African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5181

Full Length Research Paper

Microbiota sampled from a polluted stream in Recife-PE, Brazil and its importance to public health

Antonio Fernando da Purificaçao Junior
  • Antonio Fernando da Purificaçao Junior
  • Genetic, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Lívia Caroline Alexandre de Araujo
  • Lívia Caroline Alexandre de Araujo
  • Biological Science, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Ana Catarina de Souza Lopes
  • Ana Catarina de Souza Lopes
  • Department of Tropical Medicine, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Marcela de Araujo Sobral
  • Marcela de Araujo Sobral
  • Department of Antibiotics, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Glaucia Manoella de Souza Lima
  • Glaucia Manoella de Souza Lima
  • Department of Antibiotics, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Marcia Vanusa da Silva
  • Marcia Vanusa da Silva
  • Department of Biochemistry, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Maria Tereza dos Santos Correia
  • Maria Tereza dos Santos Correia
  • Department of Biochemistry, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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Maria Betania Melo de Oliveira
  • Maria Betania Melo de Oliveira
  • Department of Biochemistry, Federal University of Pernambuco (UFPE), Recife, PE, Brazil.
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  •  Received: 03 May 2017
  •  Accepted: 07 July 2017
  •  Published: 28 July 2017


Pollution of water bodies can cause environmental and public health problems. The Cavouco stream is a tributary of the Capibaribe River, one of the main rivers in the state of Pernambuco, Brazil, and receives a high pollution load from residential, laboratory and hospital effluents. The aim of the present study was to perform phenotypic and molecular characterization in this stream, and evaluate the water quality using microbiological parameters. Water was collected from five sampling points, and bacterial species were identified using biochemical and molecular methods through 16S rRNA gene sequence analysis. Total and thermotolerant coliforms were also quantified. Fermenting Gram-negative bacilli from the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis), non-fermenting bacilli (Pseudomonas aeruginosa and Pseudomonas putida) and Gram-positive bacilli (Bacillus cereus, Bacillus licheniformis, Bacillus pumilus and Staphylococcus hominis) were identified. A total of 25 bacterial isolates were phenotypically identified. All phenotypic identifications were confirmed by molecular analysis, except for S. hominis, which was molecularly identified as Exiguobacterium. Regarding water quality, all analyzed samples were positive for total and thermotolerant coliforms. The results obtained suggest that the Cavouco stream presents a potential risk for transmission of water-borne diseases, because of the presence of pathogenic bacteria. In addition, the current state of the stream also threatens the conservation of its native species.

Keywords: Public health, Enterobacteriaceae, thermotolerant, Bacilli.


Aquatic ecosystems have suffered significant changes due to multiple environmental effects, resulting from the release of large quantities of effluent without prior treatment (International Joint Commission, 2015; USGS, 2015). This discharge can cause physical,  chemical  and biological deterioration, and endangers both the resident aquatic organisms and public health. Scientific, technological and epidemiological advances have provided new tools for the assessment of water quality, both  for  human  consumption  and environmental purposes (Tanchou, 2014).
In 2015, the Centers for Disease Control and Prevention (CDC) reported that approximately 780 million people had no access to drinking water around the world. The consumption of contaminated water and lack of basic sanitation is estimated to cause 842,000 deaths per year worldwide, and 1,000 children under the age of five years die every day (WHO, 2014; WHO and UNICEF, 2015). Contaminated water carries pathogens that cause diarrhea, gastrointestinal disorders and systemic diseases, approximately 70% of diarrheal diseases could be avoided by improving basic sanitation (WHO, 2014). According to the Brazilian Ministry of Health, 6,715 deaths caused by diarrhea or gastroenteritis, presumably resulting from infection, were recorded between 2010 and 2015 (BRASIL, 2015).
Some studies have used traditional methods of selective isolation and cultivation to characterize the microbial communities of the affected environments (Skariyachan et al., 2013). However, taxonomic classification by these methods can be difficult because of variations in phenotypic characteristics (Woo et al., 2008). For this reason, molecular methods that allow fast and reliable confirmation of microbial identity have been developed (Ramírez-Castillo et al., 2015). Among these, methods using 16S rRNA gene sequencing are predominant. This gene is used as a phylogenetic marker because its sequences are highly conserved (Srinivasan et al., 2015). 
The aim of the present study was to evaluate the water quality by the isolation and identification of representative bacterial species present in this environment, using biochemical and molecular methods.


Study area
The Cavouco stream, located at latitude 8°2'52.05"S and longitude 34°57'10.33" W, state of Pernambuco (UFPE), Brazil, is approximately 6 km long, and flows into the right margin of the Capibaribe River, one of the main rivers of state. Along its course, it receives pollutants from residential, laboratory and hospital waste, which reduces the water quality and threatens the aquatic life (Araujo and Oliveira, 2013; Freitas et al., 2016). Water samples (200 mL) were collected from five points (Figure 1) along the stream, according to the methods of Araújo and Oliveira (2013), and stored between 1 and 4°C until subsequent bacteriological analysis.
Isolation and phenotypic identification of bacterial isolates
For bacterial isolation, 50 µL  of  water  was  inoculated  onto  eosin  methylene blue (EMB) agar and 5% bovine blood agar (to count colony forming units), and incubated at 37°C for 24 to 48 h. Gram staining was then performed to make presumptive identifications of the bacteria found according to the technique described by Koneman and Winn (2006).
Gram-negative isolates were preliminarily identified using the following biochemical tests: glucose, lactose and sucrose fermentation, hydrogen sulfide production, motility, indole production and citrate, lysine and urea degradation. Species identification was confirmed using the Kit API 20E (Biomérieux), according to the manufacturer’s instructions.
Gram-positive isolates were preliminarily identified through colony morphology, hemolysis in blood agar, and presence/absence and position of spores, visualized through Gram staining. Species identification was confirmed using an automated system (BD Phoenix™ Automated Microbiology System).
Analysis of total and thermotolerant coliforms
For analysis of total and thermotolerant coliforms, water samples were collected from the water surface at a depth of 30 cm, from each of the five sampling points. The samples were stored between 1 and 4°C until analysis.
The presence and number of total and thermotolerant coliforms were determined using the multiple-tube fermentation method, and the results were expressed as most probable number (MPN) per 100 mL of sample, according to APHA (2015).
Molecular identification using 16S rRNA gene sequence analysis
The bacterial samples were inoculated into 5 mL brain heart infusion (BHI) broth for 24 h at 37°C for to DNA extraction. Chromosomal DNA was extracted using the phenol-chloroform method (Sambrook and Russell, 2001). DNA quality was evaluated by electrophoresis in a 0.8% agarose gel using 0.5× TBE buffer and run at 100 V for 1 h; gels were analyzed using a UV transilluminator and photographed. DNA concentration was quantified using a Nanodrop spectrophotometer (Thermo Scientific).
The 16S rRNA gene was amplified by polymerase chain reaction (PCR), using the primers fD1 (5´-AGAGTTTGATCCTGGCTCAG-3´) and rD1 (5´-AAGGAGGTGATCCAGCC-3´) (Weisburg et al., 1991). PCR was performed in a final volume of 25 μL, containing 1× buffer, 200 µM dNTPs, 1.5 mM MgCl2, Taq DNA-polymerase (1 U/µL; Invitrogen), 10 pmol of each primer, and 10 ng of DNA template. The PCR was performed using a thermocycler (C1000 Thermal Cycler – BioRad), and the PCR program consisted of 95°C for 5 min, 30 cycles at 95°C for 45 s, primer annealing at 54°C for 45 s, extension at 72°C for 2 min, and a final extension at 72°C for 5 min.
PCR products were purified using the Pure Link purification kit (Invitrogen) according to the manufacturer’s instructions, and sequenced using the Big Dye Kit (Applied Biosystems) on an automated DNA sequencer (ABI 3100). The 16S rRNA gene sequences obtained was compared with sequences deposited in the GenBank database (NCBI). The dendrogram was constructed using multiple sequence alignment, based on genetic distances, maximum parsimony, and maximum likelihood, using Molecular Evolutionary Genetic Analysis 5.2 software (MEGA5).
Bacterial library of Cavouco stream – UFPE
Identified bacteria were stored in 80% glycerol (150 µL of glycerol and 850 µL of bacterial culture) at -80°C, and under pure mineral oil (Isofar) at ambient temperature. The isolates were labeled according to: C (Cavouco), P (sampling point), the number of sampling points, and the number of isolations.


Microscopic and biochemical identification of bacterial colonies
Inoculated blood agar presented innumerable colony forming units (CFU), except for water samples collected at the stream source (Point 1), for which only a few colonies were observed. Due to EMB medium selectivity, these plates presented an average of 200 CFU, with a lower number of CFUs observed for Point 1, and higher number for point 5 EMB plates presented colonies with metallic green sheen, dark center, some with bright or pink edges, and mucoid appearance. In blood agar, some colonies were shiny or gray, with or without beta-hemolysis, and with irregular edges. Microscopic examination of Gram-stained colonies revealed  the  presence  of  Gram-negative and positive bacilli. Some Gram-positive bacilli were observed to be arranged in chains, filaments and spherical bodies. Approximately, 22 and 25 colonies were selected from the most frequent colonies present on EMB medium, and from blood agar, respectively.
A total of 25 isolates were identified phenotypically, belonging to seven genera (Bacillus, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas and Staphylococcus). Enterobacteria were present in all sampling points. Proteus mirabilis was highly prevalent among Gram-negative species (with eight isolates mostly from point 5), followed by Escherichia coli (six isolates), and Klebsiella pneumoniae (five isolates, from points 2, 3, and 4). Among the Gram-positive bacilli, the genus Bacillus was prevalent, with two species (Bacillus licheniformis and Bacillus pumilus) identified from point 4, and one (Bacillus cereus) from point 1. Another Gram-positive species, S. hominis, was identified from point 4 (Table 1).
Presence of total and thermotolerant coliforms
All analyzed samples were positive to total and thermotolerant  coliforms,  presenting   an   MPN  >  1.4 ×  103/100 mL.
Molecular identification using 16S rRNA gene analysis
The 16S rRNA gene sequences (approximately 1.500 bp) for the 25 selected isolates were aligned, and compared with sequences deposited in the GenBank database. The obtained sequences were found to have a high degree of genetic similarity (98-99%) with deposited sequences for the same species, confirming their phenotypic identification. Of the 25 isolates, only S. hominis did not display concordance between the phenotypic and molecular identification. This isolate was identified phenotypically as belonging to the genus Staphylococcus, but through molecular analysis, it revealed 99% similarity with the genus Exiguobacterium (Table 1). The phylogenetic tree showed clustering of 16S rRNA gene sequences obtained from the studied isolates with those from GenBank, confirming molecular identification (Figure 2).
Construction of the bacterial library of Cavouco
The bacterial library of Cavouco currently includes 21 Gram-negative and four Gram-positive species isolated from five sampling points, stored in frozen stocks under mineral oil. These are the first bacterial isolates of this environment, and the first representatives of the Bacterial Library of Impacted Environments of UFPE.



The analyses showed high water contamination of the Cavouco stream, located in Recife-PE, Brazil, with all samples containing thermotolerant coliforms. The presence of coliforms is a parameter evaluated for water quality monitoring programs and indicates the presence of potentially pathogenic microorganisms (WHO, 2014).
In the present study, the quantification of microbiological parameters indicated that all samples were unfit for human consumption and recreational use. The observed coliform levels were higher than those considered safe by the US Environmental Protection Agency (EPA), 2011 and the Brazilian Environmental Council (Normative Resolution N° 357/2005) (CONAMA, 2005), representing risk for human health.
Indications of significant pollution in the studied regions of the Cavouco stream had been previously observed. Araujo and Oliveira (2013) analyzed Cavouco stream water, and found changes in the levels of dissolved oxygen, ions and ammonia, as well as values of the Index of Water Quality for the Protection of Aquatic Life (IQAPVA) indicating low capacity for the maintenance of aquatic life.
Enterobacteria were present at all sampling points in the present study. Several studies have shown that most enterobacteria  can  cause  infections  because   of   their ability to survive in hostile environments and their ability to develop resistance to antimicrobial drugs (Irenge et al., 2015; Tajbakhsh et al., 2015; Patel et al., 2016).
Several pipes located along the margins of the Cavouco stream discharge residential, hospital, and laboratory effluents into the stream, which explains the presence of bacteria of fecal origin. In addition, the existence of pastures for animal grazing and the development of other activities, together with the poor conservation state of the local riparian forest, could contribute to this microbiological water pollution.
Previous studies of affected environments have reported similar results. Rodrigues et al. (2009), evaluated the water quality of the Piracuama River, located in the state of São Paulo (SP), Brazil, and identified enterobacteria of genera Citrobacter, Edwardsiella, Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella and Shigella. A study performed on a contaminated river in India also identified the presence of important pathogens for public health, namely Edwardsiella spp., Enterobacter spp., E. coli, Morganella spp., Proteus spp., Pseudomonas spp., Serratia spp. and Staphylococcus spp. (Skariyachan et al., 2013), with fecal coliforms being the most common pathogens in contaminated rivers and streams (Kim et al., 2013; Liu et al., 2015).
Escherichia coli was isolated from several sampling points of stream, is used as a specific indicator of fecal contamination in tropical and temperate regions (Páll et al., 2013). This species is considered one of the main causes of diarrhea in adults and children in developing countries, infection most often occurring through contact with contaminated water (Walker et al., 2007; Isidean et al., 2011) and is an important cause of urinary tract and wound infections and pneumonia in immunosuppressed hospitalized patients, and meningitis in newborn children (Wijetunge et al., 2015; Martelius et al., 2016).
Proteus spp. has been described as an important infectious agent in hospital environments (Chen et al., 2014; Murray et al., 2015). Bacteria from the genus, are commonly found in the environment, especially in locations with water pollution and soils with degraded material (Drzewiecka, 2016). A study performed in hospitals located in northeast Brazil, the same region as the present study, isolated P. mirabilis, which produces extended spectrum beta-lactamase (ESBL), have been reported to be an important cause of nosocomial infection in worldwide (Abreu et al., 2011). In the present study, this species was isolated in an area where there is discharge of hospital waste.
The genus Klebsiella is widely distributed in nature and in the gastro intestinal tract of humans and animals.. K. pneumonia can also be found in the oropharynx of hospitalized patients, constituting a source of pulmonary infections, and usually occurring in patients with debilitating conditions such as alcoholism and diabetes (Distel et al., 2013). This species can also infect the urinary tract, cutaneous wounds, and blood, causing bacteremia, meningitis in infants, hepatic abscess, and urinary tract infections (Siu et al., 2012). This species has gained importance due to the development and inter-species and intra-species dis-semination of several antimicrobial resistance mechanisms, namely, the production of beta-lactamases such as ESBLs and KPC (K. pneumoniae Carbapenemase), which degrade beta-lactam antibiotics, frequently detected in hospitalized patients in Recife, PE, Brazil (Lopes et al., 2010; Cabral et al., 2012; Melo et al., 2014).
The presence of the genus Bacillus in the Cavouco stream is worrisome. The presence of Bacillus in food in amounts higher than 10⁶ cells per gram indicates multiplication, and indicates a high health risk (Germano and Germano, 2003). Another interesting aspect was  the observed diversity of Bacillus species. This might indicate that the environmental conditions (nutrients, temperature, humidity, oxygen concentration, and pH) were favorable for the multiplication and maintenance of these species at the studied site.
Bacillus species can sporulate, and the resistance conferred by these spores constitutes an important problem for the epidemiology of associated infections. Because the Cavouco stream is a tributary of the Capibaribe River, which is used as a water source for local agriculture and fishing, the presence of Bacillus could contribute to food contamination.
Of the five sampling points, points 3 and 4 presented the highest microbial diversity, whereas point 5 yielded only one species, P. mirabilis. The absence of diversity at point 5, which receives the discharge of effluents from a morgue and hospital, may have been due to difficulty of isolating Gram-positive bacteria and the lack of selectivity of the blood agar used for bacterial cultures. Another possible explanation for the difficulty in isolating Gram-positive bacteria was the presence of high concentrations of toxic substances in hospital effluents, such as antibiotics, cytostatic agents, heavy metals, disinfectants and hormones, which could have a genotoxic effect on these bacteria (Jean et al., 2012; Devarajan et al., 2015).
Molecular tools were used in the present study to confirm the phenotypic identification of the 25 isolates. The 16S rRNA gene is widely used as phylogenetic marker, and it has been sequenced for a large number of bacterial lineages (Srinivasan et al., 2015). Most of these sequences are deposited in the GenBank database (Benson et al., 2012), and can therefore be compared with sequences of new isolates.
For isolates belonging to different species, their 16S rRNA gene sequences must share less than 97% similarity (Goebel and Stackebrant, 1994). The sequences obtained in the present study shared 98% or higher similarity with sequences deposited in the GenBank database, confirming the phenotypic identification of all isolates, except for S. hominis, which presented 98% similarity with genus Exiguobacterium. This can be explained by the fact that both genera have very similar morphological and biochemical characteristics, such as motility, positive catalase and urease activity, negative phosphatase and coagulase activity, presence or absence of nitrate reduction, and acid production under aerobic conditions, and both contain aerobic and facultative anaerobic species (Stieglmeier et al., 2009). These colonies are spherical, opaque, butyrous or yellow-orange, growing at temperatures varying between 20 and 45°C (Schleifer et al., 1979; Collins et al., 1983). The similar morphological and biochemical characteristics of the two genera might have contributed to its initial identification as Staphylococcus. Similar results were reported by Elmaci et al. (2015), who observed a divergence in identification at the species level for 53.3% (81/152) of the tested lactic  acid bacteria isolates, identified phenotypically using the API CHL method and 16S rRNA gene sequence analysis. Bosshard et al. (2006) observed a discrepancy at the genus and species level for 20.6% (12/58) of the tested non-fermenting Gram-negative bacterial isolates, identified using API 20 NE and 16S rRNA gene sequence analysis. Both studies attributed this divergence to the similarity of morphological and biochemical characteristics of different taxonomic groups, making identification using phenotypical methods difficult, and confirming the specificity of molecular analysis.
The present results indicate that the Cavouco stream could significantly contribute to an increase in microbial pollution, presenting a potential risk of waterborne disease transmission, as genera of pathogenic bacteria were identified. In addition, the current state the Cavouco stream also threatens the conservation of its native species. Environmental cleaning actions such as the establishment of a sewage collection and treatment systems are urgently needed to improve the water quality of this and other affected aquatic environments, such as rivers, lakes and coastal areas. The obtained data enabled the establishment of a bacteria library, which will help to understand the evolution of impacted environment, in terms of environmental quality, overtime. The present study characterized a sample of cultured bacteria isolated from the studied area. In spite of the importance and relevance of this data for public health, it does not represent the totality of organisms present in the studied area. 


The authors have not declared any conflict of interests.


Abreu AG, Marques SG, Monteiro-Neto V, Carvalho RM, Gonçalves AG (2011). Nosocomial infection and characterization of extended-spectrum β-lactamases-producing Enterobacteriaceae in Northeast Brazil. Rev. Soc. Bras. Med. Trop. 44(4):441-446.


APHA-American Public Health Association. Standard Methods for the Examination of Water and Wastewater (2015). Multiple tube fermentation technique for members of the coliform group. American Public Health Association, 22 ed. Washington, DC, USA.


Araujo MC, Oliveira MBM (2013). Monitoramento da qualidade das águas de um riacho da Universidade Federal de Pernambuco, Brasil. Rev. Ambient. Água 8(3):247-257.


Benson DA, Karsch-Mizrachi I, Clark K, Lipman DJ, Ostell J, Sayers EW (2012). GenBank. Nucleic Acids Res. 40 (D1):D48-D53.


Bosshard PP, Zbinden R, Abels S, Böddinghaus B, Altweeg M, Bötter EC (2006). 16S rRNA Gene Sequencing versus API 20 NE System and the VITEK 2 ID-GNB Card for identification of non-fermenting gram-negative bacteria in the clinical laboratory. J. Clin. Microbiol. 44(4):1359-1366.


BRASIL Ministério da Saúde (2015). DATASUS, informações de Saúde- Brasil. 



Cabral AB, Melo RCA, Maciel MA, Lopes AC (2012). Multidrug resistance genes, including bla (KPC) and bla (CTX)-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil. Rev. Soc. Bras. Med. Trop. 45:572-578.


Centers for Disease Control and Prevention- CDC (2015). Global Water, Sanitation, and Hygiene (WASH). 



Chen P, Zhang L, Meng B (2014). Correlation between urinary stones and urinary tract infections. Zhonghua Liu Xing Bing Xue Za Zhi. 35:597-599.


Collins MD, Lund BM, Farrow JAE, Schleifer KH (1983). Chemotaxonomic study of an alkalophilic bacterium, Exiguobacterium aurantiacum gen. nov., sp. nov. J. Gen. Microbiol. 129:2037-2042.


Conselho Nacional do Meio Ambiente (CONAMA) (2005). Resolução 357. Diário Oficial da União, n. 53, 17 de março de 2005, Seção 1. 



Devarajan N, Laffite A, Ngelikoto P, Elongo V, Prabakar K, Mubedi JI, Piana PT, Wildi W, Poté J (2015). Hospital and urban effluent waters as a source of accumulation of toxic metals in the sediment receiving system of the Cauvery River, Tiruchirappalli, Tamil Nadu, India. Environ. Sci. Pollut. Res. Int. 22:12941-12950.


Distel C, Jacobson S, Tille PM (2013). Alcohol induced diabetic ketoacidosis exacerbated by an acute respiratory infection with Klebsiella pneumoniae. Clin. Lab. Sci. 26(2):68-71.


Drzewiecka D (2016). Significance and roles of Proteus spp. bacteria in natural environments. Microb. Ecol. 72(4):741-758.


Elmaci SB, Tokath M, Dursun D, Özçelik F, Åžanlibaba P (2015). Phenotypic and genotypic identification of lactic acid bactéria isolated from traditional pickles of the Çubuk region in Turkey. Folia Microbiol. 60:241-251.


Environmental Protection Agency- EPA (2011). National Characteristics of Drinking Water Systems Serving 10,000 or Fewer People. 



Freitas JHES, Santana KV, Nascimento ACC, Paiva SC, Moura MC, Coelho LCCB et al. (2016). Evaluation of using aluminum sulfate and water-soluble Moringa oleifera seed lectin to reduce turbidity and toxicity of polluted stream water. Chemosphere 163:133-141.


Germano PML, Germano MIS (2003). Higiene E Vigilância Sanitária de Alimentos, Qualidade das Matérias Primas, Doenças transmitidas por alimentos e Treinamento de recursos humanos, 2nd ed. Varela, São Paulo, Brasil. 655p.


Goebel BM, Stackebrandt E (1994). Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments. Appl. Environ. Microbiol. 60(5):1614-1621.


International Joint Commission (2015). The Impact of Urban Development on Water Quality. 



Irenge LM, Kabego L, Kinunu FB, Itongwa M, Mitangala PN, Gala JL, Chirimwami RB (2015). Antimicrobial resistance of bacteria isolated from patients with bloodstream infections at a tertiary care hospital in the Democratic Republic of the Congo. S. Afr. Med. J. 105(9):752-755.


Isidean SD, Riddle MS, Savarino SJ, Porter CK (2011). Porter A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression. Vaccine 29:6167-6178.


Jean J, Perrodin Y, Pivot C, Trepo D, Perraud M, Droguet J, Tissot-Guerraz F, Locher F (2012). Identification and prioritization of bioaccumulable pharmaceutical substances discharged in hospital effluents. J. Environ. Manage.103:113-121.


Kim JY, Lee H, Lee JE, Chung MS, Ko GP (2013). Identification of human and animal fecal contamination after rainfall in the Han River, Korea. Microbes Environ. 28:187-194.


Koneman EW, Winn WC (2006). Color Atlas and Textbook of Diagnostic Microbiology, 6th edn. Williams & Wilkins, Philadelphia: Lippincott-Raven Publishers. 1565p.


Liu WC, Chan WT, Young CC (2015). Modeling fecal coliform contamination in a tidal Danshuei River estuarine system. Sci. Total. Environ. 502:632-640.


Lopes AC, Veras DL, Lima AM, Melo RD, Ayala J (2010). bla(CTX-M-2) and bla(CTX-M-28) extended-spectrum beta-lactamase genes and class 1integrons in clinical isolates of Klebsiella pneumoniae from Brazil. Mem. Inst. Oswaldo Cruz. 105:163-167.


Martelius T, Jalava J, Kärki T, Möttönen T, Ollgren J, Lyytikäinen O (2016). Nosocomial bloodstream infections caused by Escherichia coli and Klebsiella pneumoniae resistant to third-generation cephalosporins, Finland, 1999-2013: Trends, patient characteristics and mortality. Infect. Dis.(Lond). 48:229-234.


Melo RCA, Barros EM, Loureiro NG, de Melo HR, Maciel MA, Lopes AC (2014). Presence of fimH, mrkD, and irp2 virulence genes in KPC-2-producing Klebsiella pneumoniae isolates in Recife-PE, Brazil. Curr. Microbiol. 69:824-831.


Murray EC, Marek A, Thomson PC, Coia JE (2015). Gram-negative bacteraemia in haemodialysis. Nephrol. Dial. Transplant. 30(7):1202-1208.


Páll E, Niculae M, Kiss T, Åžandru CD, Spînu M (2013). Human impact on the microbiological water quality of the rivers. J. Med. Microbiol. 62:1635-1640.


Patel CB, Shanker R, Gupta VK, Upadhyay RS (2016). Q-PCR based culture-independent enumeration and detection of Enterobacter: an emerging environmental human pathogen in riverine systems and potable Water. Front. Microbiol. 7:172.


Ramírez-Castillo FY, Loera-Muro A, Jacques M, Garneau P, Avelar-González FJ, Harel J, Guerrero-Barrera AL (2015). Waterborne pathogens: Detection methods and challenges. Pathogens 4(2):307-334.


Rodrigues JDD, Jorge AOC, Ueno M (2009). Avaliação da qualidade das águas de dias áreas utilizadas para recreação do Rio Piraciama-SP. Revista Biociências. 15:88-94.


Sambrook J, Russell DW (2001). Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory; 3 ed.


Schleifer KH, Meyer SA, Rupprecht M (1979). Relatedness among coagulase-negative staphylococci: Deoxyribonucleic acid reassociation and comparative immunological studies. Arch. Microbiol. 122:93-101.


Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY (2012). Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect. Dis. 12:881-887.


Skariyachan S, Lokesh P, Rao R, Kumar AU, Vasist KS, Narayanappa R(2013).A pilot study on water pollution and characterization of multidrugresistant superbugs from Byramangala tank,Ramanagara district, Karnataka, India. Environ. Monit. Assess. 185:5483-5495.


Srinivasan R, Karaoz U, Volegova M, MacKichan J, Kato-Maeda M, Miller S, Nadarajan R, Brodie EL, Lynch SV (2015). Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS ONE 10:e0117617.


Stieglmeier M, Wirth R, Kminek G, Moissl-Eichinger C (2009). Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms. Appl. Environ. Microbiol. 75(11):3484-3491.


Tajbakhsh E, Tajbakhsh S, Khamesipour F (2015). Isolation and molecular detection of Gram negative bacteria causing urinary tract infection in patients referred to Shahrekord hospitals, Iran. Iran Red Crescent Med. J. 17(5):e24779.


Tanchou V (2014). Review of Methods for the Rapid Identification of Pathogens in Water Samples—ERNCIP Thematic A = πr2 Chemical & Biological Risks in the Water Sector—Task 7, Deliverable 1. Institute for the Protection and Security of the Citizen Publications Office of the European Union, 



USGS-Science for a Changing World (2015). The Effects of Urbanization on Water Quality: Waterborne Pathogens. 



Walker RI, Steele D, Aguado T, Ad Hoc ETEC Technical Expert Committee (2007). Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 25:2545-2566.


Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991). 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703.


WHO (2014). Water Sanitation and Health. 


WHO and UNICEF (2015). Lack of sanitation for 2.4 billion people undermining health improvements. 



Wijetunge DS, Gongati S, DebRoy C, Kim KS, Couraud PO, Romero IA, Weksler B, Kariyawasam S (2015). Characterizing the pathotype of neonatal meningitis causing Escherichia coli (NMEC). BMC Microbiol. 15(1):211.


Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen KY (2008). Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bactéria in clinical microbiology. Clin. Microbiol. Infect. 14:908-934.