VIRULENCE AND ANTIMICROBIAL RESISTANCE OF ESCHERICHIA COLI STRAINS ISOLATED FROM THE WATER OF THE AMAZON RIVER, STATE OF AMAPÁ

Objective: We investigated the identification of E. coli strains in water samples from the Amazon River in the state of Amapá to determine the antimicrobial susceptibility profile and verify the presence of virulence factors and resistance genes. Reference theoretical: Antimicrobial resistance is a global public health concern, and the presence of resistant bacteria in water samples represents an additional risk for the spread of these microorganisms, with great impact on society in general, especially after the discovery of pathogenic strains of Escherichia coli. Method: The bacterial isolates were submitted to the technique of isolation of colonies in selective medium and identification by biochemical tests. Isolates identified as Escherichia coli were submitted to the antimicrobial sensitivity test and the polymerase chain reaction (PCR) technique to search for virulence genes and drug resistance. Results and conclusion: The results showed that the strains of E. coli isolated from the water samples of the Amazon River showed high rates of antimicrobial resistance. In addition, virulence gene markers were detected: eagg, eaeA, stx1, stx2, as well as resistance markers: qnrB and qnrS. Index of resistance to multiple antimicrobials MARI ranged from 0.1 to 0.6. All collection sites of water isolates has the presence of strains with multiresistance. Research implications: This study revealed the importance of monitoring water quality and implementing control measures to prevent the spread of resistant and potentially pathogenic bacteria.


INTRODUCTION
Water is an essential resource for the urban processes of human or economic activities, and water deficit is increasingly frequent, causing a worldwide problem.In Brazil, the Amazon region has the highest surface water availability; however, the action of man in watersheds has interfered in aquatic ecosystems and led to qualitative and quantitative water loss (Salas Salvadó et al., 2020).Thus, the use of water is diverse, such as domestic supply, industrial supply, agriculture, recreation and leisure, landscape harmony and preservation of fauna and flora.
In this sense, water is the natural resource of remarkable importance for the maintenance of life, participating and dynamizing all ecological cycles.In the Northern region of Brazil, the scarcity of technologies that meet the local context and that can replace direct launches in rivers and lakes is an environmental problem of large cities, in addition to the lack of collecting network and adequate treatment of sewage (Uddin, 2022).In these cities, domestic exhaustion is the main cause of water resources contamination, becoming a major challenge in mitigating the degradation of the aquatic ecosystem.
These wastes give water the unhealthy character that increases the degree of vulnerability of the population (close urban and peri-urban), which, depending on the frequency of contact with water, incur greater risks associated with various pathogenic microorganisms (Momberg et al., 2022).As an additional challenge, several microorganisms become more virulent and resistant to antimicrobials such as Escherichia coli, Gram-negative bacillus of the Enterobacteriaceae family, known to cause severe infections in humans and warm-blooded animals, easily spread by fecal-oral, contact, water or food contamination (Sarowska et al., 2019).
Generally, infections caused by E. coli are associated with the gastrointestinal tract and can cause different diarrhea and vomiting episodes, being pathogenic strains known as extras intestinals, causing infections and/or more serious lesions, such as urinary tract infections (UTIs), bloodstream infections, meningitis, peritonitis and, in some cases, hospital sepsis (Sarowska et al., 2019;Naylor et al., 2018).
Therapeutic options vary depending on the type of infection, and accurate detection of E. coli is essential to indicate the need for adaptation of the drug treatment of the affected patient.However, it is not always possible to identify the types of bacterial strains involved in the process of infection, and the treatment is basically performed with the application of broadspectrum antibiotics to contain the symptoms of virulence, which may cause bacterial resistance (Naylor et al., 2018) Thus, antimicrobial resistance is a global public health concern, with great impact on society in general, especially after the discovery of pathogenic strains of E. coli capable of producing enzymes of β-lactamase type, which may hydrolyze and/or destabilize the βlactamic rings of antibiotics (Loureiro et al., 2016;Holmes et al. 2016;Sinha, Parli, 2020).Moreover, the identification of E. coli isolates from water samples from the Amazon River is of great importance to understand the microbial ecology of the region.
In this context, the state of Amapá does not have a cloacal sewage network, with all domestic effluents released directly into the water body (Amazon River), a condition conducive to increased contamination by biological agents from effluents, which favors the proliferation of waterborne diseases, thus making this environment outside the quality standards required by the Brazilian legislation (Salas Salvadó et al., 2020).
Furthermore, it is in urban centers that aquatic environments suffer strong anthropogenic influence, generally neglected in studies of isolation, identification, virulence and antimicrobial resistance (Hyde et al., 2019).Additionally, since these sites are a source of contamination, they are rich in microorganisms that have chemicals with active properties such as extracellular enzymes and organic acids capable of becoming more virulent and resistant pathogenic strains (Omeyke et al., 2019).
Therefore, knowledge of the diversity of microorganisms of the Amazon River, especially isolates of E. coli, can produce important information on the profile of virulence and resistance, as well as instigate research on new agents capable of fighting these strains of bacteria.In addition, aquatic environments have been pointed out as promising sources for the search for new molecules with bioactive properties, and such environments are likely to house a wide variety of strains with promising antimicrobial bioactivity (Akpotu et al., 2017).
In this study, we investigated the isolation and identification of E. coli strains in water samples from the Amazon River in the state of Amapá to determine the antimicrobial susceptibility profile and to verify the presence of virulence factors and resistance genes.

Aquatic Environment From A One Health Perspective
The water on Earth involves oceans, seas, rivers, lakes, lagoons, lagoons and glaciers, and is also found underground and in the atmosphere.The development of civilizations, such as Egypt, Mesopotamia, China and India, were linked to the availability of water for growing food, personal hygiene and navigation.Water is fundamental for humanity, it is what maintains life on planet Earth, sustains biodiversity, food production and supports all natural cycles (TUNDISI; TAKAKO, 2020).
The state of Amapá, in the northern region of Brazil, is bathed by the largest river in the world, the Amazon River, thus having great economic importance, as in addition to fishing activities, its waters are used as a source of collection and supply of water and sewage.by the Amapá water and sewage company (CAESA) located in the municipality of Macapá (COSTA, et al., 2019).The same river that supplies water for domestic use is used as a sewage network, with a lack of basic sanitation and sewage treatment, and is also used for inadequate waste disposal (PINHEIRO et al., 2008;VIANA, 2022).
The water made available for consumption by the population is collected in the natural reservoir, through treatment points to later be distributed and used in households.The health of the population is closely linked to the availability of quality water.Today's water contamination is a consequence of human action and has become a major problem, especially in urbanized areas.It is necessary to control pollution to protect health and guarantee a balanced environment (COSTA et al., 2019).
Single health has an approach that integrates aspects involving human health, animal health, plant health and the environment.The WHO (2018) classifies it as: "an approach to designing and implementing programs, policies, legislation and research in which multiple sectors communicate and work together to achieve better public health outcomes".Therefore, the single health guidelines go much further than theoretical aspects and play practical roles in the health of humans, animals, plants and the environment.

Characteristics Of Enterobacteria
Enterobacteriaceae is a family of microorganisms that has very high clinical importance, being the largest group of isolates related to community and hospital infections.They have a multilayer cell wall made of peptidoglycan, and on the outside, they have liposaccharides, phospholipids and lipoproteins in their composition (OLIVEIRA, 2013).Enterobacteria are microorganisms widely present in the environment, and have characteristics such as the fermentation of glucose, the reduction of nitrate to nitrite and the non-formation of spores.They inhabit the gastrointestinal tract of living beings and can cause enteric diseases (MORALES-LÓPEZ et al., 2019).Present in the environment and in the organisms of humans and animals, enterobacteria represent the group of bacteria of greatest clinical interest, isolated from hospital samples and water from urban and rural communities.
They play an important role in the propagation of RAM genes that can be transferred to pathogenic organisms, increasing the risk of infections.Many RAM genes significant in clinical settings may have originated from nonpathogenic bacteria.Resistance to ampicillin, cotrimoxazole, tetracycline, chloramphenicol, nalidixic acid, and ciprofloxacin has been reported in developing countries.Likewise, there is evidence of AMR in enterobacteria found in children in different environments: cities in the Amazon and regions (DANG et al., 2018).

Area of study and collection sites
The descriptive study of virulence patterns and antimicrobial susceptibility of isolates of water samples was conducted in three municipalities in the state of Amapá (Macapá, Santana and Mazagão), in the period from 2022 to 2023.Amapá is located in the extreme north of Brazil, in the Brazilian Amazon region, bordered by the state of Pará, to the west and south; by French Guiana, to the north; by the Atlantic Ocean to the northeast; by the mouth of the Amazon River, to the east; and by Suriname, to the northwest (Bacelar, 2019).In addition, the study collection sites were characterized, being denominated PCM1, PCM2 and PCM3 (Macapá collection sites), PCS (Santana collection site) and PCMZ (Mazagão collection site) (Figure 1).In addition, the coordinates of Sewage treatment (ST) and the points of Water abstraction (WA) were inserted to the study as a base of location and proximity of water collections, and the coordinates were recorded through the application "My coordinates" as (Table 1).A collection was performed in each of the sites of Macapá and two in each site of Mazagão and Santana.The sites were selected due to high concentration of activities and human interaction, economic activity such as fishing and tourism, recreational and leisure time.Specifically in the city of Macapá, the collection took into account sites related to the water supply of the city, which occurs by the capture of this resource by the Water and Sewage Company of Ámapa -CAESA, located on the edge of Macapá.
The Amazon River is used as the main source of supply of the capital of the state of Amapá, and the PCM1 is the closest representative of where water is collected by the city.The PCM2, located on the edge of Macapá, place with intense flow of human activities of recreation and fishing.The PCM3 was located near the port of the "Pedrinas", place of flow of vessels that make the interstate journey between Amapá and islands of the state of Pará.

Collection and Conservation of Samples
The collected water samples were packed in sterilized polystyrene bottles, stored in thermal vats and taken to the Special Laboratory of Applied Microbiology (SLAM) of the Federal University of Amapá (UNIFAP) for isolation and subsequent identification.

Laboratory Analysis
The laboratory activities complied with biosafety protocols and ensured compliance with applicable ethical and legal regulations, being divided into two stages: 1 -Isolation of bacterial colonies performed according to the SLAM/UNIFAP Standard Operating Procedures, and methodology described by Young et al., (2005). 2 -Identification of bacterial species and evaluation of antimicrobial sensitivity and resistance tests and research of virulence genes in the National Reference Laboratory for Bacterial Enteroinfections of the Oswaldo Cruz Institute (LABENT/FIOCRUZ-RJ).
The samples were tested for total coliforms and Escherichia coli by the ONPG-MUG Colilert (IDEXX®) assay.We added 100 mL of the sample in sterilized Nasco Bag and the reagent substrate of colilert, homogenized until complete dissolution, being added to the Quanti-TraySealer, sealed in Quanti-TraySealer sealer (IDEXX®) and incubated in an oven (Olidefcz®) at 37 ºC for 24 h.
After 24 hours, the cards were read.Wells with yellow color indicate positive for total coliforms and fluorescence when subjected to UV Light at 365 nm, characteristics of the presence of E. coli.Then, with the aid of automatic pipettes, 10 µl of random well samples were taken and sown in the culture medium Coliforms Chromogenic Agar (CCA) ISO 9308® (KASVI Laboratorios Conda S.A., Spain©), with supplementation of Ceftriaxone (2 μg/mL) adapted from the protocol of Jacob et al., (2009), by the technique of inoculation by exhaustion and subsequent incubation in an oven (Olidefcz®) at 37 ºC for 24 h.A total of 37 bacterial colonies were selected for identification, antimicrobial sensitivity test and research of virulence and resistance genes.

Biochemical tests for the identification of enterobacteria
The biochemical tests use culture media and specific reagents to detect metabolites resulting from bacterial activity, assisting in their identification.

Susceptibility to antimicrobials
Antimicrobial susceptibility was performed by disk diffusion method, also known as Kirb-Bauer, using the Mueller-Hinton Agar -MH (Sigma-Aldrich, Inc.St. Louis, MO, USA © ) prepared and sterilized according to the manufacturer's instructions.
The isolates were resuspended in 0.9% saline until reaching turbidity at 0.5 of the McFarland scale.Then, the inoculum were sown throughout the Mueller-Hinton agar plate, using the swab.The discs with the drugs were added, and the plates were read after 18 h of incubation at 37 ºC with the aid of a caliper.
Multiresistant isolates that showed resistance to at least one antimicrobial of three different classes were considered, according to the criteria proposed by Magiorakos et al., (2012).The Multiple Antimicrobial Resistance Index (MARI) was performed, which follows the equation: MARI = drugs to which the isolate is resistant / drugs tested in the isolate, in which values > 0.2 indicate the ability of this microorganism to be a reservoir of resistance (Krumperman, 1983).

Bacterial DNA extraction
Bacterial DNA was extracted with the PureLink® commercial kit (Invitrogen), according to the manufacturer's guidelines.

Molecular detection of virulence genes
Virulence genes were detected by multiplex polymerase chain reaction (PCR), applying the protocol of Omar e Barnard (2014).Each reaction was prepared with a total volume of 20 µl, with 2.5 µl of 10 PCR buffer (New EnglandBioLabs, Ipswich, MA, USA), 5 pmol of each MWG-Biotech primer (AG, Ebersberg, Germany), deoxynucleotide triphosphates 34 at a concentration of 200 µM (Promega) 1 U/µl Taq DNA polymerase (New EnglandBioLabs) and 50 ng of genomic DNA.The amplifications were performed in the thermal cycler Veriti (Applied Biosystems, Foster City, CA, USA), under the following conditions: 95 ºC for 15 min.;followed by 30 cycles at 94 ºC for 45 s, 55 ºC for 45 s, 68 ºC for 2 min.and then final extension at 72 ºC for 5 min.PCR products were submitted to electrophoresis in 2% agarose gel (Sigma) stained with ethidium bromide for reading in transilluminator Image Quant, for interpretation of the results.The nucleotide sequence for gene characterization is shown in Table 2.

Molecular detection of resistance genes
The resistance genes were detected by PCR, researching genes associated with resistance to Fluoroquinolones and Cephalosporin, mediated or not by mobile genetic elements.Among the genes related to quinolone resistance, qnrA, qnrB, qnrS in multiplex reactions and cephalosporin resistant genes (blaCMY) in simplex reactions were investigated.The sequence of nucleotides used in the characterization of resistance genes is shown in Table 3.The PCR technique is considered an efficient and fast strategy for the detection of various microorganisms, as well as for epidemiological studies in the evaluations of genes associated with virulence and antimicrobial resistance.PCR, simplex and multiplex, can be used as an alternative method for the simultaneous detection of target sequences in the same sample.

Data Analysis
The results of phenotypic tests and antimicrobial sensitivity tests, as well as the virulence and resistance genes were inserted, analyzed and systematized in spreadsheets, graphs and tables, through the Microsoft 365 Office Excel application for creating a database.To determine the Index of multiple antibiotics resistance (MAR) in isolates that exhibited resistance against the action of three or more antimicrobials tested, in the analysis of each strain the following formula was applied: x / y, where 'x' indicates the sum of antibiotics to which the isolate was resistant and 'y' indicates the number of antibiotics tested against the isolate.Results greater than 0.2 indicate that the microorganism is a potential reservoir of resistance determinants (Kruperman, 1983).The bacteria used in this study were isolated from the water of the Amazon River in the state of Amapá, and Law N. 11,794 of 2008, which deals with animal experimentation, establishes in its art.2º, applying to animals of species classified as phylum Chordata, subphylum Vertebrata, observed environmental legislation (Brazil, 2008), where the legal provision does not include bacteria in the mandatory authorization to conduct studies, which exempts this study.In view of the legal framework of biodiversity, embodied by Law N.

RESULTS AND DISCUSSION
From the quantity of 37 water samples sent to LABENT/FIOCRUZ for confirmation of bacterial species, in 21, there was growth of enterobacteria.Of this quantitative, 24 enterobacteria were identified.Of these, 16 samples did not show growth in specific medium for enterobacteriales.Eighteen strains of Escherichia coli, four strains of Klebisiella.pneumoniae, one strain of Enterobacter spp.and one strain of Chromobacterium violaceum.In all collection sites PCM1, PCM2, PCM3 (Macapá), PCS (Santana) and PCMZ9 (Mazagão), there was the presence of E. coli.Strains of K. pneumoniae were also found in the three municipalities, at sites PCM1, PCS, PCMZ.C. violaceum at PCM1, as shown in Table 4.
The isolates submitted to antimicrobials Ciprofloxacin -CIP (100%) and Meropenem -MEM (100.0%), were resistant and sensitive in this order.The antimicrobials Levofloxacin -LEV (100%) and Imipenem -IMP (100%) were also resistant and sensitive respectively.Isolates from sites PCM1, PCM2 and PCMZ were the most resistant to nalidixic acid -NAL.All of the PCM2, PCM3 and PCS present resistance to Ceftazidime -CAZ.All isolates of PCMZ were resistant to Streptomycin -STR, according to the susceptibility profile of the isolates observed in Table 5.

Virulence and resistance in water
The virulence eagg, eaeA, stx1, stx2 genes were found in water isolates at the sites PCM1, PCM2 and PCM3, in the municipality of Macapá.The eagg gene refers to the presence of E. coli EAEC (enteroagregative) with virulence factor due to the presence of Shigella enterotoxin (ShET1), responsible for Shigelose, which is an acute infection of the intestine with symptoms including fever, nausea, vomiting, tenesmus and diarrhea, with possibility of bloody feces.This strain was found in isolates from the PCM1 collection site.
The eaeA gene is associated with the presence of E. coli EHEC (enterohemorrhagic), capable of causing acute bloody diarrhea and was present in isolates at PCM2 site.The stx1 is also a gene associated with the presence of E. coli EHEC (enterohemorrhagic), and was found in PCM2.The resistance profiles of 13 (72.2%) of these strains were multiresistant.The MARI, water resistance index, ranged from 0.1 to 0.7, according to Table 6.
Table 6 -Phenotypic and molecular profile of antimicrobial resistance and virulence of each Escherichia coli isolate E. coli was the most prevalent bacteria found in 18 of the water isolates.This is significant because this bacterium is a common indicator of fecal contamination and its presence in water may indicate contamination by human or animal feces.The presence of E. coli at multiple collection points suggests a possible source of widespread contamination.In addition to E. coli, other enterobacteria were also found in water isolates, such as Klebsiella pneumoniae found in 4 isolates, Enterobacter spp in 1 isolate and Chromobacterium violaceum in 1 isolate.E. coli is used as bacteriological indicator organism of the quality and fecal pollution of surface waters for being highly abundant, easy to grow and widely associated with the intestinal tract of aquatic organisms, understanding of the environmental impact on water resources and their organisms.Although bacteria that indicate fecal contamination may not directly cause human diseases, their prevalence at elevated levels in recreational and domestic waters is associated with the possibility of an increased risk of gastrointestinal diseases (Kichana et al., 2022).
The widespread distribution of Escherichia coli in water can be attributed to the flow of environments with poor sanitation practices, such as open defecation.The consumption of water contaminated with this microorganism leads to the emergence of waterborne diseases and infections, which later may result in morbidity and mortality in children and adults (Mok, 2021).
The Gram-negative bacteria belonging to families of the order Enterobacterales are potentially pathogenic, and their presence in water isolates is worrying because they are known to cause a wide range of infections in humans and animals.Moreover, several strains of these bacteria are virulent and can cause hospital infections, as well as develop resistance to multiple antimicrobials, which represents a significant public health problem (Yusuf et al., 2021).
It is important to emphasize that the development of resistance to multiple antimicrobials in gram-negative bacilli, including enterobacteria, is a global trend.This means that these bacteria are becoming more difficult to treat due to their resistance to different types of antibiotics, which limits the available treatment options (Amâncio, et al, 2021).
Therefore, the presence of these enterobacteria in water isolates, together with the occurrence of resistance to multiple antimicrobials, highlights the importance of control measures and monitoring of water quality.The adoption of appropriate water treatment practices and responsible use of antibiotics are essential to prevent infections and limit the spread of resistant bacteria (De Souza, et al, 2021).
The study presents the susceptibility profile of isolates of E. coli found in the water of the Amazon River of three mini-cilia of the state of Amapá (Macapá, Santana and Mazagão) in relation to different antimicrobials.The results reveal important information about susceptibility, as nalidixic acid -NAL (88.9%), ceftazidime -CAZ (77.8%), sulfamethoxazoletrimethoprim -SXT (66.7%), ciprofloxacin -CIP (100%) and levofloxacin -LEV (100%) showed a high resistance rate of isolates.This is worrying, as these antibiotics are often used in the treatment of infections caused by E. coli.
In general, these results highlight the presence of E. coli strains in the collected water that present resistance to important antimicrobials, such as Nalidixic Acid and Ceftazidime.At the same time, there are still effective treatment options, such as Cefoxitin, Chloramphenicol, Streptomycin, Nitrofurantoin and Meropenem.Thus, the study brings the need to monitor antimicrobial resistance in these bacteria in the implementation of appropriate control measures, as well as in the responsible use of antimicrobials to avoid aggravating the problem of bacterial resistance (Mello et al., 2020;Evagelista Sobrinho, et al, 2020).
Regarding the phenotypic and molecular profile of antimicrobial resistance and virulence of each E. coli isolate, the study evidenced the virulence genes eagg, eaeA, stx1 and stx2 found in water isolates collected at sites PCM1, PCM2 and PCM3.These genes are associated with different types of E. coli with virulent potential.The eagg gene is related to the presence of E. coli EAEC (enteroagregative), which produces ShET1 enterotoxin and can cause symptoms such as fever, nausea, vomiting, tenesm and bloody diarrhea.The genes eaeA, stx1 and stx2 are associated with E. coli EHEC (enterohemorrhagic), which can cause acute bloody diarrhea (Amin, 2022).
The presence of these virulence genes indicates a greater potential of these E. coli strains in causing diseases in humans.In addition, the resistance genes qnrB and qnrS were detected in the water isolates.These genes are associated with drug resistance of the class of fluoroquinolones, in this case, Ciprofloxacin.The presence of these genes indicates that E. coli strains found in water have important antimicrobial resistance mechanisms, limiting the options for effective treatment of these infections (Śliwka, et al, 2023;Amin, 2022).
Additionally, the multiple antimicrobial resistance index (MARI) ranged from 0.1 to 0.6.Isolates with a higher MARI indicate greater resistance to multiple drugs.It is important to note that most water isolates presented an MARI above 0.4, which indicates a high rate of multidrug resistance, a significant datum of antimicrobial resistance in these strains (Śliwka, et al, 2023).
Although commensal bacteria are generally considered harmless, they can act as a reservoir for many antimicrobial resistance genes (ARGs).Increasing evidence indicates that ARGs can be easily transferred between microbial species, including between commensal species and pathogens, mainly through transformation and conjugation events (Dungan;Bjorneberg, 2021).
Moreover, E. coli is commonly used in the monitoring of antimicrobial resistance that act against the activity of Gram-negative and Gram-positive bacteria.Aquatic environments may allow the opportunity to exchange genetic materials, such as antibiotic-resistant genes among bacteria without pathogenicity, being this transfer of resistant genes to bacteria with pathogenic degree a very worrying factor (DeFlorio-Barker et al., 2018).
Thus, it is evident that the contamination of rivers is intimately linked to waste and pollutants discharged, causing aquatic contamination, being E. coli often detected in surface waters as etiological agents of waterborne infections, and resistant strains identified in developed and developing countries (DeFlorio-Barker et al., 2018).
Additionally, bacteria with resistance and virulence genes from human-animal activity and interaction have circulated in waters that are provided to human communities, through water treatment plants considered large reservoirs of bacteria that include virulent bacterial strains, with heavy metals and bioactive ingredients that can promote the development of the emergence of resistance genes, being the aquatic environments identified as critical sites of development of RAM (Jeamsripong et al., 2023).
Thus, it is important to evaluate water sources of potentially unhealthy origin, so that the risks to human health associated with access and procurement of water can be predicted (Cunha et al., 2005).Therefore, the research showed that the waters of the Amazon River were associated with an increased risk of gastrointestinal disease associated with recreation and human consumption.The microbial quality of water is an important issue, especially in developing countries, where many people still rely on water sources, whether raw, partially treated, or fully drinkable (Kichana et al., 2022).
Thus, the results of the study highlight the presence of E. coli strains with virulence genes and antimicrobial resistance, representing a challenge for the treatment of infections caused by these bacteria (Pinto et al., 2020).Adequate control measures and responsible use of antimicrobials are essential to prevent the spread of these resistant strains and minimize risks to public health.

FINAL CONSIDERATIONS
Based on the data presented on antimicrobial resistance and virulence of E. coli strains isolated from water samples from the Amazon River, there is a worrying presence of strains with pathogenic characteristics and resistant to multiple antimicrobials.The virulence genes detected, such as eagg, eaeA, stx1 and stx2, indicate the presence of E. coli strains with great potential to cause serious infections in humans.
These genes are associated with different types of E. coli, such as EAEC (enteroagregative) and EHEC (enterohemorrhagic), which can cause symptoms such as bloody diarrhea, fever and other gastrointestinal problems.In addition, the detection of the qnrB and qnrS resistance genes reveals that these strains of E. coli have mechanisms of resistance to important antimicrobials, such as fluoroquinolones.This limits effective treatment options and can result in more difficult-to-control infections.
Thus, these results highlight the importance of monitoring and controlling the water quality of the Amazon River, as well as promoting the responsible use of antimicrobials in humans and animals.Prevention and education strategies are essential to prevent the spread of these resistant strains and reduce public health risks.Therefore, antimicrobial resistance and virulence of E. coli strains represent a significant challenge and require coordinated actions to mitigate health risks and preserve antimicrobial efficacy.

Figure 1 -
Figure 1 -Map of Brazil (A), State of Amapá (B), municipalities of Macapá (C), Santana and Mazagão and their respective collection sites.Source: Collection data, 2023

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River, State of Amapá ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.3 | p.1-17 | e04424 | 13,123 of May 20, 2015, regulated by Decree N. 8,772 of May 11, 2016, which provides for the rules and conditions for access to samples of genetic heritage and associated traditional knowledge, for research and technological development purposes, this study is registered with the National System of Genetic Heritage Management and Associated Traditional Knowledge (SISGEN), under N. ACC3633, managed by the Ministry of the Environment (Brazil, 2016).

Table 1 -
Coordinates of collection site Research data collection, 2023 PCM1, PCM2, PCM3 (Collection points in Macapá); PCS (Collection point in Santana); PCMZ (Collection point in Mazagão) The biochemical test used for E. coli was by sources of carbon glucose, agar TSI, SIM citrate, Motility test, Indole test and Methyl red test.

Table 2 -
Sequence of nucleotides used in the characterization of virulence genes.Virulence and Antimicrobial Resistance of Escherichia Coli Strains Isolated from The Water of The Amazon River, State of Amapá ___________________________________________________________________________ Rev. Gest.Soc.Ambient.| Miami | v.18.n.3 | p.1-17 | e04424 | 2024.8 CTG AAT CCC CCT CCA TTA TG

Table 3 -
Sequence of nucleotides used in the characterization of resistance genes.

Table 4 -
Enterobacteria present in water at collection sites.

Table 5 -
Susceptibility Profile of Escherichia coli isolates

Table 6 -
Phenotypic and molecular profile of antimicrobial resistance and virulence of each Escherichia coli