Species distribution and resistance profile of medical importance bacteria isolated from lesions of cats with sporotrichosis

Sporotrichosis is an infection with zoonotic potential caused by the Sporothrix schenckii complex. It is widely distributed worldwide. The cutaneous form is the most common presentation of the disease. The cat is one of the main animals affected, having numerous fungal cells in its lesions, becoming an important fungi disseminator. Due to the type of skin lesion caused by the fungus, secondary bacterial infections commonly occur. The aim of the present study was to learn about the most common bacterial agents involved in feline lesions with sporotrichosis, as well as their resistance profile to antimicrobial agents. The faster the treatment of bacterial and fungal infections is controlled, the faster the lesions heal. Consequently, the transmission of the disease is controlled. Samples of skin lesions or nasal discharge from 18 domiciled or semi-domiciled cats diagnosed with sporotrichosis were collected. Cats were evaluated for secondary bacterial infections. The samples were processed to isolate aerobic bacteria that were later identified by MALDI TOF. Susceptibility was assessed using the disk diffusion method for a total of 17 antimicrobial agents. The main species identified were Staphylococcus aureus and Staphylococcus felis. Proteus mirabilis has also been identified in some samples. Among these microorganisms, two strains of S. aureus were identified as resistant to methicillin. The TSA's results showed sensitivity to most of the tested antimicrobials. Staphylococcus was the most identified genus in skin lesions and nasal secretions of cats with sporotrichosis. Most microorganisms were sensitive to fluoroquinolones and aminoglycosides. Penicillins and cephalosporins showed less potential for action on these bacteria, which showed greater resistance to these classes.


Introduction
Sporotrichosis is a zoonosis considered hyperendemic in the state of Rio de Janeiro/Brazil Gremião et al., 2017;Schubach et al., 2004). Transmission is mainly through biting, scratching, and contact with the exudate present in lesions of infected cats de Souza et al., 2018). The main agent of the disease are fungi from the S. schenckii complex (Barros et al., 2011). The main agent isolated from cat lesions in Brazil is Sporothrix brasiliensis (Boechat et al., 2018a;Oliveira et al., 2011;Rodrigues et al., 2013). Numerous fungal cells can be isolated from the surface of the ulcerated lesions of cats. This is important for the transmission by direct contact from one animal to another. The animal infected by the fungus can spread it in the environment through contact with plants or soil (da Cruz, 2013;Schubach et al., 2008).
In cats, the disease has different clinical forms. Skin lesions are the most common manifestation of this disease, with the appearance of nodules, gums, and ulcers (Scott et al., 1996). It is believed that lesions in the disease favor secondary infections by opportunistic bacteria (Houghton et al., 2005). According to Cavalcanti e Coutinho (2005) , fungal skin diseases can predispose the animal to secondary bacterial infections. This secondary infection can worsen the lesion, increasing the time necessary for the closing and healing of lesions (Huang et al., 2014;Yeboah-Manu et al., 2013).
Staphylococcus spp. are resident members of the cutaneous microbiota and mucous membranes of men and animals.
However, several species are also opportunistic pathogens that can cause serious skin diseases (Morris et al., 2008), and some studies have reported the possibility of exchanging genetic material between animal and human staphylococcal species capable of conferring antimicrobial resistance (Ferreira et al., 2011;Frank et al., 2009;Hemeg, 2021). No previous studies demonstrate the main bacteria related to secondary infection in sporotrichosis in cats or their relationship with the healing process. With this in mind, this study aimed to perform the identification and resistance profile of aerobic bacteria isolated from cutaneous lesions and nasal secretions of cats with sporotrichosis.

Methodology
Samples processing and bacterial identification: Cats were selected (n = 18), regardless of gender, race, and age, who had ulcerated lesions and/or nasal discharge (rhinotracheitis), diagnosed with sporotrichosis. Animals were domiciled or stray cats of the city of Volta Redonda -RJ/Brazil.
All of them had access to the street. The samples were collected during a period corresponding to July 2017 to March 2018.
All cats were diagnosed with Sporotrichosis by a cytological examination and culture. Samples selected presented concomitant neutrophilic inflammation or the presence of phagocytosis by neutrophils, macrophages, or eosinophils, which suggest the diagnosis of pyoderma. Samples were obtained from the cutaneous lesions or nasal secretion of the affected animals.
Samples of ulcerated lesions or nasal secretions were collected using a sterile swab with transport Stuart medium (ABSORVE, China). In animals that had multiple lesions, only one of the lesions was chosen to collect the material. Only one swab was collected from each animal. Swabs were transported to the Microbiology and Parasitology Laboratory at Severino Sombra University (USS) -Vassouras -Rio de Janeiro/Brazil, and processed within 24 hours.

Identified microorganisms
-Isolates were obtained in 100% of the collected swabs (n = 18). Twenty different microorganisms were identified, mainly (90% -18/20) as Staphylococcus genus. Among these, half (9/18) were identified as S. felis, 44.4% (eight) S. aureus, and 5.6% (one) S. pseudintermedius. Two Gram-negative isolates could be identified from two different animals as Proteus mirabilis (10% -2/20). The results are summarized in Table 1. * The subscribed numbers represent the animals which more than one bacterial strain was isolated from the samples collected. Source: Authors.

Susceptibility profile
-Of the 18 bacterial isolates of gram-positive cocci belonging to the genus Staphylococcus 100% (18/18) were sensitive to ciprofloxacin, ofloxacin, gentamicin, and tobramycin, 94.4% (17/18) were sensitive to levofloxacin (one isolate was considered susceptible, using increased exposure) and amikacin. Two S. aureus isolates showed resistance to methicillin, five to penicillin G, and one isolate of S. felis was resistant to penicillin G. Both Proteus mirabilis isolates demonstrated susceptibility to ampicillin, ceftriaxone, meropenem, amoxicillin + clavulanic acid, ampicillin + sulbactam, ofloxacin, tobramycin, ciprofloxacin, levofloxacin, and gentamicin. On the other hand, both isolates were resistant to cefazolin and cefovecin. The results of the susceptibility tests are compiled in Table 2 and Table 3

Discussion
Zoonotic sporotrichosis caused by S. brasilienses is hyperendemic in Rio de Janeiro (Boechat et al., 2018b;Gremião et al., 2017). In cats, the most common form of sporotrichosis are multiple nodules and skin ulcers frequently associated with nasal mucosa lesions and respiratory signs. Also, low responses to treatment with itraconazole, the drug of choice for this disease, are commonly reported (Pereira et al., 2010). In this context, our study was the first to investigate bacteria present in Research, Society andDevelopment, v. 10, n. 6, e15810615377, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i6.15377 cat sporotrichosis. Also, the first to evaluate the antimicrobial susceptibility pattern of these bacteria. This can contribute to its treatment.
Studies on feline pyoderma are poorly reported in the literature compared to canine pyoderma. Few studies assess bacteria isolated from clinical lesions (Medleau & Blue, 1988;Scott et al., 2001). Pyoderma usually occurs secondary to other processes such as trauma, drugs, or illness that induces immunosuppression and a diversity of other dermatological problems (Mueller, 1999). Clinical lesions of pyodermas are quite variable and include pustules, furuncles, crusts, linear, and nodular ulcerative granulomatous lesions (Yu & Vogelnest, 2012). Therefore, may resemble the lesions caused by sporotrichosis. Due to lesions' characteristics, there is an imbalance in the cutaneous microbiota. We believe in the possibility of secondary bacterial infections in the lesions of cats affected by sporotrichosis.
Many of the animals from the present study had multiple bacterial cells in the cytological examination. Thus, it is assumed that the clinician should consider treatment with associated antibiotic therapy.

Most of the microorganisms isolated in this study belong to the genus Staphylococcus spp. A recent study showed that
Staphylococcus is more isolated in sick cats than in healthy cats (Older et al., 2017). S. felis and S. aureus were the most prevalent species. Both species have already been isolated from the healthy skin and with skin lesions described in the literature (Baptiste et al., 2005;Devriese et al., 1984).
Although previous studies have indicated that Staphylococcus skin infections are less common in cats (Anette Loeffler et al., 2005;Rich, 2005), possibly due to decreased adherence to corneocytes (Lu & McEwan, 2007;Woolley et al., 2008), this study raises the possibility of a predisposition to Staphylococcus infection after skin trauma caused by sporotrichosis in cats. Abraham et al. (2007) isolated 14 S. aureus (58% of 24 coagulase-positive Staphylococcus isolates) in 48 cats diagnosed with some inflammatory skin disease (ISD -Inflammatory Skin Disease), a smaller number of isolates of this species was obtained in healthy cats in the same study. This is in agreement with what our study found.
A greater amount of coagulase-negative Staphylococcus was isolated, a result also observed by Abraham et al. (2007) that isolated 98% of the coagulase-negative Staphylococcus from 48 cats diagnosed with ISD. Among coagulase-negative Staphylococcus, nine were S. felis, which have been reported causing otitis externa, cystitis, abscesses, wounds, and other skin infections (Igimi et al., 1989). Some studies have demonstrated the importance of infection by S. felis in cat lesions caused by flea-bite related hypersensitivity (Patel et al., 2002). S. felis may have played a role in skin disease since there was a systemic response to the antimicrobial therapy employed. Recently a study examined a single isolate of S. felis from a cat's subcutaneous wound and identified virulence factors such as biofilm and proteolytic enzymes (Kwaszewska et al., 2015). S. felis was the most isolated species in healthy cats and cats classified as having some disease in a study by Bierowiec et al. (2019).
Proteus mirabilis was the only gram-negative bacilli identified. Infections by this genus are sometimes neglected (O'Hara et al., 2000). Due to fecal elimination and free access to the street and, consequently, contact with the soil and unhealthy environments, these microorganisms assume typically opportunistic behavior, as described in animals and humans (O'Hara et al., 2000). Devitalization of tissues and injuries are considered predisposing factors for the occurrence of infections by P. mirabilis (Quinn et al., 2005), making the animal a carrier of lesions caused by sporotrichosis predisposed to these infections.
The highest resistance level was observed for Penicillin G, with 33.3% of the 18 isolates of Staphylococcus spp. This is the expected result since the production of β-lactamases by this genus is frequent. Similar rates were observed elsewhere (Qekwana et al., 2017). In recent years, several studies have demonstrated the acquisition of multidrug resistance by showed resistance to cefoxitin in this study. The TSA result shows a high degree of sensitivity to fluoroquinolones and aminoglycosides. However, it is necessary to monitor the isolation profiles and susceptibility to antimicrobials in veterinary practice, to optimize the cure rate and the rational use of antibiotics.
The number of samples collected and the lack of minimum inhibitory concentration for the tested antimicrobials were some limitations of the present study. The mecA gene for confirmation of bacteria methicillin could also be very clarifying.
Further studies with more cats may clarify the role of bacteria in the feline sporotrichosis.

Conclusion
Staphylococcus spp. was the most identified genus in skin lesions and nasal secretions of cats with sporotrichosis, with S. aureus and S. felis being the most prevalent species. Fluoroquinolones and aminoglycosides were more sensitive, with penicillins and cephalosporins being the antimicrobials that showed greater resistance. It is essential to carry out the resistance profile of the bacteria identified. This should be mandatory for responsible antibiotic therapy. Studies that identify virulence and phenotypic and genotypic resistance factors of bacteria associated with lesions of cats with sporotrichosis are necessary to understand the interaction of bacteria with fungi of the S. schenckii complex.