SYTTY
UNDESIRABLE MICROBIAL BIOMASS IN DRINKING WATER DISTRIBUTION SYSTEM
Project leader: Prof. Mirja Salkinoja-Salonen, University
of Helsinki, Department of Applied Chemistry and Microbiology,
P.O.Box 56, FIN-00014 Helsinki University, Finland, tel.+358-9-19159300,
fax.+358-9-70859301,
e-mail: Mirja.Salkinoja-Salonen@helsinki.fi
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Researchers:
Jaakko Puhakka, Department of Water and Environmental Engineering,
Tampere University of Technology, P.O.Box 541, 33101 Tampere, tel.+358-3-3652966,
fax.+358-3-3652869, e-mail: Puhakka@cc.tut.fi
Pertti Vuoriranta, Department of Water and Environmental Engineering,
Tampere University of Technology, P.O.Box 541, 33101 Tampere tel.+358-3-3652852,
fax.+358-3-3652869, e-mail:PVuorira@cc.tut.fi;
Terhi Ali-Vehmas, Department of Clinical Veterinary Sciences, Pharmacology
and Toxicology P.O.Box 57, 00014 Helsinki University, tel.+358-9-19149666,
fax.+358-9-19149636, e-mail: Terhi.Alivehmas@helsinki.fi
Financing SYTTY-organization: TEKES
KEY WORDS: biofilm, risk class, water works, disinfection,
drinking water
EXTENDED ABSTRACT
The disinfection persistent gram negative rods in drinking water identified
as species of Sphingomonas
T. Ali-Vehmas1, R. Koskinen1, M. Laurikkala2, I. Tsitko3, P. Käempfer4,
E. Kostyal3, F. Atroshi1, and M. Salkinoja-Salonen3
1Department of Clinical Veterinary Sciences, Pharmacology and Toxicology,
PO BPox 57, and 3Department of Applied Chemistry and Microbiology, PO Box
56, 00014 University of Helsinki, Finland
2Institute of Water and Environmental Engineering, PO Box 541, Tampere
University of Technology, 33101 Tampere, Finland
4Inst. F. Angew. Mikrobiol., Justus-Liebig Universität, Senckenbergstr
3, D-35390 Giessen, Germany
1 Summary
Members of the genus Sphingomonas are known to possess highly bioreactive sphingolipids and of potential to cause opportunistic infections. We isolated Sphingomonas from biofilms in drinking water distribution system in Finland, Sweden and Hungary from plates grown at 15oC for 10 days. The isolates were characterized by chemotaxonomic, biochemical and phylogenetic methods. Over 60% of the isolates were designated to the species S. aromaticivorans, S. subterranea, S. xenophaga, and S. stygia. One-third of the isolates was not identifiable as any of the 24 validly described species of Sphingomonas and may represent one or several new species. 90% of the isolates grew at 5ºC and may therefore proliferate in the Nordic drinking water pipeline (2ºC to 12º C). Up to 90% of the isolates were able to grow also at 37ºC on TSBA and 40% of the isolates on blood agar.
2 Introduction
The bacteriological quality of drinking water may deteriorate in the distribution network (Colbourne 1985). The quality of drinking water that finally reaches a consumer may differ greatly from the quality of water at the waterworks. Water intended for drinking and household purposes should be free of microbes involving potential health hazard. The new European Council Directive (European Commission, 1998) on the quality of water intended for human consumption requires colony count at 22ºC and 37ºC for bottled water only. At 37ºC slow growing bacteria may be overgrown by rapidly growing organisms, such as gamma-proteobacteria, and remain undetected.
We analysed biofilms from drinking water distribution net of different locations in Finland, Hungary and Southern Sweden. Sphingomonas were commonly found when 10 d incubation time at 15ºC was used for isolation. The aim of this study was to identify the drinking water Sphingomonas isolates to species level. We found four known species and 1/3 of the isolates not assignable to a valid species.
3 Materials and Methods
The biofilms were collected from drinking water distribution net of the distributing water works in Helsinki, Tampere and Budapest and from taps of residential buildings in a small town in south Sweden. Biofilms were disintegrated by sonication with water obtained from the samples and plated out on tryptic soy agar (BBL) or R2A at 15ºC after 10 days of incubation. Whole cell fatty acid methyl esters (FAME) were prepared from 3 d cultures as described (Pirttijärvi et al. 1996) and analysed using the aerobic TSBA library version 3.9 (MIDI Inc., Newark, DE, USA). Biochemical tests in microtitre plates were done as described (Kämpfer et al. 2000). Growth was tested on plates in a cooled precision incubator (Innova 4230, New Brunswick Scientific, N.J.). Riboprinting was conducted as described Busse et al., (2000).
4 Results
Psychrophilic (15ºC) gram negative bacteria were isolated from drinking water distribution net. A major part of these were identified by whole cell fatty acid analysis as members of the genus Sphingomonas. The dominant fatty acid in the Sphingomonas isolates was octadecenoic acid (the range 41-56%). All isolates contained a major amount of 2-hydroxy-tetradecanoic acid (about 6-24% of total) but no 3-hydroxy-fatty acid. This fatty acid composition is typical of genus Sphingomonas.
Over 60% of the Sphingomonas isolates were in whole cell fatty acid (FAME) analysis similar or close Sphingomonas subarctica, S. xenophaga, S. stygia, S. aromaticivorans and S. subterranea (the last two species cannot be distinguished by FAME). Biochemical features (65 in total) of the isolates were compared to the features of all validly described Sphingomonas species. The strains of Biotype 1 (Table 1) were most similar to the type strain of Sphingomonas aromaticivorans. Biotype 2 (Table 1) isolates were similar in to Sphingomonas subterranea, and in FAME they were similar to each other but not close to S. subterranea. The strains similar in FAME to S. subarctica, differed profoundly from it in the biochemical properties. The remaining about one-third of the Sphingomonas isolates were not assignable to any of the 24 validly described Sphingomonas species (Anonymous, 2000) by their contents of FAME or the outcome of 65 different biochemical traits.
Ribopatterns of the drinking water Sphingomonas from each biotype were compared to those of all validly described Sphingomonas species (Busse et al., 2000). The results from ribotyping confirmed the results from whole cell fatty acid analysis and phenotypic properties, i.e about one-third of the drinking water isolates were S. aromaticivorans and another 1/3 were S. subterranea, S. stygia or S. xenophaga. The remaining isolates represent Sphingomonas species not yet described.
Table 1 shows that of the 17 carbohydrates tested D-cellobiose, D-glucose, D-mannose, maltose, L-rhamnose, D-sucrose and D-xylose were utilized by most (ca. 90%) isolates. Out of the 20 organic acids tested acetate, DL-3-hydroxybutyrate, pyruvate and suberate were utilized by most (>90%) isolates. None of the isolates utilized 4-aminobutyrate, beta-alanine, L-histidine, L-serine or L-tryptophane and less than 50% used other amino acids or alditols. Organic phosphates were hydrolysed by most (>90%) isolates. The phenotypic properties indicate that the common feature of the drinking water isolates was the utilization of cellulosic or hemicellulosic building blocks.
Table 1 shows that ca. 90% of the isolates grew at +5ºC indicating that the drinking water pipeline in Finland (2ºC to 12ºC) permits growth of these Sphingomonas species. Over 80% of the isolates also grew at +37ºC and one-third of the isolates, including all those identified as S. xenophaga, grew at +37ºC on blood agar. The drinking water Sphingomonas thus may have a potential for growth in man or warm-blooded animals.
5 Discussion
Sphingomonas species are a widely distributed in nature and have previously been isolated from soil, subsurface sediments, plants and water (Laskin & White, 1999). Sphingomonas have been recovered from sea water, sea ice, river water, polluted ground water, mineral water, 'sterile water' used in hospitals and drinking water (Laskin & White;1999, Bowman et al. 1997; Männistö et al. 1999, 2000; Vachee et al. 1997). The widespread distribution of Sphingomonas can be explained by their ability to survive and grow at low temperature, at low nutrient concentration (Salkinoja-Salonen et al., 1998) and in toxic chemical environment (Männistö et al., 1999, 2000). We found S. aromaticivorans, S. subterranea, S. xenophaga and S. stygia in drinking waters of several water works and several countries.
The survival of Sphingomonas in aerosols may explain (Salkinoja-Salonen et al. 1999) their ability to colonize hospital environments such as mechanical ventilators, catheters, bronchofiberoscopes and other medical devices (Lemaitre et al. 1996; Mieszala et al. 1997; Hsueh et al. 1998).
All species of Sphingomonas contain glycosphingolipids in their cell envelope. Glycosphingolipids characteristically contain 2-hydroxy-tetradecanoic acid (Laskin & White, 1999). All drinking water isolates described in this paper contained this signature fatty acid indicating they contain glycosphingolipid. Glycosphingolipids of Sphingomonas have been shown to induce tumor necrosis factor and other monokine production in human mononuclear cells (Krziwon et al. 1995), stimulate phagocytosis and phagosome-lysosome fusion (Miyazaki et al. 1995), activate the human complement system (Wiese et al. 1996) and inhibit protein kinase C and possibly function as endogenous modulators of cell function and as second messengers. These factors may partly explain the pathogenic features of hospital Sphingomonas infections.
Sphingomonas are widely known to produce viscous polysaccharide (Laskin & White, 1999). The slime production may be linked to their ability to adhere to pipeline, especially at low temperatures. Environmental factors such as a decrease in oxygen availability have been shown convert sessile mats of Sphingomonas into freely swimming planktonic bacteria (Pollock and Armentrout 1999) which may lead to aerosolisation in environments like shower and kitchen. In addition Sphingomonas species have been shown to corrode copper pipes in stagnant water (Arens et al. 1995).
6 Conclusion
Considering the potentially pathogenic features of Sphingomonas their presence in drinking water distribution net is not desirable. Sphingomonas in drinking water environment may be more common than has been understood so far and deserves further study. A full manuscript on these results has been submitted (Koskinen et al., 2000).
Acknowledgement. We thank A. Hongell for technical assistance, J. Nuutinen for help with FAME analyses, P. Vuoriranta and J.A. Puhakka for cooperation: TEKES, CIMO, Helsinki Water, Water Works of Tampere City and the Academy of Finland gave financial support. We thank R. Rylander and L. Beijer for cooperation.
7 References
Anon.(2000). Bacterial Nomenclature up-to-date, http://www.dsmz.de/bactnom/bactname/htm
Arens, P., Tuschewitzki, G.-J., Wollmann, M., Follner, H., and Jacobi, H. ( 1995). Indicators for microbiologically induced corrosion of copper pipes in a cold-water plumbing system. Zbl.Hyg. 196: 444-454.
Bowman, J., McCammon, S., Brown, M., Nichols, D., and McMeekin, T. (1997). Diversity and association of psychrophilic bacteria in Antarctic Sea ice. Appl. Env. Microb. 63: 3068-3078
Busse, H.-J., Kainz, A., Tsitko, I. & Salkinoja-Salonen MS. 2000. Riboprints as a tool for rapid preliminary identification of Sphingomonads. Syst. Appl. Microbiol. SAM 1897
Colbourne, J.S. (1985). Materials usage and their effects on the microbiological quality of water supplies. J. Appl. Bacteriol. Symp. Suppl. 59: 47S-59S.
European Commision (1998). European directive on water intended for human consumption. Council Directive 98/83/EC 3 November, 1998. The Off. J. Eur. Communities L 330/32
Hsueh, P-R., Teng, L-J., Yang, P.-C., Chen, Y.-C., Pan, H.-J., Ho, S.-W., and Luh, K.-T. (1998). Nosocomial infection caused by Sphingomonas paucimobilis: Clinical features and microbiological characteristics. Clin. In. Diseases 26: 676-81.
Koskinen, R., Ali-Vehmas, T., Kämpfer, P., Laurikkala, M., Tsitko, I., Kostyal E., Atroshi, F., & Salkinoja-Salonen, M.S. (2000). Characterization of Sphingomonas isolates from the Finnish and Swedish drinking water distribution system. J. Appl. Microbiol., submitted
Koskinen, R., M. Laurikkala, I. Tsitko, T. Ali-Vehmas, and M. Salkinoja-Salonen, 2000b: Ribotyping as a method for identification of environmental mycobacteria related to drinking water safety. Environmental Microbiology (manuscript in preparation).
Krziwon, C., Zähringer, U., Kawahara, K., Weidemann, B., Kusumoto, S., Rietschel, E., Flad, H.-D., and Ulmer, A.J. (1995). Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells. Inf. and Imm. 63: 2899-2905
Kämpfer, P., Rainey, F.A., Andersson, M.A., Nurmiaho-Lassila, E.-L., Ulrych, U., Busse, H.-J., Weiss, N., Mikkola, R. & Salkinoja-Salonen, M.S. 2000. Frigoribacterium gen. nov., a new psychrophilic genus of the family Microbacteriaceae with the proposal of F. faeni sp. nov., . Int. J. Syst. Evol.Microb. (formerly: Int. J. Syst. Bacteriol.), 50:353-363.
Laskin, A.I.and White, D.C. (editors, 1999). Special issue on the genus Sphingomonas. J. Ind. Microbiol. & Biotechnol. 23: 231-408.
Lemaitre, D., Elaichouni, A., Hundhausen, M., Claeys, G., Vanhaesebrouck, P., Vaneechoutte, M., and Verschraegen, G. (1996). Tracheal colonization with Sphingomonas paucimobilis in mechanically ventilated neonates due to contaminated ventilator temperature probes. J. of Hospital Inf. 32: 199-206.
Mieszala, M., Kubler, J., and Gamian, A. (1997). Immunochemical characterization of lipopolysaccharide from glucose-nonfermenting gram-negative clinical bacterial isolate. Acta Biochim. Polon. 44: 293-300.
Miyazaki, Y., Oka, S., Yamaguchi, S., Mizuno, S., and Yano, I. (1995). Stimulation of phagosytosis and phagosome-lysosome fusion by sphingolipids from Sphingomonas paucimobilis. J. Biochem. 118: 271-277.
Männistö, M., Salkinoja-Salonen, M.S. and Puhakka, J.A. (2000). In situ polychlorophenol bioremediation potential of the indigenous bacterial community of boreal groundwater. Water Research, submitted.
Männistö, M., Tiirola, M., Salkinoja-Salonen, M., Kulomaa, M., and Puhakka, J. (1999). Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater. Arch. Microbiology 171: 189-197.
Pirttijärvi, T.S.M., Graeffe, T.H., and Salkinoja-Salonen, M.S. (1996). Bacterial contaminants in liquid packaging boards: assesment of potential for food spoilage. J. Appl. Bacteriol. 81: 445-458
Pollock, T.J. and Armentrout, R.W. (1999). Planktonic/sessile dimorphism of polysaccharide-encapsulated sphingomonads. J. Ind. Microbiol. Biotechnol. 23: 436-441.
Salkinoja-Salonen MS, Andersson MA., Mikkola R , Paananen A, Peltola
J , Mussalo-Rauhamaa H., Vuorio R. , Saris N-E , Grigorjev P., Helin
J., Koljalg, U. & Timonen T. (1999). Toxigenic microbes in indoor environment:
identification, structure and biological effects of the aerosolizing toxins.
In: Bioaerosols, Fungi and Mycotoxins: Health affects, Assessment, Prevention
and Control, ed. by E. Johanning. Eastern New York Occupational and Environmental
Health Center, Albany New York, USA, pp 359-374.
Salkinoja-Salonen, MS, Kähkönen, MA, Wittmann, C., Peltola,
RJ. (1998). Microbial activity in cold climate. In: Prospective terrestrial
environment and ground water pollution research conference. Proceedings,
Göteborg 15-18.11.1998, ed. by Karsten Pedersen. European Science
Foundation & MISTRA, Göteborg, 1999, pp. 117-125.
Vachee, A.,Vincent, P., Struijk, C.B., Mossel, D.A.A.and Leclerc, H. (1997). A study of the fate of the autochtonous bacterial flora of still mineral waters by analysis of restriction fragment length polymorphism of genes coding for rRNA.Syst. Appl. Microb. 20:492-503.
Wiese, A., Reiners, J.,Brandenburg, K.,Kawahara, K.,Zähringer, U., and Seydel, U. (1996). Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: Membrane potential, porin function, and complement activation. Biophys. J. 70: 321-329.
8 Co-operation
Domestic cooperation:
Helsinki Water (Jarmo Kaartinen, director; Tapani Vakkuri, manager;
Juha Hämäläinen, chemist; Eira Toivanen, chemist)
Tampere City Water Works (Esko Haume, director)
International cooperation:
National Institute of Hygiene, Dept. of Water Hygiene, Budapest,
Hungary (Matyas Borsanyi, director), Lousiana State University, Dept. of
Microbiology, Baton Rouge LA (prof. Frederick A. Rainey), Inst. F. Angew.
Mikrobiol., Justus-Liebig Universität, Germany (Dr. P. Kämpfer),
Universität des Saarlandes, Germany (Dr. C. Wittmann), German Collection
of Microorganisms and Cell Cultures, Germany (Dr. P. Schumann), Institut
für Mikrobiologie und Genetik, Universität Wien, Austria (Dr.
H.-J. Busse), Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische
Universität, Austria (Dr. A. Kainz).
| Table 1. Phenotypic
features of Sphingomonas isolates from Finnish and Swedish drinking
water distribution systems.
+ all strains positive, - all strains negative, (+) majority (>50%) of strains positive, (-) majority (>50%) of strains negative. |
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Biotype 5 | ||||||||||||
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| Substrate utilization:1 | ||||||||||||||||||
| n-acetyl-D-glucosaminide |
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| L-arabinose |
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| p-arbutin |
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| D-cellobiose |
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| D-fructose |
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| D-galactose |
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| Gluconate |
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| D-glucose |
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| D-mannose |
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| Maltose |
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| Melibiose |
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| L-rhamnose |
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| D-sucrose |
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| Salicin |
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| D-trehalose |
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| D-xylose |
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| Acetate |
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| Propionate |
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| cis-aconitate |
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| Adipate |
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| Azelate |
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| Citrate |
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| Fumarate |
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| Glutarate |
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| DL-3-hydroxybutyrate |
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| DL-lactate |
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| L-malate |
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| Pyruvate |
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| Suberate |
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| L-alanine |
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| L-aspartate |
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| L-leucine |
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| L-ornithine |
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| L-phenylalanine |
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| L-proline |
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| 4-hydroxybenzoate |
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| Hydrolysis of:2 | ||||||||||||||||||
| Esculin |
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| pNP-b-D-galactopyranoside |
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| PNP-b-D-glucuronide |
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| Bis-pNP-phosphate |
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| PNP-phenyl-phosphonate |
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| PNP-phosphorylcholine |
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| L-proline-pNA |
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| Growth temperature: | ||||||||||||||||||
| on TSBA +5°C, 20d |
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| +10°C, 20d |
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| +15°C, 10d |
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| +37°C, 3d |
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| on Blood agar +37°C, 3d |
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| 1 Substrates
that were not utilized by any isolate included D-ribose, adonitol, i-idositol,
maltitol, D-sorbitol, putrescine, trans-aconitate,
4-aminobutyrate, itaconate, mesaconate, oxoglutarate, á-alanine, L-histidine, L-serine, L-tryptophane, 3-hydroxybenzoate, and phenylacetate. |
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| 2 Substrates
that were hydrolysed by all isolates included pNP-alfa-D-glucopyranoside,
pNP-beta-D-glucopyranoside,
2-deoxythymidine-5´-pNP-phosphate, L-alanine-pNA, and L-glutamate-gamma-3-carb.-pNA. |
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