Identification and resistance profile of gram positive bacteria from aquatic environment

The Meia Ponte River – Goiás/Brazil, is responsible for benefiting about 2 million people in Goiás State. However, the increase in pollution with the disposal of sewage, chemicals and drug remains have contributed to the increase in bacterial resistance and the exchange of resistance genes. The objective of this study was to isolate, identify and analyze the resistance profile of gram-positive bacteria present in raw water and sediment of the Meia Ponte River – Goiás. The samples were collected from four sampling points and two collections were carried out, one in the dry season and the other in the rainy season. The isolated bacteria were identificated, then the antibiogram was performed. A total of 75 strains were isolated, 72.0% (54/75) of Streptococcus spp., 12.0% (9/75) of Staphylococcus spp., 9.3% (7/75) of Bacillus spp. and 6.7% (5/75) of Enterococcus spp. Furthermore, 52.0% (39/75) of the isolated strains were from raw water and 48.0% (36/75) were isolated from the sediment. Among the samples, strains of Staphylococcus spp. and Bacillus spp. showed greater resistance to antimicrobials, on the other hand, Enterococcus spp. showed less resistance. Some strains of Bacillus spp. and Streptococcus spp. presented multidrug resistant, Staphylococcus spp. showed multidrug resistant and some pan-drug resistant. In the correlation of Spearman Staphylococcus spp. and Streptococcus spp. isolated, were the ones that presented the most significant correlations (p < 0.05). Thus, the study shows the importance of ascertaining the resistance profile of this group of bacteria that aquatic environment.


Introduction
In the mid-1940s, the fight against infections began using antibiotics and over the years this drug has been applied in agricultural, industrial and veterinary sectors. The indiscriminate use of antimicrobials contributes to the increase in resistant bacteria, including the dissemination of their resistance genes in the environment (Shao et al., 2018).
Sewage discharged into rivers is associated with an increase in resistant bacteria in the environment, due to the fact that these medications are not fully metabolized by the body, leaving traces present in both urine and feces of living beings. Other factors that also contribute to the topic addressed are the medicines applied in agriculture that end up contaminating soils and rivers, as well as industries despising them in the aquatic environment (Gogoi et al., 2018;Ruiz-Aguirre et al., 2017). In addition to these reported compounds, numerous others also influence the increase in environmental contamination, being called emerging contaminants (ECs). Thus, ECs have caused environmental impacts, increased bacterial resistance and generated public health problems (Gogoi et al., 2018;Mohapatra & Kirpalani, 2019).
The increase in antibiotic resistance genes (ARGs) in environmental bacteria is associated with selective pressure of antibiotics in water, together with the acquisition of resistance genes present in the environment from animals and/or humans, causing the exchange of genetic material in aquatic sites (Maruzani et al., 2020;Wang et al., 2020).
The aquatic environment is considered the largest disseminator and deposit of ARGs, this is due to pollution and the "stress" that occurs on the bacteria in the presence of antibiotics in the environment (Amarasiri et al., 2019;Dang et al., 2017). The ARGs sharing occurs through mobile genes as conjugated plasmids and transposons, integron, among others; thus facilitating horizontal transfer and exchange of genes among bacteria (Dang et al., 2017;Sobisch et al., 2019).
The presence of resistant gram-positive bacteria in aquatic sites is of concern, as some are signs of fecal contamination, such as Enterococcus. Others are pathogenic to humans, such as Staphylococcus (Karkman et al., 2018), in which the acquisition of resistance genes makes it difficult to find effective antimicrobials against infections (Rahmani et al., 2020). Other gram-positive bacteria considered important are Streptococcus spp., which are responsible for being pathogenic and for developing severe infections in humans (Zhang et al., 2018), and Bacillus spp. that are known for spore formation, resistance to environmental changes and can cause food poisoning in the population (Ruiz-Aguirre et al., 2017).
The Meia Ponte River is one of the main water resources in the Goiás State -Brazil, being responsible for benefiting about 2 million people from Goiás and contributing to agriculture, livestock, water supply, recreation, among others. With the urbanization and with one of the tributaries running through the metropolitan region of Goiânia city, it caused an increase of pollution, interfering with its water quality (Bailão et al., 2020;Coelho et al., 2021).
Because it is an essential condition of the water supply network of Goiás and knowing that the presence of resistant bacteria that location can harm the health of the population, it becomes necessary to research and investigate the presence of these microorganisms in this aquatic environment. Thus, the aim of the study was to isolate, identify and verify the resistance profile of gram-positive bacteria taken from raw water and sediment from the aquatic environment.

Methodology
This study deals with an experimental research (Köche, 2016;Pereira et al., 2018) for its methodological basement.

Study area and sample collection
The collection of raw water and sediment samples were made in four sample points following the basin of the Meia The collection of raw water and sediment followed the National Guide for Collection and Preservation of Samples (Brandão et al., 2011), 100 mL of raw water and 20 g of sediment from each sampling point were collected in sterile flasks, in addition to recording the water temperature with the aid of a thermometer at the time of each collection. After collection, the samples were stored and refrigerated on ice (temperature between 2 °C to 8 °C), and these samples were to be processed within a maximum of 24 hours after collection.

Bacterial count and isolation
The 20 g of sediment samples from each collection point were added in 0.5% Tween solution (200 mL of solution) and incubated in a shaker (BIOCOMPARE®) for 2 hours at 30 ºC, later, 200 µL of this solution was seeded in petri dishes using the scanning technique and incubated for 48 hours at 37 ºC. The raw water samples were homogenized, 200 µL of these samples were seeded in petri dishes using the scanning technique and the plates were incubated for 48 hours in a bacteriological incubator at 37 ºC (APHA, 2017).
The petri dishes had specific culture media for isolation and quantification of gram-positive bacteria, using the Azide Dextrose Broth Agar (ADBA) and Enterococcus Confirmatory Agar (ECA), both from the company HIMEDIA®. The bacterial isolation and quantification process followed the protocol (APHA, 2017), being necessary to quantify the Colony Forming Units (CFU) present on the plates, select the morpho-colonial characteristics of each culture medium used, following Research, Society and Development, v. 10, n. 13, e226101321182, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21182 4 the respective leaflets of the HIMEDIA® to carry out the isolations of these bacteria. The preservation of pure colonies, in turn, were preserved in 2 mL cryogenic tubes, containing 20% glycerol in medium BHI (Brain Heart Infusion) BD® at -4°C.

Bacterial identification
The identification followed the Manual of Clinical Microbiology for the Control of Infection Related to Health Care (ANVISA, 2013). To phenotypic identification was used tests: Gram stain, catalase test, verification of spore and filament production, motility were performed, hydrogen sulfide (H2S) production, indole production, 5% NaCl tolerance test, esculin bile test, blood agar growth and hemolysis and DNAse test.

Antibiogram
The antimicrobial susceptibility test of disk diffusion method followed the National Sanitary Surveillance Agency

Statistic
For data tabulation and descriptive statistics, Microsoft Excel 2016 was used. To verify the association and correlation, Spearman's correlation test was used in STATISTICA software 7.0 version (StatSof©) (StatSoft I, 2012).

Results
The The water temperature during the collections in the dry and rainy period of all sampling points had an average of 23.9 °C, being an elevated temperature compared to the established criterion of < 20 °C (WHO, 2017). Table 1 shows the results of counting the CFU of the culture media from the two collections. Staphylococcus spp. they were the only bacteria that presented PDR, showing 55.6% (5/7).
The association/correlation among all sample data, collection, point, culture medium and antibiotic resistance/sensitivities found was verified, these are described in Table 3 In Table 3. some data were extremely important for the research, for example, when comparing antibiotics and stitches, and also among samples and antibiotics.
Data only within each bacterial species were also listed.

Discussions
The water temperature investigation process serves as a quality parameter, and the increase in temperature is associated with pollutants and proliferation of microorganisms in the aquatic environment (WHO, 2017). In addition, the occurrence of temperature changes among the dry and rainy seasons may be associated with the flow of waste in the water brought in by the rains (Adesakin et al., 2020).
On the media chosen for isolation, it was observed (Table 1), in general, the highest bacterial count for sediment samples when compared to raw water samples. This increased amount of strains found in the sediment reports a bacterial accumulation, demonstrating that the flow of sediment into the water body can generate an increase in the amount of fecal and bacterial contamination (Pandey et al., 2018).
There was more CFU in the culture media in the rainy season when compared to the dry season, occurring due to the fact that in the rainy season the runoff brings animal feces, pollutants and soil contaminants to rivers and lakes, multiplying the amount of these microorganisms in the water (Chen et al., 2017).
The work published in 2017 (Gomes et al., 2017), also performed analyzes in the water in the same aquatic environment of the present study, collections carried out in 2015/2016 and in periods of rain and drought, found Bacillus spp.
13.6% (14/103) and Enterococcus spp. 20.4% (21/103). Comparing the previous data with the present work, it is a smaller number of the total of isolates, however, many strains of Streptococcus spp. were isolated and in the study in 2017 the bacterium was not found. The explanation for this distinction is because the cited authors (Gomes et al., 2017), used a no specific culture media and in this current research has chosen to use specific culture media for gram-positive bacteria.
Another study carried out in the Apies River in South Africa showed an increase in the quantification of Enterococcus spp. in sediment samples using specific isolation medium for this bacterium, the indication of this change being the association with the accumulation of fecal bacteria in the sediment, where this location is described as a "protection" for these microorganisms (Ekwanzala et al., 2017).
Researchers (Semedo-Lemsaddek et al., 2018) Another study in Dhaka, Bangladesh with mineral water samples analyzed in different climatic seasons found resistance of Staphylococcus spp. During the study, there is an association among the resistance to cefixime of these bacteria and the access of this microorganism with the population, warning of contamination by Staphylococcus spp. in water accessible to the population, consolidating the importance of inspection and research to assess these resistant microorganisms (Aditi et al., 2017).
According to the authors (Kaur et al., 2020) Phenotypic results of MDR presented by some isolated bacteria are being related to findings of high doses of antibiotics found in sewage of a hospital in São Paulo -Brazil. This disposal found in sewers may be related to the exacerbated use of antibiotics in hospitals, because it is an environment with a patient with several and distinct infections, thus influencing the spread of bacterial resistance (Brito et al., 2020;Carvalho et al., 2021).
The strong association in the antibiotic-antibiotic correlation is characterized by being of similar antimicrobial classes (El-Din et al., 2021), being ERI and AZI, for example, belonging to the class of macrolides that have the function of preventing the protein synthesis of the bacteria (Munita & Arias, 2016). The author (Olivas, 2013), explains that S. aureus has resistance to the efflux pump and this can generate multiple resistances to antimicrobials, such as: TET, CIP, GEN, PEN, among others; may explain these various correlations arising from Staphylococcus spp. presented in the statistics.

Conclusion
The presence of gram-positive bacteria with resistance in the Meia Ponte River was already expected because it is a place with several different bacterial strains (animal and human), in addition to the presence of pollution and sewage disposal at the site, however, the results found for MDR and even PDR were alarming for being a water resource used by countless individuals in the state of Goiás.
Still, it is necessary to collect more data on the spot and carry out research /tests, such as genotyping to find possible resistance genes in the isolated bacteria and comparing with the phenotypic resistance found. With these data collected in this research, it can serve to alert the population that uses this water and raise awareness about possible problems with infections caused by these resistant bacteria.