Original Article

The BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water


Background and Objectives: Public health protection requires timely evaluation of pathogens in potable water to minimize outbreaks caused by microbial contaminations. The present study was aimed at assessing the microbiological quality of water obtained from Shantinagar (a rural area in the South Goa region of Goa, India) using 5-Bromo-4-Chloro-3-Indoxyl β-D-glucuronide-Sorbitol MacConkey agar (BCIG-SMAC) medium and, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for differential detection and quantification of viable Escherichia coli cells in water samples.
Materials and Methods: Membrane filtration method was used for both BCIG-SMAC medium and PMA-qPCR methods. To determine the efficiency of detection of viable cells, we first evaluated the PMA treatment protocol and established the standard calibration curves using previously reported primers.
Results: PMA-qPCR detected as low as 7 femtograms of DNA of E. coli per qPCR reaction whereas the limit of detection (LOD) of BCIG-SMAC medium was 1.8 CFU/100mL. A total of 71 water samples spanning 2017-2018 have been analyzed using BCIG-SMAC medium and PMA-qPCR, of which 95.77% (68/71) and 7.04% (5/71) were found to be total E. coli and E. coli O157:H7, respectively. PMA-qPCR study showed the viable counts of total viable E. coli cells ranging from 3 CFU/100mL to 8.2×102 CFU/100mL. The total E. coli CFU/100mL quantified by PMA-qPCR significantly exceeded (paired t-test; P<0.05) the number on BCIG-SMAC medium.
Conclusion: The present study indicates that the microbiological quality of environmental water samples analyzed do not comply with the regulatory standard. Therefore, special attention is warranted to improve the overall portable quality of water in the perspective of public health.

1. UNICEF Somalia - Water, Sanitation and Hygiene - Priority issues. Retrieved August 2019. https://www.unicef.org/somalia/water-sanitation-and-hygiene
2. Ramírez-Castillo FY, Loera-Muro A, Jacques M, Garneau P, Avelar-González FJ, Harel J, et al. Waterborne pathogens: detection methods and challenges. Pathogens 2015; 4: 307-334.
3. Deshmukh RA, Joshi K, Bhand S, Roy U. Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview. MicrobiologyOpen 2016; 5: 901-922.
4. March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 1986; 23: 869-872.
5. Deshmukh RA, Bhand S, Roy U. A novel molecular quantitative method for rapid and sensitive detection of Escherichia coli from roof-harvested rainwater. Anal Methods 2019; 11: 3155-3167.
6. Maheux AF, Bissonnette L, Boissinot M, Bernier JL, Huppé V, Picard FJ, et al. Rapid concentration and molecular enrichment approach for sensitive detection of Escherichia coli and Shigella species in potable water samples. Appl Environ Microbiol 2011; 77: 6199-6207.
7. Wang S, Levin RE. Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. J Microbiol Methods 2006; 64: 1-8.
8. Oliver DM, Bird C, Burd E, Wyman M. Quantitative PCR profiling of Escherichia coli in livestock feces reveals increased population resilience relative to culturable counts under temperature extremes. Environ Sci Technol 2016; 50: 9497-9505.
9. Li B, Liu H, Wang W. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli. BMC Microbiol 2017; 17: 215.
10. Nocker A, Camper AK. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 2006; 72: 1997-2004.
11. Alvarez G, González M, Isabal S, Blanc V, León R. Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide. AMB Express 2013; 3: 1.
12. Elizaquível P, Sánchez G, Selma MV, Aznar R. Application of propidium monoazide- qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water. Food Microbiol 2012; 30: 316-320.
13. Hellein KN, Kennedy EM, Harwood VJ, Gordon KV, Wang SY, Lepo JE. A filter-based propidium monoazide technique to distinguish live from membrane-compromised microorganisms using quantitative PCR. J Microbiol Methods 2012; 89: 76-78.
14. Nocker A, Cheung CY, Camper AK. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 2006; 67: 310-320.
15. Taskin B, Gozen AG, Duran M. Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification. Appl Environ Microbiol 2011; 77: 4329-4335.
16. Nocker A, Sossa-Fernandez P, Burr MD, Camper AK. Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 2007; 73: 5111-5117.
17. Li B, Chen JQ. Real-time PCR methodology for selective detection of viable Escherichia coli O157: H7 cells by targeting Z3276 as a genetic marker. Appl Environ Microbiol 2012; 78: 5297-5304.
18. Khan IU, Gannon V, Kent R, Koning W, Lapen DR, Miller J, et al. Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds. J Microbiol Methods 2007; 69: 480-488.
19. Picard FJ, Gagnon M, Bernier MR, Parham NJ, Bastien M, Boissinot M, et al. Internal control for nucleic acid testing based on the use of purified Bacillus atrophaeus subsp. globigii spores. J Clin Microbiol 2009; 47: 751-757.
20. Walker DI, McQuillan J, Taiwo M, Parks R, Stenton CA, Morgan H, et al. A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters. Water Res 2017; 126: 101-110.
21. Ibekwe AM, Grieve CM. Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR. J Appl Microbiol 2003; 94: 421-431.
22. Luna GM, Dell'Anno A, Pietrangeli B, Danovaro R. A new molecular approach based on qPCR for the quantification of fecal bacteria in contaminated marine sediments. J Biotechnol 2012; 157: 446-453.
23. Edberg SC, Rice EW, Karlin RJ, Allen MJ. Escherichia coli: the best biological drinking water indicator for public health protection. Symp Ser Soc Appl Microbiol 2000; (29): 106S-116S.
24. Khatri N, Tyagi S. Influences of natural and anthropogenic factors on surface and groundwater quality in rural and urban areas. Front Life Sci 2015; 8: 23-39.
25. Lenart-Boroń A, Wolanin A, Jelonkiewicz E, Żelazny M. The effect of anthropogenic pressure shown by microbiological and chemical water quality indicators on the main rivers of Podhale, Southern Poland. Environ Sci Pollut Res Int 2017; 24: 12938-12948.
IssueVol 13 No 5 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v13i5.7427
Coliforms; Detection; Escherichia coli; Pathogens; Propidium monoazide; Public health

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Deshmukh R, Bhand S, Roy U. The BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water. Iran J Microbiol. 2021;13(5):624-631.