Determination of Pathogens in Greywater

  • Adel Ali Saeed Al-Gheethi
  • Efaq Ali Noman
  • Radin Maya Saphira Radin Mohamed
  • Amir Hashim Mohd Kassim
Part of the Water Science and Technology Library book series (WSTL, volume 87)


There are many methods used for isolation and the enumeration of pathogenic organisms. The direct methods depend on the culture medium and microscopic examination. However, these techniques are not effective for all pathogenic organisms in the environment since many organisms require a specific condition to grow on the culture medium. Therefore, the using of enrichment methods might exhibit more efficiency in the determination of pathogens from the wastewater samples. The main challenge in the microbiological assessment of greywater lies in finding the most effective method to detect the presence or absence of pathogens which are available in low concentrations. In this chapter, traditional methods including direct culture and enrichment methods are reviewed.


Culture methods Greywater Pathogens Antibiotics resistant bacteria 



The authors wish to thank the Ministry of Higher Education (MOHE) for supporting this research under FRGS vot 1574 and also the Research Management Centre (RMC) UTHM for providing grant IGSP U682 for this research.


  1. Al-Gheethi AA, Norli I, Lalung J, Azieda T, Ab Kadir MO (2013) Reduction of faecal indicators and elimination of pathogens from sewage treated effluents by heat treatment. Caspian J Appl Sci Res 2(2):29–45Google Scholar
  2. Al-Gheethi AA, Abdul-Monem MO, AL-Zubeiry AHS, Al-Amery R, Efaq AN, Shamar AM (2014) Effectiveness of selected wastewater treatment plants in Yemen for reduction of faecal indicators and pathogenic bacteria in secondary effluents and sludge. Water Pract Technol 9(3):293–306CrossRefGoogle Scholar
  3. Al-Gheethi AA, Aisyah M, Bala JD, Efaq AN, Norli I (2015a) Prevalence of antimicrobial resistance bacteria in non-clinical environment 4th International Conference on Environmental Research and Technology (ICERT 2015) on 27–29 May 2015 at Parkroyal Resort, Penang, MalaysiaGoogle Scholar
  4. Al-Gheethi AA, Lalung J, Efaq AN, Bala JD, Norli I (2015b) Removal of heavy metals and β-lactam antibiotics from sewage treated effluent by bacteria. Clean Technol Environ Policy 17(8):2101–2123CrossRefGoogle Scholar
  5. Al-Gheethi AA, Mohamed RM, Efaq AN, Amir HK (2016) Reduction of microbial risk associated with greywater utilized for irrigation. Water Health J 14(3):379–398CrossRefGoogle Scholar
  6. APHA (1999) Coliforms-total, faecal and E. coli, Method 8074, m-Endo. Standard methods for the examination of water and wastewater, American Public Health Association, American Water Works Association, Water Environment Federation. 9221 B, 9222 B, 9225B, 9230C. Adopted by U.S. EPA. DOC316.53.001224Google Scholar
  7. Audra M, Andrew J (2003). Fate of a representative pharmaceutical in the environment. Final Report Submitted to Texas Water Resources InstituteGoogle Scholar
  8. Benson JH (2005) Microbiological applications: laboratory manual in general microbiology, 7th edn. WCB/McGraw-Hill Co., New York, USAGoogle Scholar
  9. Bhat P, Rajan D (1975) Comparative evaluation of desoxycholate citrate medium and xylose lysine desoxycholate medium in the isolation of Shigellae. Am J Clin Pathol 64(3):399–404CrossRefGoogle Scholar
  10. Brichta-Harhay DM, Arthur TM, Bosilevac JM, Guerini MN, Kalchayanand N, Koohmaraie M (2007) Enumeration of Salmonella and Escherichia coli O157: H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. J Appl Microbiol 103(5):1657–1668CrossRefGoogle Scholar
  11. Brichta-Harhay DM, Guerini MN, Arthur TM, Bosilevac JM, Kalchayanand N, Shackelford SD, Koohmaraie M (2008) Salmonella and Escherichia coli O157: H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: an evaluation of prevalence and bacterial loads by immunomagnetic separation and direct plating methods. Appl Environ Microbiol 74(20):6289–6297CrossRefGoogle Scholar
  12. Broekema NM, Van TT, Monson TA, Marshall SA, Warshauer DM (2009) Comparison of cefoxitin and oxacillin disk diffusion methods for detection of mecA mediated resistance in Staphylococcus aureus in a large scale study. J ClinMicrobiol 47:217–219Google Scholar
  13. Cassar R, Cuschieri P (2003) Comparison of Salmonella chromogenic medium with DCLS agar for isolation of Salmonella species from stool specimens. J ClinMicrobiol 41(7):3229–3232Google Scholar
  14. Castillo A, Villarruel-López A, Navarro-Hidalgo V, Martínez-González NE, Torres-Vitela MR (2006) Salmonella and Shigella in freshly squeezed orange juice, fresh oranges, and wiping cloths collected from public markets and street booths in Guadalajara, Mexico: incidence and comparison of analytical routes. J Food Protect 69(11):2595–2599CrossRefGoogle Scholar
  15. Choi YW, Hyde KD, Ho WH (1999) Single spore isolation of fungi. Fungal Divers 3:29–38Google Scholar
  16. Chunduri NS, Madasu K, Goteki VR, Karpe T, Reddy H (2012) Evaluation of bacterial spectrum of orofacial infections and their antibiotic susceptibility. Ann Maxillofac Sur 2(1):46CrossRefGoogle Scholar
  17. Collee J, Duguid JP, Fraser AG, Marmion BP (1989) Practical medical microbiology, 13th edn. Longman-FE. Ltd, LondonGoogle Scholar
  18. Dudley DJ, Guentzel MN, Ibarra MJ, Moore BE, Sagik BP (1980) Enumeration of potentially pathogenic bacteria from sewage sludges. Appl Environ Microbiol 39(1):118–126Google Scholar
  19. Dunn C, Martin WJ (1971) Comparison of media for isolation of Salmonellae and Shigellae from fecal specimens. ApplMicrobiol 22(1):17–22Google Scholar
  20. El-Lathy AM, El-Taweel GE, El-Sonosy MW, Samhan FA, Moussa TA (2009) Determination of pathogenic bacteria in wastewater using conventional and PCR techniques. Environ Biotechnol 5:73–80Google Scholar
  21. El-Taweel GE, Ali GH (2000) Evaluation of roughing and slow sand filters for water treatment. Water Air Soil Pollut 120(1):21–28CrossRefGoogle Scholar
  22. Emine S, Kambol R, Zainol N (2010) Morphological characterization of soil Penicillium sp. strains—potential producers of statin. In: Biotechnology Symposium IV, 1–3 Dec 2010, Universiti Malaysia Sabah, Sabah, MalaysiaGoogle Scholar
  23. Erickson MC, Islam M, Sheppard C, Liao J, Doyle MP (2004) Reduction of Escherichia coli O157: H7 and Salmonella enterica serovar enteritidis in chicken manure by larvae of the black soldier fly. J Food Protect 67(4):685–690CrossRefGoogle Scholar
  24. Friedler E, Kovalio R, Galil NI (2005) On-site greywater treatment and reuse in multi-storey buildings. Water Sci Technol 51(10):187–194Google Scholar
  25. Gilchrist JE, Campbell JE, Donnelly CB, Peeler JT, Delaney JM (1973) Spiral plate method for bacterial determination. Appl Microbiol 25(2):244–252Google Scholar
  26. Götz F, Bannerman T, Schleifer KH (2006) The genera Staphylococcus and Micrococcus. In: The prokaryotes. Springer, US, pp 5–75CrossRefGoogle Scholar
  27. Guarro J, Gene J, Stchigel AM (1999) Developments in fungal taxonomy. Clin Microbiol Rev 12(3):454–500Google Scholar
  28. HPA (2004) Health protection agency, enumeration of Staphylococcus aureus by membrane filtration. National Standard Method W 10 Issue 3 (2004).
  29. Iveson JB (1973) Enrichment procedures for the isolation of Salmonella, Arizona, Edwardsiella and Shigella from faeces. Epidemiol Infect 71(2):349–361Google Scholar
  30. Jørgensen F, Bailey R, Williams S, Henderson P, Wareing DRA, Bolton FJ, Humphrey TJ (2002) Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods. Int J Food Microbiol 76(1):151–164CrossRefGoogle Scholar
  31. Kelly S, Cormican M, Parke L, Corbett-Feeney G, Flynn J (1999) Cost-effective methods for isolation of Salmonella enterica in the clinical laboratory. J Clin Microbiol 37(10):3369Google Scholar
  32. King S, Metzger WI (1968) A new plating medium for the isolation of enteric pathogens I. Hektoen Enteric Agar. Appl Microbiol 16(4):577–578Google Scholar
  33. Létourneau V, Nehmé B, Mériaux A, Massé D, Cormier Y, Duchaine C (2010) Human pathogens and tetracycline-resistant bacteria in bioaerosols of swine confinement buildings and in nasal flora of hog producers. Int J Hygiene Environ Health 213(6):444–449CrossRefGoogle Scholar
  34. Lewis K (2008) Multidrug tolerance of biofilms and persister cells. In: Bacterial Biofilms. Springer, Berlin, Heidelberg, pp 107–131Google Scholar
  35. Maddocks S, Olma T, Chen S (2002) Comparison of CHROM agar Salmonella medium and xylose-lysine-desoxycholate and Salmonella-Shigella agars for isolation of Salmonella strains from stool samples. J Clin Microbiol 40(8):2999–3003CrossRefGoogle Scholar
  36. Morello JA, Granato PA, Mizer HE (2003) Laboratory manual and work book in microbiology application to patient care, 7th edn. The McGraw − Hill CompaniesGoogle Scholar
  37. Morris RF, Fulton WC (1970) Heritability of diapause intensity in Hyphantria cunea and correlated fitness responses. Can Entomol 102(8):927–938CrossRefGoogle Scholar
  38. Muniesa M, Blanch AR, Lucena F, Jofre J (2005) Bacteriophages may bias outcome of bacterial enrichment cultures. Appl Environ Microbiol 71(8):4269–4275CrossRefGoogle Scholar
  39. Nagvenkar GS, Ramaiah N (2009) Abundance of sewage-pollution indicator and human pathogenic bacteria in a tropical estuarine complex. Environ Monit Assess 155(1–4):245CrossRefGoogle Scholar
  40. Niederstebruch N, Sixt D (2013) Standard nutrient agar 1 as a substitute for blood-supplemented Müller-Hinton agar for antibiograms in developing countries. Eur J Clin Microbiol Infect Dis 32(2):237–241CrossRefGoogle Scholar
  41. Noman EA, Al-Gheethi AA, Rahman NNNA, Nagao H, Kadir MA (2016) Assessment of relevant fungal species in clinical solid wastes. Environ Sci Pollut Res 23(19):19806–19824CrossRefGoogle Scholar
  42. Nye KJ, Fallon D, Frodsham D, Gee B, Graham C, Howe S, Warren RE (2002) An evaluation of the performance of XLD, DCA, MLCB, and ABC agars as direct plating media for the isolation of Salmonella enterica from faeces. J ClinPathol 55(4):286–288Google Scholar
  43. Ogbulie JN, Adieze IE, Nwankwo NC (2008) Susceptibility pattern of some clinical bacterial isolates to selected antibiotics and disinfectants. Polish J Microbiol 57:199–204Google Scholar
  44. Ongeng D, Muyanja C, Geeraerd AH, Springael D, Ryckeboer J (2011) Survival of Escherichia coli O157: H7 and Salmonella enterica serovar Typhimurium in manure and manure-amended soil under tropical climatic conditions in Sub-Saharan Africa. J Appl Microbiol 110(4):1007–1022CrossRefGoogle Scholar
  45. Pant A, Mittal AK (2008) New protocol for the enumeration of Salmonella and Shigella from wastewater. J Environ Eng 134(3):222–226CrossRefGoogle Scholar
  46. Park JE, Ahn TS, Lee HJ, Lee YO (2006) Comparison of total and faecal coliforms as faecal indicator in eutrophicated surface water. Water Sci Technol 54(3):185–190CrossRefGoogle Scholar
  47. Patra AK, Acharya BC, Mohapatra A (2009) Occurrence and distribution of bacterial indicators and pathogens in coastal waters of Orissa. Indian J Mar Sci 38(4):474–480Google Scholar
  48. Petkar A, Alali WQ, Harrison MA, Beuchat LR (2011) Survival of Salmonella in organic and conventional broiler feed as affected by temperature and water activity. Agric Food Anal Bacteriol 1:175–185Google Scholar
  49. Pollock HM, Dahlgren BJ (1974) Clinical evaluation of enteric media in the primary isolation of Salmonella and Shigella. Appl Microbiol 27(1):197–201Google Scholar
  50. Prescott H (2002). Laboratory exercises in microbiology, 5th edn. The McGraw-Hill CompaniesGoogle Scholar
  51. Promputtha I, Jeewon R, Lumyong S, McKenzie EHC, Hyde KD (2005) Ribosomal DNA fingerprinting in the identification of non sporulating endophytes from Magnolia liliifera (Magnoliaceae). Fungal Divers 20:167–186Google Scholar
  52. Rodrigues V, Ramaiah N, Kakti S, Samant D (2011) Long-term variations in abundance and distribution of sewage pollution indicator and human pathogenic bacteria along the central west coast of India. Ecol Indic 11(2):318–327CrossRefGoogle Scholar
  53. Shaban AM, El-Taweel GE (1999) Prevaience of Listeria and Listeria monocytogenes in certain aquatic environments in Egypt. Egypt J Microbiol 34(1):67–78Google Scholar
  54. Stone GG, Oberst RD, Hays MP, McVey S, Chengappa MM (1994) Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure. J Clin Microbiol 32(7):1742–1749Google Scholar
  55. Sugumar G, Mariappan S (2003) Survival of Salmonella sp. in freshwater and seawater microcosms under starvation. Asian Fish Sci 16(3/4):247–256Google Scholar
  56. Synnott AJ, Kuang Y, Kurimoto M, Yamamichi K, Iwano H, Tanji Y (2009) Isolation from sewage influent and characterization of novel Staphylococcus aureus bacteriophages with wide host ranges and potent lytic capabilities. Appl Environ Microbiol 75(13):4483–4490CrossRefGoogle Scholar
  57. Tao XQ, Lu GN, Dang Z, Yang C, Yi XY (2007) A phenanthrene-degrading strain Sphingomonas sp. GY2B isolated from contaminated soils. Process Biochem 42(3):401–408CrossRefGoogle Scholar
  58. Thompson JM, Gündoğdu A, Stratton HM, Katouli M (2013) Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin-resistant Staphylococcus aureus (MRSA). J Appl Microbiol 114(1):44–54CrossRefGoogle Scholar
  59. U.S. EPA (2003) Control of pathogens and vector attraction in sewage sludge; 40 CFR Part 503. U.S. Environmental Protection Agency, Cincinnate, OH 45268Google Scholar
  60. U.S. EPA (2006a) Method 1103.1, E. coli in water by membrane filtration using membrane-thermotolerant Escherichia coli Agar (mTEC). U.S. Environmental Protection Agency. Office of Water (4303T) 1200 Pennsylvania Avenue, N W. Washington, USAGoogle Scholar
  61. U.S. EPA (2006b) Method 1106.1: Enterococci in water by membrane filtration using membrane-Enterococcus-Esculin Iron Agar (mE-EIA). U.S. Environmental Protection Agency. Office of Water (4303T) 1200 Pennsylvania Avenue, N W. Washington, USAGoogle Scholar
  62. Vila J, Gascon J, Abdalla S, Gomez J, Marco F, Moreno A, De Anta TJ (1994) Antimicrobial resistance of Shigella isolates causing traveler’s diarrhea. Antimicrob Agent Chemother 38(11):2668–2670CrossRefGoogle Scholar
  63. Walsh F (2013) Investigating antibiotic resistance in non-clinical environments. Front Microbiol 4:19Google Scholar
  64. Warren BR (2006) Improved sample preparation for the molecular detection of Shigella sonnei in foods (Doctoral dissertation, University of Florida)Google Scholar
  65. Wiggins BA (1996) Discriminant analysis of antibiotic resistance patterns in faecal streptococci, a method to differentiate human and animal sources of faecal pollution in natural waters. Appl Environ Microbiol 62(11):3997–4002Google Scholar
  66. Zhang S (2005) Development of a biosensor for the rapid detection of Salmonella Typhimurium in milk. PhD thesis, Auburn University, AlabamaGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2019

Authors and Affiliations

  • Adel Ali Saeed Al-Gheethi
    • 1
  • Efaq Ali Noman
    • 2
    • 3
  • Radin Maya Saphira Radin Mohamed
    • 1
  • Amir Hashim Mohd Kassim
    • 1
  1. 1.Department of Water and Environmental Engineering, Faculty of Civil and Environmental EngineeringMicro-Pollutant Research Centre (MPRC), Universiti Tun Hussein Onn Malaysia (UTHM)Parit Raja, Batu PahatMalaysia
  2. 2.Faculty of Applied Sciences and Technology (FAST)Universiti Tun Hussein Onn Malaysia (UTHM)PagohMalaysia
  3. 3.Department of Applied Microbiology, Faculty Applied SciencesTaiz UniversityTaizYemen

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