grande do Sul to Rio de Janeiro in mid-June, where most of the time they are dead, and when found alive, they are usually weakened due to their lack of experience in hunting for food, which causes them to get lost from the group during migration (BRANDÃO, 2013). The weakening, physical debilitation and stress generated by starvation, pollution, collision with vessels and other human actions, decrease the immunity of these young birds, leaving them predisposed to develop infections, mainly caused by microorganisms belonging to their natural microbiota or those that usually occur with asymptomatic conditions in healthy penguins (GEEVERGHESE, 2013;MINISTÉRIO DO MEIO AMBIENTE, 2010).
Although the Magellanic penguin is the most common penguin species in temperate regions, with an estimated population of 1300000 breeding pairs in 2010, the main breeding colonies in Argentina have been experiencing a population decline every year. Studies in Rio Grande do Sul have found 19500 carcasses in the coastal regions of the state in just one year, with 97% of them being young animals. These alarming data show that the population of Magellanic penguins has been drastically declining annually (VANHONI et al., 2018).
The International Union Conservation of Nature has listed the Magellanic penguin as near threatened, highlighting the need for information gathering to increase conservation and protection efforts to the species. These actions are mainly carried out by teams at marine animal rehabilitation centers located along the coastal regions from northern Brazil to Argentina. These teams rescue stranded animals from coastal areas and send them to the rehabilitation center where they are treated and may have three possible outcomes. One is that the patient does not respond to treatment and dies, the second is that the patient responds to treatment but remains unfit to release, so it is kept in captivity for environmental education and species conservation, the third, and the most desirable outcome, is that the treatment is effective for the penguin and it can be reintroduced into the ocean without further debilitation (GEEVERGHESE, 2013;VANHONI, 2018).
The aim of the study was to isolate microbiological agents from Magellanic penguin samples from rehabilitation centers in the state of Santa Catarina.

MATERIALS AND METHODS
During the months of May to July, the laboratory "Medivet: Centro de Diagnósticos Veterinários" located in Joinville, Santa Catarina, received nine samples with requests for bacterial culture, eight requests for Salmonella spp. research, and two requests for fungal culture.   All samples that showed bacterial growth grew on both chromogenic agar and MacConkey agar, indicating they were Gram-negative bacteria, which was confirmed by bacterioscopy. Gram- The second most prevalent bacterial agent in the bacterial cultures was Pseudomonas sp., growing in three out of the nine samples taken. Widmer (2016) found Pseudomonas aeruginosa growth in six out of nine cultures from Humboldt penguins, mainly in lesions of the oral cavity and paranasal sinuses. It is a strict aerobic Gram-negative bacillus that is oxidase positive and has polar flagella, which makes it motile. It is found in the environment, including soil and water, and also forms part of the natural microbiota of the skin and mucous membranes of most animals (WIDMER, 2016). It is considered an opportunistic pathogen and can causes local infections in a variety of birds species, mainly due to the reduction of normal microflora, immunosuppression, generalized weakness, systemic diseases or mucosal lesions. They primarily infect the oropharynx and respiratory tract, but can also cause enteritis, peritonitis, bumblefoot and sepsis.
Another enterobacteria that commonly causes weakness in penguins is Escherichia coli, which showed positivity in two of nine samples analyzed in this study. They are short facultative anaerobic bacilli that, although part of the enteric microbiota of most animals, including penguins, infection with it becomes of great importance due to its ability to cause sepsis and sudden death in immunosuppressed animals. This is one of the possible illness found in these penguins (OSÓRIO et al., 2017).
All bacterial agents for which the antibiogram was performed showed resistance to at least one antibiotic. The one that showed the most resistance were cefalotin and amoxicillin, both showing resistance in ten out of the eleven samples tested, followed by penicillin, which showed resistance in nine agents, and cephalexin, which showed resistance in eight agents out of the eleven tested. Cefalotin and cephalexin belong to the cephalosporin group, whose results in this present study disagree with the results presented by the study of Silva et al., (2013) that investigated the resistance profile of agents related to bumblefoot disease in Magellanic penguins, where the cephalosporin class of drugs was the one that showed the most activity against bacterial strains.
The study agrees with Silva et al., (2013) regarding the resistance of Escherichia coli, for which sensitivity to cephalosporin was null, similar to the findings in the present study, where both positive samples for Escherichia coli showed resistance to both cefalotin and cephalexin. The present study also agrees with Silva et al., (2013) regarding the penicillin class of antibiotics, which showed the highest resistance to bacterial agents at 63,3%. In the present study, the first and third antibiotics that showed the most resistance were also from the penicillin class, with the first being amoxicillin with 90,90% (10 out of the 11 samples) and the third being penicillin with 81,81% resistance (9 out of the 11 samples).
The antibiotics that showed the highest sensitivity were gentamicin, which showed resistance in only two bacterial agents out of the eleven tested, followed by amikacin and ciprofloxacin, which showed resistance in three out of the eleven bacterial agents tested for antibiotic susceptibility. Amikacin and gentamicin belong to the aminoglycoside class, while ciprofloxacin belongs to the fluoroquinolone class (WOLLECK, 2022). Wolleck (2022), who studied the genotypic and phenotypic characterization of Escherichia coli resistance to antimicrobials in penguins, found that aminoglycosides were the second least resistance class of antibiotics, with an average resistance of 35,76%. In the present study, both samples of Escherichia coli showed sensitivity to both amikacin and gentamicin, and overall, the aminoglycoside class showed the highest sensitivity, with 22,75% resistance. Regarding ciprofloxacin, Wolleck (2022) showed 78,2% resistance for Escherichia coli in your study, but both samples of this bacterium in the present study were sensitive to the antibiotic and ciprofloxacin was the second most sensitive antibiotic overall, demonstrating 27,27% resistance (3 out of 11 samples), tied with amikacin and behind only gentamicin, which showed 18,18% resistance (2 out of 11 samples).
Nine out of the eleven tested bacterial agents demonstrated multidrug resistance, being resistant to three or more classes of antibiotics. Meregalli and Dewes (2019), in their study on antibiotic resistance in the microbiota of penguins, found that Magellanic penguins present more antimicrobial resistance than Antarctic penguins. Wolleck (2022), in their study on antimicrobial resistance in Escherichia coli isolates, found multidrug resistance in 48 out of 55 isolated samples.
In the present study, one of the two Escherichia coli samples showed multidrug resistance, although the other also showed resistance to all tested antibiotics in the penicillin and cephalosporin classes.
This multidrug resistance may be a result of human actions on the environment, such as contamination of rivers and oceans (MEREGALLI; DEWES, 2019). Anthropogenic pressure, such as coastal effluent discharges and urban surface seepage, is one of the main factors contributing to contamination of the aquatic environment, increasing the likelihood of marine life acting as vectors and reservoirs of multidrug-resistant bacteria (ELIOPULOS et al., 2022).
By using antimicrobials and developing resistance, the reservoirs of these resistance determinants remain in the host and the environment, and the movement of these genes that confer resistance occurs among the microorganisms present in the environment and the microbiota of humans and animals. Thus, it can be observed that microbial multiresistance is a complex problem involving humans, animals, and the environment (WOLLECK, 2022). Hence, the concept of One Health arises, which seeks to analyze problems from a viewpoint that prioritizes the health of humans, animals, and the environment in order to achieve the collective well-being of all individuals in the ecosystem. The World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) consider One Health to be an important tool for controlling microbial resistance, given that humans dump waste containing resistant bacteria into the environment, animals consume antimicrobials daily as growth promoters, and they can also act as vectors of resistant bacteria (WOLLECK, 2022).
ESBLs are a group of enzymes derived from beta-lactamases that confer resistance to bacteria against cephalosporins, penicillins, and monobactams. However, these enzymes are encoded by genes present in plasmids that can carry resistance genes to other antibiotics such as aminoglycosides, trimethoprim, sulfonamides, tetracycline, and chloramphenicol. Therefore, ESBL-producing strains are usually multidrug-resistant, but they typically remain susceptible to carbapenems, cefamycins, and the action of specific beta-lactamase inhibitors such as clavulanic acid (GIURIATTI, 2017).
ESBL production is one of the main resistance mechanisms in enterobacteria, and in the present study, two out of the seven samples tested were positive for ESBL (Figure 1). The isolates belonging to the Klebsiella genus, according to the literature, produce the greatest variety of these enzymes because they are good vectors for plasmids and allow for the evolution of genes that encode ESBL more rapidly than other enterobacteria (FRANCISCO, 2018). The research for Salmonella spp. in commercial and wild birds, such as the Magellanic penguins, is of great importance because these birds act as reservoirs, being able to eliminate the bacteria in various environments, infecting other birds and thus spreading the disease (MATOS et al., 2014). Salmonella spp. are known to cause severe food intoxication in various animals. In wild birds, the Salmonella genus is known to cause mortality and has a high potential for dissemination, especially in penguins, as they are free-living migratory birds that can come into contact with other animals and humans, which facilitates transmission (MATOS et al., 2014). In SS medium, Salmonella spp. grows with a black center due to the production of hydrogen sulfide, which can be detected due to the presence of sodium thiosulfate and ferric sulfate in the agar (ANVISA, 2004).
In the present study, seven cultures were made for the research of Aspergillus spp. was isolated in 11 animals, and 10 of these were classified as Aspergillus fumigatus.
The sample in the present study was collected from the contents of the air sacs, as penguins do not have an epiglottis and diaphragm, making them easily susceptible to respiratory infections, especially immunosuppressed animals due to conditions of stress, trauma, malnutrition, vitamin A deficiency, and ingestion of oil due to contamination of the seas they face during their migratory period or rehabilitation process (OLIVEIRA et al., 2013;XAVIER et al., 2006).

CONCLUSION
The majority of bacterial agents isolated in this study are part of the natural microbiota of penguins, however, these agents can cause diseases in penguins, especially in young and immunosuppressed animals.
The observation of multidrug resistance to the most commonly used antibiotics in therapy alerts to the impact of pollution of seas and oceans on marine life and highlights the importance of performing antibiotic susceptibility testing for better therapeutic outcomes.
This study was limited by access only to laboratory analyses, with no data on the history, physical examination, and other diagnoses of the rescued animals, in addition, the short period of time in which the study was conducted limited the understanding of the role of Magellanic penguins as sentinels in infections caused by environmental microorganisms. Therefore, new studies need to be conducted encompassing the entire clinical examination of the rescued animals, to determine if these agents are indeed the causes of the infection. Additionally, longer-term studies are necessary to gather more data and obtain more precise findings for the various microbiological analyses on these animals.