Antimicrobial-resistant enterobacteria in surface waters with fecal contamination from urban and rural communities

Abstract INTRODUCTION: Inadequate wastewater treatment and fecal contamination have a strong environmental impact on antimicrobial resistance (AMR). This study evaluated the profile of AMR enterobacteria and fecal contamination from four surface waters: Jiquiriça-Brejões River and Cabrito, Tororó, and Abaeté Lagoons. METHODS: We analyzed AMR β-lactamase genes using the polymerase chain reaction method and fecal contamination using Coliscan®. RESULTS: We found high levels of fecal contamination, β-lactamase producers, and AMR genes (blaOXA-48, blaSPM, and blaVIM) in all waterbodies. CONCLUSIONS: Poor sanitation evidenced by fecal contamination and human activities around these surface waters contributed to the distribution and increase in AMR enterobacteria.

are the main contributors to human morbidity and mortality owing to their high transmissibility from person to person, from animals to humans, and to the environment. Therefore, AMR has become a major threat to human health 6 . In rural environments, AMR occurs because of the use of antibiotics for agricultural purposes and occurs in livestock through contamination of the soil and adjacent rivers 7 . In urban areas, high population density and inadequate basic sanitation have altered the microbiologic balance of aquatic environments, making them reservoirs of AMR bacteria and a potential source of community infections 8 . Here, we evaluated the fecal contamination of surface waters in rural and urban areas and determined the AMR of isolated Enterobacteriaceae.

Study sites
The rural river Jiquiriça-Brejões (JB) is located in Jenipapo, municipality of Ubaíra, Bahia, Brazil. The urban water sources, located in three areas of the capital, Salvador, in Bahia, Brazil are as follows: Cabrito Lagoon (CL), Tororó Lagoon (TL), and Abaeté

Water sampling
We collected water samples (n=48) every 3 months, from October 2016 to August 2017. At each time point, 400 mL of water was collected in sterile glass vials at a depth of approximately 30 cm below the surface. The vials were transported in thermal boxes with dry ice until microbiological analysis.

Fecal contamination
We identified coliforms using the Coliscan Easygel ® kit (Microbiology Laboratories, Goshen, IN, USA), following the manufacturer's instructions. The count limit established in this study was 1,000 CFU/mL. According to the standards of bathing conditions determined by the Brazilian Federal Environmental Council (CONAMA; Ordinance No. 274/00), waterbodies exceeding 25 CFU/mL total coliforms and 20 CFU/mL total Escherichia coli are considered unsuitable for drinking and recreational use.
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (VITEK-MS ® , Biomérieux, France) was employed to identify bacterial species. The AMR susceptibility profile was determined using the VITEK-2 ® automated system for Enterobacteriaceae (Biomérieux, France). Subsequently, bacterial isolates were stored in the tryptic soy broth medium supplemented with glycerol (20%) at −80 °C for future analysis.

Detection of AMR encoding genes
Enterobacter cloacae, E. coli, and Klebsiella pneumoniae isolates were analyzed for AMR. Frozen isolates were re-cultured on TSA for 18-24 h at 36 ± 1 °C, and colonies (±5) were resuspended in 100 μL of sterile distilled water for DNA extraction. Each isolate was incubated at 95 °C for 5 min and then centrifuged at 12,000 rpm for 2 min. The supernatant was transferred to another cryotube and stored at −20 °C until use.
The β-lactamase AMR identification (bla CTX-M , bla SHV , bla TEM , bla KPC , bla VIM , bla NDM , bla SPM , and bla OXA-48 ) was performed using the conventional polymerase chain reaction (PCR) method using TopTaq Master Mix® (Qiagen, USA), in accordance with several different protocols. A standard annealing temperature was used for all primers.

Statistical analysis
The data were tabulated using Microsoft Excel (Microsoft Corporation, USA) and analyzed with EpiInfo™ software (Centers for Disease Control, USA) to summarize descriptive statistics, such as frequency distributions, means, and standard deviations. The median count of coliforms and E. coli isolates were compared using the nonparametric Kruskal-Wallis test, followed by Dunn's multiple comparison post hoc test using GraphPad Prism™ 9.0.0 (GraphPad Software, USA). P values < 0.05 were considered significant.
The average total coliform (TC) count for JB was 367.0 CFU/mL. At the urban sites of CL, TL, and AL, the average TC counts were 736.0, 149.4, and 154.3 CFU/mL, respectively. The average E. coli count in JB was 164.2 CFU/mL, while that in CL, TL, and AL were 405.7, 4.8, and 9.0 CFU/mL, respectively ( Table 1). We did not observe a significant difference between the rural and urban areas; however, among the urban sites, there was a significant difference between CL, AL, and TL. AL and TL are near tourist areas and undergo water treatment and local maintenance, though it is inefficient to make the water usable to humans. In contrast, CL   ----2  8  --2  3   SAM, MEM, HODGE +  ----2  8  --2  3   SAM, ETP, HODGE +  ----13  50  --13  20   GEN  ------2  22  2  3   Susceptible  1  6  ----2  is located in a poor neighborhood in a region lacking maintenance or water treatment.

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Fecal contamination, especially from human sources, is an important route for the dissemination of AMR enterobacteria and microbiota modification of waterbodies 8 . Our findings indicate that the rural and urban water sources we examined in Brazil are widely contaminated with human feces and, according to CONAMA (ordinance n o 274/00), these water sources are inappropriate for human consumption and recreational use. In rural areas, both human and animal feces may contribute to microbiota modification and, therefore, to the selection of resistant species 7,10 . In urban areas, high population density around waterbodies and lack of adequate sanitation influence the degree of contamination 4,11 . A previous study 1 demonstrated that the human fecal content in CL was similar to that of raw sewage in Cleveland, Ohio, USA, using tracking of microbial source and DNA of human-indicative Bacteroides species. These findings require increased attention from local authorities and residents, as the waterbodies evaluated in this study serve as environmental reservoirs of AMR 12,8 .
We found AMR genes, even though there was no evidence of direct disposal of hospital waste in these waterbodies. The identified bacteria have an important association with human diseases (e.g., gastrointestinal colonization) and serve as a source of infection. The WHO has described them as a major public health concern, especially when associated with AMR 5 . In rural areas, we identified resistance genes, such as bla CTX-M, bla  , and blaVIM, using PCR; they may correlate with the spread of β-lactam-resistant bacteria in pig pens and via soil and domestic sewage. Our findings reinforce the presence of AMR in northeast Brazil, consistent with the results of studies in other rural environments 10,13 .
The urban settings in developing countries have high population density and are less efficient in sanitation 12 . Therefore, it was not surprising that we observed a higher AMR profile diversity in urban areas than in rural areas. Moreover, the presence of carbapenemase-producing and ESBL-positive bacteria is associated with worse prognosis of human infections. This type of resistance is frequently related to healthcare problems and many communityacquired infections 14 . Overall, in urban areas, our genotypic analysis revealed that the most frequent cephalosporinase gene was bla CTX-M (22%) and carbapenemase gene was bla OXA-48 (33%). Studies have frequently reported these enzymes, probably owing to their easy dissemination, as they reside in mobile vectors, such as plasmids and transposable elements 15 .
Communal activities and poor infrastructure for sanitation pollute the surrounding aquatic environments. Therefore, rural and urban waterbodies are important reservoirs for the dissemination and selection of AMR enterobacteria and potential sites of acquiring severe and non-treatable human infections. Furthermore, poor sanitation aggravates this problem, especially in urban settings in developing communities. Consequently, more people are prone to become ill and infected with difficult-to-treat microorganisms.