Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil

The aim of this study was to evaluate the species distribution, antibiotic-resistance profile and presence of enterotoxin (SE) genes in staphylococci isolated from the Dilúvio stream in South Brazil. Eighty-eight staphylococci were identified, 93.18% were identified as coagulase-negative (CNS) and 6.82% coagulasepositive (CPS). Fourteen Staphylococcus species were detected and the most frequently were Staphylococcus cohnii (30.48%) and S. haemolyticus (21.95%). Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin. None of the investigated strains showed gentamicin and ciprofloxacin resistance. The strains were tested for the presence of sea, seb, sec, sed and see genes by PCR and only CNS strains (43.18%) showed positive results to one or more SE genes. The scientific importance of our results is due to the lack of data about these topics in polluted waters in Brazil. In conclusion, polluted waters from the Dilúvio stream may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community. In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination and poor sanitation conditions.


INTRODUCTION
Microbiological water contamination is one of the biggest public health problems in the world.The World Health Organization (WHO) estimates that around one billion people drink unsafe water and approximately three million people die each year from waterborne diseases (Omari and Yeboah-Manu 2012).The Dilúvio is a polluted stream that crosses the city of Porto Alegre, the state capital of Rio Grande do Sul, the 10 th most populous city in Brazil, with approximately 1,500,000 inhabitants.The Dilúvio stream flows into the Guaíba Lake, the main source of water supply to Porto Alegre which is also used for water-based sports, such as sailing, kayaking, canoeing, powerboating and windsurfing (Devos 2009).
Staphylococcus species are ubiquitously distributed in nature (Varnam and Evans 1996) and resistant strains have been isolated from diverse sources, such as foods, animals and healthy and ANA P. BASSO et al. hospitalized individuals (Malik et al. 2005, Sorum and L'Abee-Lund 2002, Wisplinghoff et al. 2004).Members of the Staphylococcus genus can express a wide range of virulence factors, such as: cell wall components, proteases, coagulase, hemolysins, enterotoxins, toxic-shock syndrome (TSS) and exfoliatins toxin (Novick et al. 2001).
Staphylococcal Enterotoxins (SEs) are part of the pyrogenic proteins associated to many human diseases (Pinchuk et al. 2010).These proteins are resistant to thermal inactivation and to gastrointestinal proteases, causing emesis and diarrhae, and/or can act as superantigens (Balaban andRasooly 2000, Pinchuk et al. 2010).Although more than 20 different enterotoxins were identified, only the classical enterotoxins, like SEA, SEB, SEC, SED and SEE were well studied (Goto et al. 2007).These enterotoxins are generally produced by Staphylococcus aureus, but other species, such as S. intermedius, S. hyicus, S. xylosus and S. epidermidis can also express them (Bhatia and Zahoor 2007).
In Brazil, numerous studies have been published about staphylococci, but so far there are no studies that evaluate staphylococci in polluted water.Therefore, the aim of this study was to evaluate staphylococci species distribution, antibiotic resistance profile and enterotoxin genes frequency in Staphylococcus sp.isolated from polluted water from the Dilúvio stream in South Brazil.
DETECTION OF STAPHYLOCOCCAL ENTEROTOXINS GENES DNA extractions and polymerase chain reaction (PCRs) for five classical staphylococcal enterotoxins genes (sea, seb, sec, sed and see) were performed as previously described by Moura et al. 2012. The S. aureus ATCC 13565, ATCC 14458, ATCC 19095, ATCC 23235 and ATCC 27664 strains were used as positive controls for sea, seb, sec, sed and see genes, respectively.
Staphylococcus are a genre of bacteria widespread in nature, being found in the air, soil, water, food products and animals, including human beings (Faria et al. 2009, Hou et al. 2012, Martins et al. 2013).In this study the frequency to CNS species was higher than CPS, this result agrees with Serapicos (2008) and Faria et al. (2009) who also demonstrated an elevated frequency of CNS in wastewater.The presence of staphylococi species in polluted waters of the Dilúvio stream can be justified, since members of the Staphylococcus genus commonly inhabit the intestinal tract of humans and warm-blooded animals and they contaminate the surface waters through inadequate septic tanks, wastewater effluents, and fecal discharges (Orrett and Shurland 1998).
The distribution of staphylococci species isolated from water samples obtained from different sites of the Dilúvio stream is shown in Table I.A wide variety of staphylococci species was isolated from Sites B, C, D and E, while from Site A, no staphylococci were isolated.The region of Site A is considered the headwaters of the Dilúvio stream located in the Saint Hilaire Park, which is the area with the lowest anthropic influence.Site B is located in an urbanized area, where many residential homes are not connected to a public sanitary sewer system.In addition, Site B also has domestic animals, such as cows, horses, chickens and pigs, which are used for food or labor, and the feces of these animals drain directly into the stream, being a recognized source of contamination.Sites C, D and E were located in a dense urban area, with hospitals, schools, restaurants, universities and residential homes.The occurrence of staphylococci species in these sites, can be explained due to the fact that the sanitary sewer system of many places remain connected to the urban fluvial channel, contributing significantly to the contamination of the Dilúvio's stream (Nachtigall et al. 2013).
Table II shows the antibiotic resistance of Staphylococcus species isolated from polluted water.Resistance to erythromycin was verified in 37.50% of the strains, followed by 27.27% to penicillin, 12.50% to clindamycin, 6.81% to trimethoprim-sulfamethoxazole, 5.68% to chloramphenicol and 2.27% to norfloxacin.None of the investigated Staphylococcus strains showed gentamicin and ciprofloxacin.Isolates resistant to two or more classes of antimicrobials were classified as multiresistant and accounted for 10.2% of all isolates.
The Dilúvio's stream receives contaminants from both natural and anthropogenic sources.In this study, staphylococci strains showed resistances to antibiotics commonly used in human and veterinary medicine to treat infectious diseases caused by Gram-positive bacteria, like penicillin and erythromycin.Penicillin is the antibiotic of choice in most staphylococcal infections, and clindamycin and erythromycin are alternative  agents in patients who are allergic to penicillin (Dryden 2010).Previous studies, also reported antibiotic resistance in staphylococci isolated from wastewater, polluted and drinking water.Kessie et al. (1998) also identified CNS resistant to chloramphenicol and erythromycin isolated from polluted water.Serapicos (2008) observed a high frequency of staphylococci resistance to lincomycin, eritrhomicyn and clindamycin, while a low frequency of isolates were resistant to penicillin in water collected from a wastewater treatment plant.Faria et al. (2009) have encountered a high frequency of CNS resistant to erythromycin, beta-lactams, tetracycline, clindamycin and erythromycin in wastewater and drinking water.In sea water, freshwater and sediments samples from the Lebanon coast, resistant staphylococci were also isolated, where 51% of them were resistant to at least one tested antimicrobial (Harakeh et al. 2006).
Multiresistant staphylococci were also detected in potable water from Pakistan and from the United States (Armstrong et al. 1981, Samra et al. 2009).
Staphylococcal enterotoxins (SE) constitute a family of biologically and structurally related toxins and the ingestion of these toxins results in gastrointestinal effects such as nausea, vomiting, diarrhea, and abdominal pain.The SE are the main cause of bacterial food poisoning in human    The scientific importance of our results is due to the lack of data about these topics in polluted water.The results, indicate that the occurrence of staphylococci antibiotic resistant and harboring enterotoxin genes in polluted waters from the Dilúvio stream, may constitute a reservoir for disseminating antibiotic-resistance and enterotoxin into the community, since Dilúvio's stream flows into the Guaiba Lake, which is the main source of water supply to Porto Alegre as well as being used for water-based sports.In addition, the detection of staphylococci in the polluted waters of the Dilúvio stream indicated a situation of environmental contamination by fecal microorganisms of human and animal origin and poor sanitation conditions.

ACKOWLEDGEMENTS
We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of the Brazilian government, for the support received.

Figure 1 -
Figure 1 -Map of the study areas and sampling sites into Dilúvio stream in Porto Alegre, RS, Brazil: Site A: headwaters in the Saint Hilaire Park; Site B: Ipiranga Av. corner Antônio de Carvalho Av.; Site C: Ipiranga Av. corner Guilherme Alves St; Site D: Ipiranga Av. corner Ramiro Barcelos St and Site E: Ipiranga Av. corner Borges de Medeiros Av.Source: Google Maps.[Accessed 05/12/2013].

TABLE II Number of antibiotic-resistant staphylococci isolates from polluted water
. ANA P. BASSO et al.