Hokajärvi, A.-M.

Ikonen, J.

Miettinen, I. T.

Räsänen, P. S.

Julkari

THE MOLECULAR DETECTION FOR THE VIABILITY OF YERSINIA CELLS 1 DURING SHOCK CHLORINATION – A PILOT SCALE STUDY  2  3  4  5  6  7 P.S. Räsänen*, J. Ikonen, A.-M. Hokajärvi and I.T. Miettinen 8  9  10 Water and Health unit, National Institute for Health and Welfare, P.O. Box 95, FI-70701 11 Kuopio, FINLAND 12  13 * Corresponding author. Tel.: +358 29 524 6449; E-mail address: pia.rasanen@thl.fi 14  15  16  17  1 INTRODUCTION 18  19 Efficient decontamination of pipeline system is an essential action in situations where a 20 drinking water distribution system is contaminated. The challenge in decontamination is 21 the fact that biofilms may protect faecal and pathogenic microbes against disinfectant after 22 contamination event.1,2 The one of the most frequently used cleaning method for drinking 23 water distribution networks is clorination.3,4,5,6 Shock-chlorination can be used for 24 purification of microbial contaminated water distribution system in waterborne outbreaks. 25 As an example, shock-chlorination was applied to clean the network after the severe 26 waterborne outbreak in the Nokia city, Finland year 2007.5,7  27      It is also important to detect pathogenic microbes from water and biofilm samples as 28 fast and efficient as possible if suspected contamination. The rapid and specific detection 29 techniques for pathogenic bacteria could save valuable time and may reduce illness cases 30 during an emergency situation. It also enables the verification of the success in the 31 cleaning procedure.8 32      The new molecular biological techniques allow detection of viability of target 33 pathogens as combination of Propidium monoazide (PMA)9 treatment with quantitative 34 polymerase chain reaction (qPCR) and Direct Viable Count (DVC) cell elongation with 35 Peptide Nucleic Acid probes and Fluorescence in situ Hybridization (PNA-FISH).10 36 Moreover, the traditional cultivation could take several days to weeks for reporting final 37 results6 when PCR and FISH based assays can provide results faster.8,11,12 In addition, the 38 FISH and PCR based techniques have been reported to be able to observe pathogenic 39 bacteria from water and biofilm samples when the traditional cultivation method was not 40 able to detect them.13, 8  41      The aim of this study was to test how a pilot water distribution network can be cleaned 42 after contamination with bacteria using shock-chlorination.  43  44  45 2 THE EXPERIMENTAL SET-UP 46  47 The pilot distribution system consisted of a 400m long PEX plastic pipeline. Water flow in 48 the system was 1.8 l/min. The quality of water and growth of the biofilms was followed for 49 a period of one month prior the contamination phase. The progress of decontamination was 50 followed with the chlorine concentration, DAPI staining and heterotrophic plate counts 1 (HPC). The chlorine concentration used for shock-chlorination was 10 mg Cl2/l. During the 2 experiment two parallel outlet water and three biofilm samples were took in each sampling 3 point. 4      The experiments were carried out using Yersinia pseudotuberculosis strain, the closest 5 relative surrogate bacteria for severe human bacterial pathogen Yersinia pestis. The effect 6 of shock-chlorination for microbiological agents was followed with new molecular biology 7 techniques such as PMA-PCR and PNA-DVC-FISH assays for the determination of 8 viability of the surrogate bacteria. A photoreactive PMA compound cannot pass through 9 the cell membrane of a viable bacterial cell. When a cell membrane is damaged, the PMA 10 gets into the cell and covalently binds to the cell genome. The attached PMA inhibits real-11 time PCR reactions which allow observing differences between viable and damaged 12 cells.9,14 The combination of DVC-FISH technique includes enrichment of samples in the 13 presence of an antibiotic.10,15 The antibiotic prevents normal cell division leading to 14 elongation of viable cells.16 A fluorescent PNA probe can bind as a sequence specific for 15 ribosomal RNA of target cells. The successful binding between the PNA probe and 16 specific target can microscopically be observed. When the fluorescent elongated cells 17 could be detected the bacterial viability state can be estimated.17 In addition, the Yersinia 18 selective cefsulodin-Irgasan-novobiocin (CIN) plate counting was used as parallel with the 19 new molecular biological techniques for detection of cultivable Yersinia cells. 20  21  22  3 RESULTS 23  24 The concentration and viability of Yersinia pseudotuberculosis cells decreased rapidly in 25 water and biofilm samples after start of the chlorination (Figure 1 and 2) as well as the 26 counts of heterotrophic plate count and total microbial counts. In biofilms, the FISH counts 27 of Yersinia were reduced by approximately 5 log and with qPCR 4 log. 28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 Figure 1 The viable/ VBNC and total cell concentration in biofilm samples. 46  47  48  49  50  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15 Figure 2  The viable/ VBNC and total cell concentration in water samples. 16  17 In water, the FISH and qPCR counts of Yersinia were reduced by approximately 7 log. The 18 new PCR and FISH techniques were able to detect Yersinia cells (total counts/ viable 19 counts) while the selective culture method could not found any vegetative bacterial cells.  20  21  22  4 CONCLUSIONS  23  24 In this study it was found out that the new qPCR and FISH based detection techniques 25 were usable for determination of viability of the Yersinia pseudotuberculosis cells. 26 According to previous studies, it has been stated that traditional FISH and PCR techniques 27 could not separate live, viable but non-cultivable (VBNC) or dead cells from live 28 culturable cells and that conventional cultivation based methods should be used for the 29 detection of live, cultivable bacterial cells.6,18 It is known that chlorine damages nucleic 30 acids19  which may affect the results of nucleic acid based FISH and qPCR methods. Over-31 all, this study indicated that the shock-chlorination appeared to be an effective technique 32 for cleaning a water distribution network after a bacterial contamination. However, it is 33 obvious that some of the Yersinia cells introduced in the pipeline may have washed away 34 from the pipeline network during the experiment. This might explain part of the rapid 35 decrease in Yersinia cells in the water and biofilm samples. Therefore it is difficult to 36 assess the true inactivation efficiency of shock-chlorination for the removal of the 37 microbial contaminant. 38  39  40 Acknowledgements 41  42 This work has been done undertaken as part of a research project which is supported by the 43 European Union within the Seventh Framework Programme (FP/2001-2011), Security 44 Theme, “Increasing the security of citizens”, contract no 217976. There hereby follows a 45 disclaimer stating that the authors are solely responsible for the work, it does not represent 46 the opinion of the Community and the Community is not responsible for any use that might 47 be made of data appearing herein. 48  49  50  1  2 References 3  4 1  U. Szewzyk, R. Szewzyk, W. Manz and K.H. Schleifer, Annu. Rev. Microbiol., 2000, 5 54, 81. 6 2 N. Trachoo, J.F. Frank and N.J. Stern, J. Food Prot., 2002, 65, 1110. 7 3  W.H. Farland and H.J. Gibb, ‘U.S. perspective on balancing chemical and 8 microbiological risks of disinfection’ in ILSI Press, ed., G.C. Craun, Washington, 9 D.C., 1993. 10 4  G.C. White, Van Nostrand Reinhold Company Inc., New York, 1986. 11 5  O. Zacheus and I.T. Miettinen, J. Water Health, 2011, 9, 763. 12 6  T. Pitkänen, J. Bräcker, I.T. Miettinen, A. Heitto, J. Pesola and E. Hakalehto, Can. J. 13 Microbiol., 2009, 55, 849. 14 7 T. Pitkänen, I.T. Miettinen, U.-M. Nakari, J. Takkinen, K. Nieminen, A. Siitonen, M. 15 Kuusi, A. Holopainen and M.-L. Hänninen, J. Water Health, 2008, 6, 365.  16 8 R. Rimhanen-Finne, M.-H. Hänninen, R. Vuento, J. Laine, T.S. Jokiranta, M. 17 Snellman, T. Pitkänen, I. Miettinen and M. Kuusi, Scand. J. Infect. Dis., 2010, 42, 18 613. 19 9 A. Nocker and A.K. Camper, Appl. Environ. Microbiol., 2006, 72, 1997. 20 10 J. Baudart and P. Lebaron, J. Appl. Microbiol., 2010, 109, 1253. 21 11  C. Almeida, N.F. Azevedo, R.M. Fernandes, C.W. Keevil and M.J. Vieira, Appl. 22 Environ. Microbiol., 2010, 76, 4476. 23 12 S. Yang and R.E. Rothman, Lancet Infect. Dis., 2004, 4, 337. 24 13 M.J. Lehtola, T. Pitkänen, L. Miebach and I.T. Miettinen, Water Sci. Technol., 2006, 25 54, 57. 26 14 A. Nocker and A.K. Camper, J. Microbiol. Methods, 2006, 67, 310. 27 15 T. Juhna, D. Birzniee, S. Larsson, D., Zulenkovz, A. Sharipo, N.F. Azevedo, F. 28 Ménard-Szczebara, S. Castagnet, C. Féliers and C.W. Keevil, Appl. Environ. 29 Microbiol., 2007, 73, 7456. 30 16 S. Larsson, L. Mezula and T. Juhna, Techneau, 2008. 31 17  L. Cerqueira, N.F. Azevedo, C. Almeida, T. Jardim, C.W Keevil and M.J. Vieira, Int. 32 J. Mol. Sci., 2008, 9, 1944. 33 18 J.E. Hobbie, R.J. Daley and S. Jasper, Appl. Environ. Microbiol., 1977, 33, 1225.  34 19 M.H. Phe, M. Dosset and J.C. Block, Water Res., 2004, 38, 3729. 35 

The Molecular Detection for the Viability of Yersinia cells during shock chlorination – a pilot scale study

http://www.julkari.fi/bitstream/handle/10024/114590/Draft_Rasanen%20Pia%20REVISED%2022nd%20Jan.pdf?sequence=1&isAllowed=y

The Molecular Detection for the Viability of Yersinia cells during shock chlorination – a pilot scale study

Abstract

Similar works

Full text

Available Versions

Julkari