Evaluation of pathogenic fungi occurrence in traumatogenic structures of freshwater fish

Introduction: Fungal infections in human skin, such as sporotrichosis, can occur after fish induced trauma. This work aimed to identify fungi in freshwater fish that are pathogenic to humans. Methods: Extraction of dental arches from Serrassalmus maculatus (piranha) and Hoplias malabaricus (wolf fish), stings from Pimelodus maculatus (mandis catfish), dorsal fin rays from Plagioscion spp. (corvina) and Tilapia spp., for culture in Mycosel agar. Some cultures were submitted to DNA extraction for molecular identification by sequencing ITS-5.8S rDNA. Results: Cultures identified most yeast as Candida spp., while sequencing also permitted the identification of Phoma spp. and Yarrowia lipolytica. Conclusions: While the search for S. schenckii was negative, the presence of fungus of the genera Phoma and Candida revealed the pathogenic potential of this infection route. The genus Phoma is involved in certain forms of phaeohyphomycosis, a subcutaneous mycosis caused by dematiaceous fungi, with reports of infections in human organs and systems. Traumatizing structures of some freshwater fish present pathogenic fungi and this may be an important infection route that must be considered in some regions of Brazil, since there are a large number of a fisherman in constant contact with traumatogenic fish.

Fungi are ubiquitous and are found in soil, water, vegetables, animals and in humans.Fungal species undergo changes in incidence and according to location, season and air moisture content, as well as other variables 1 .The growth of fungi can be divided into two phases, vegetative and reproductive.Most of these microorganisms are made up of microscopic filaments with welldefined cell walls called hyphae.Fungi were classified as plants until 1969 and then inserted into a separate kingdom called Fungi.In fact, they are phylogenetically more closely related to animals than plants and some of them are responsible for infections in several tissues.Sporothrix schenckii is an example of a fungus causing infections in humans; it causes subcutaneous mycosis with acute or chronic evolution.Fungi are installed by previous injuries, such as scratches by sharp plant or animal structures 2,3 .There are well documented infections transmitted by dog bites 4 , rats 5 and other animals.Sporotrichosis has also been documented from handling fish, originally reported by Mayorga & Caceres in 1978 6 and more recently in Brazil 7,8 .The incidence of accidents in river fishermen appears to be high, as observed in retrospective and prospective studies 9 .Poor working conditions and a lack of preventive measures are precipitating factors and the main fish species causing accidents are of commercial value and present traumatic structures or are venomous, such as mandis catfish, tilapia, traíras, piranhas and others.A large number of injuries and envenomings result in secondary fungal and bacterial infections that require further study to develop improved treatment and preventive measures against these accidents in fishing communities 10 .The objectives of this communication were to research the possible presence of pathogenic fungi in common fish present in the nets of fluvial fishermen.

Leme FCO et al -Evaluation of pathogenic fungi in fish
Considering the cases of sporotrichosis reported by one of the authors 7,8 , this work aimed to verify the existence of S. schenkii and other potentially pathogenic fungi in traumatogenic structures (stings, rays of fins and sharp teeth) of fish cited as the most hazardous by local fishermen and documented as such in previous publications 9 .The methods were applied in the same river where the cases of sporotrichosis were previously identified, in the Piracicaba River, near the Tietê River, in the central region of the State of São Paulo, Brazil.In three samples collected on three mornings of daily routine work of the colony of fishermen, two specimens each of corvina or freshwater silver croaker (Plagioscion squamosissimus), piranha (Serrasalmus maculatus), dog-fish (Acestrothychus lacustris), mandijuba or mandiamarelo catfish (Pimelodus maculatus), tilapia (Tilapia spp.) and wolf fish (Hoplias malabaricus) were obtained.
A sterile scalpel blade was used to remove traumatic structures from each fish, which were then directly inoculated into culture medium: upper and lower dental arches from Acestrothychus lacustris, Hoplias malabaricus and Serrassalmus maculatus; dorsal fin rays from Plagioscion squamosissimus and Tilapia spp.and stingers from Pimelodus maculatus (Figure 1).These steps were conducted where the fish were captured, at the fishermen's colony on the Piracicaba River, close to the junction with the Tietê River.
The cultures were duplicated and incubated at 25°C and 35°C.Cultures at 25°C, close to ambient temperature, were used to investigate fungal flora and assess possible changes in fungi morphology.Cultures at 35°C were intended to investigate the possible presence of fungal pathogens, because this is closer to human body temperature and pathogenic fungi, such as S. schenckii, show increased tolerance to heat.
The yeasts were isolated in the samples and maintained in Sabouraud Dextrose Agar (SDA) with subcultures every 15 days.During the final period, the yeasts were cultivated on CHROMagar-Candida (CHROMagar-Candida, Difco), a differential culture medium for identifying Candida albicans, Candida tropicalis, and Candida spp., by respectively developing green, blue, and pink colonies 10 .The CHROMagar-Candida plates were divided and, following colony cultivation, were incubated up to 25°C for a maximum of five days to observe staining 10 .

Ethical considerations
The study was approved by Botucatu Medical School Animal Research Ethics Committee.DNA extraction permitted high concentrations to be obtained, making it necessary to dilute samples for use in PCR reactions.The PCR reactions were all successful, demonstrating amplicons of approximately 400-650bp, as shown in Figure 3.These amplicons were purified and subjected to gene sequencing analysis to obtain the nucleotide sequence to compare with NCBI genomic databases (National Center for Biotechnology Information) using BLAST (Basic Local Alignment Search Tool for nucleotide).
Nucleotide sequence analyses presented consistency with fungi of the genus Phoma spp.and Yarrowia lipolytica, the latter is used in genetic engineering experiments and is currently reported as nonpathogenic, while the former is a pathogenic fungus associated with diseases in various organs and human systems, primarily the skin.Phoma belongs to the group of phaeohyphomycosis 11-14 .

Figure 2
Figure 2 is a macroscopic view of the isolated colonies.Evaluation by CHORMagar enabled most of the yeasts to be classified.One isolate obtained from Tilapia was classified as C. tropicalis (blue color; Figure 2).DNA extraction permitted high concentrations to be obtained, making it necessary to dilute samples for use in PCR reactions.The PCR reactions were all successful, demonstrating amplicons of approximately 400-650bp, as shown in Figure3.These amplicons