2.1 Parks selection

We selected at northern El Virrey Park, at southern Timiza Park, at eastern Nacional Park and at western Simon Bolívar Park. In each one, four items were sampled: fecal samples, canine fur, environment samples that were near populated zones and elements in recreational areas. A total of 50 samples were taken distributed in ten samples for each park except on Simon Bolivar Park where twenty samples were collected because its bigger extension.

2.2 Cultures procedure and biochemical testing

2.3 Identification

For Gram-negative bacteria, identification was done using macroscopic description and biochemical test, Gram-positive stains followed the same procedure with one exception: the agglutination test for Staphylococcus aureus on cases not determined. For fungus samples, the first step was doing the macroscopic description followed by the microscopic identification of sexual or asexual structures and its genre.

3.1 Bacterial isolations from fecal samples

From 50 fecal samples taken, 50 isolations were performed, of which 11 genus and 5 species of bacteria were identified (Table 2, 3).

3.2 Bacterial isolations from recreational items

From 50 samples taken from recreational items, 76 bacterial isolations were performed, of which 11 genus and 9 species were identified. Isolations of Gram-positive bacilli were reported with a prevalence of 17 % (Tables 4, 5).

Two Staphylococcus aureus (label as A and B, obtained from El Virrey park) and one isolation of Staphylococcus spp. (obtained from Simón Bolívar park) showed oxacillin resistance. E-test for vancomycin was applied in both S. aureus and one of them showed susceptibility to vancomycin (3 µg/ml) and the other one showed an intermediate pattern (8 µg/ml) (Table 6).

3.3 Isolations from environmental material

3.4 Fungal isolations from canine hair

From 50 samples taken from canine hair, 94 isolates were performed, of which 13 genera were identified. Yeast colonies and fungal colonies with sterile mycelium were reported with a prevalence of 8.5% and 4.2% respectively (Table 10).

4.1 Bacterial isolation from fecal samples

The high prevalence of Escherichia coli is because this specie is part of the normal intestinal microbiota of mammals like dogs, however most strains of E. coli are non- pathogenic, while those that are pathogenic can cause infections such as travelers diarrhea, abdominal cramps, vomit, urinary tract infections among others ¹⁵. Like E. coli, genus Citrobacter spp, Enterobacter spp, Edwardsiella spp, Pantoea spp, Klebsiella, Plesiomonas spp, Proteus spp, Salmonella spp, and Yersinia spp. belong to the Enterobacteriaceae family, within which it has been reported species that normally are part of the intestinal microbiota of some animals or which are isolated from the feces of them ².

Additionally, a portion of the species belonging to the mentioned genera, such as Klebsiella pneumoniae and Salmonella enteritidis reported here, have medical importance because they generate pathologies in humans related to bacteremia, urinary and lung or respiratory infections, gastroenteritis, meningitis, septicemia and other infections, mainly in people with immunodeficiency ^16,17. In the case of S. enteritidis, human infections occur mostly by eating contaminated foods. It is possible to happen the same with a dog, which becomes in the vector of the pathogen and as in this particular case where S. enteritidis was isolated from a feces sample ¹⁸.

A few species of Aeromonas spp, can be isolated from some animals and occasionally from human feces, or may also be isolated from freshwater ². It has been reported that some species of this genus, such as A. hydrophila can cause chronic diarrhea or gastroenteritis, mainly in infants, from enterotoxin production ¹⁹.

4.2 Bacterial isolations from recreational and environmental material

Recreational items are constantly manipulated by people who are in contact with other elements of the area or with organic materials such as fallen leaves, plants, water, including soil, in which microorganisms form biofilms of one or more species, like Staphylococcus epidermidis and S. aureus, which can explain the prevalence presented ¹⁵. In this situation, both species showed the highest prevalence in both type of samples, recreational items and environmental material. S. aureus (see Figure 1) and S. epidermidis can be found normally in the human skin, however S. aureus is found in a lesser proportion, and both S. aureus and S. epidermidis are species of high clinical importance, because these can cause nosocomial infections, skin infections with a variable severity and in some cases infections by ingestion of contaminated food, being more vulnerable people who have immunodeficiency ^2,20. In addition, the strain of S. aureus (B) reported, isolated from El Virrey Park and which showed an intermediate pattern to vancomycin (VISA), have a high clinical importance because these strains represent a considerable threat as potential pathogens that can cause difficulties during treatment ²¹.

Figure 1 Mannitol salt agar test for Staphylococcus spp. obtained from a sample from El Virrey Park. Author: Camila Andrea Camacho 

Moreover the species Rahnella aquatilis showed the same prevalence percentage as the genus Staphylococcus spp. in the environmental samples, possibly because R. aquatilis is isolated from soil, freshwater and in some cases from small invertebrates ^22,23. However, it has been reported unusual cases of bacteremia, sepsis and other infections produced by R. aquatilis in wounded patients, elderly or immunocompromised ²³.

The bacterial genera and species and its prevalence reported here, can be related with the contact not only people have but also other organisms such as birds, rodents, dogs, etc. with the recreational items and organic sources or material nearby which are full or covered with microorganisms as the biofilms mentioned before (24). For example, Salmonella gallinarum is a species that is mainly spread through birds and can cause gastrointestinal infections ²⁵. Also, it is important to point out the genus Streptococcus spp. which has saprophytic species that are normally isolated from skin, mouth and respiratory tract of humans. Yet, some species are of clinical importance as they can produce diseases like pneumonia, meningitis, tonsillitis and other infections ^2,26. Other species reported are potential pathogens because they can produce many infections by its ingestion (in the case of Yersinia enterocolitica) or can also be considered as opportunistic microorganisms, such as Ochrobactrum anthrophi, although its pathogenicity is uncommon and is mainly related to nosocomial infections and immunocompromised patients ^27,28.

The antimicrobial susceptibility test results of all isolates showed a considerable high percentage of resistance to ampicillin (44.4%) and, although not as high, trimethoprim (21.1%), being ampicillin only used in the Gram-negative bacilli isolations and trimethoprim in both Gram-negative bacilli and Gram-positive cocci. The high percentage of resistance to both antibiotics can be seen specifically in the feces samples results, where isolations reach the 72% and 55% ampicillin and trimethoprim resistance, which can be associated to the uncontrolled supply of antibiotics to dogs and possible exposure to resistant bacteria in places such as veterinary clinics, where there have been reported a high percentage of multi-resistant strains of potentially pathogenic bacteria that can infect dogs ^2,29.

4.3 Fungal isolations from environmental material and canine hair

In both type of samples, the genus Penicillium spp. showed the highest prevalence, probably because is a cosmopolitan genus and it is isolated from the environment, mainly from the soil, thus generating the prevalence result in both environmental material samples and canine hair samples (24). Few cases have been reported where some species of Penicillium spp. cause infection or disease in humans, where the reported ones have generally been in immunocompromised patients ^24,30

The genus Cladosporium spp. can be found widely distributed in the environment (like Penicillium spp,) and some species can produce diseases like black tinea, onychomycosis, phaeohyphomycosis, among other, in healthy people ³¹. Other genera that can produce “feohifomicosis” in healthy and immunocompromised patients are Alternaria spp, (see Figure 2) Bipolaris spp, Phoma spp. and Nigrospora spp. ^31,32. Also, some of the species of the genus Fusarium spp. (see Figure 3) and Trichoderma spp, can cause hyalohyphomycosis ³².

Figure 2 Conidia from Alternaria spp. obtained from an environmental sample, observed through a light microscope with 100x. Author: Diego Gonzalez Lozano 

Figure 3 Characteristic septate conidia from Fusarium spp. observed through a light microscope with 100x. Author: Diego Gonzalez Lozano 

The genus Mucor spp, had a high prevalence compared to the other fungi genera in samples of environmental material, because it is a genus that is distributed in soil and decaying organic material ³¹. Mucor spp, as Rhizopus spp. (see Figure 4) and Absidia spp, can cause infections or mucormycosis, affecting eyes and respiratory structures ^31,33. Additionally, it has been reported the genus Chrysonilia spp, with a relevant prevalence, where it is important to point out a case of occupational asthma reported in Paris caused by a constant exposure to C. sitophila in a healthy grown man without relevant medical history related to respiratory diseases ³⁴.

Figure 4 Asexual structure of Rhizopus spp. and coenocyte hypha with rhizoids observed through a light microscope with 100x. Author: Diego Gonzalez Lozano 

A large proportion of fungal isolates from environmental material are normally found distributed in the environment, carrying out the decomposition of organic matter, and in some cases they don’t represent a threat to human health, except in people with underlying disorders that impair their immune response which makes them vulnerable to infections by opportunistic microorganisms ³¹. In the canine hair samples we reported mainly environmental genera without relevant dermatophyte genus being identified, probably because the samples were taken from a long part of hair or from the outermost part of the dog’s hair, in which fungi found in the environment can adhere as the dog have contact with the soil, plant material, water, etc. bearing in mind that the results of fungal identification in both types of samples are similar.