Ilkka T. Miettinen

Detection and Typing of Campylobacter jejuni and Campylobacter coli and Analysis of Indicator Organisms in Three Waterborne Outbreaks in Finland

ABSTRACT Waterborne outbreaks associated with contamination of drinking water by Campylobacter jejuni are rather common in the Nordic countries Sweden, Norway, and Finland, where in sparsely populated districts groundwater is commonly used without disinfection. Campylobacters, Escherichia coli, or other coliforms have rarely been detected in potential sources. We studied three waterborne outbreaks in Finland caused by C. jejuni and used sample volumes of 4,000 to 20,000 ml for analysis of campylobacters and sample volumes of 1 to 5,000 ml for analysis of coliforms and E. coli, depending on the sampling site. Multiple samples obtained from possible sources (water distribution systems and environmental water sources) and the use of large sample volumes (several liters) increased the chance of detecting the pathogen C. jejuni in water. Filtration of a large volume (1,000 to 2,000 ml) also increased the rate of detection of coliforms and E. coli. To confirm the association between drinking water contamination and illness, a combination of Penner serotyping and pulsed-field gel electrophoresis (digestion with SmaI and KpnI) was found to be useful. This combination reliably verified similarity or dissimilarity of C. jejuni isolates from patient samples, from drinking water, and from other environmental sources, thus confirming the likely reservoir of an outbreak.

Norovirus outbreaks from drinking water.

Norovirus contamination calls for viral monitoring of drinking water.

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland

ABSTRACT Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 × 105 and 3.9 × 105 CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.

Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and Caliciviruses in Drinking Water-Associated Biofilms Grown under High-Shear Turbulent Flow

ABSTRACT Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

Disinfection by-products in Finnish drinking waters

Disinfection by-products (DBPs) were measured in plant effluents of 35 Finnish waterworks, which utilized different treatment processes and raw water sources. DBPs were measured also from the distribution systems of three waterworks. Di- and trichloroacetic acids, and chloroform were the major DBPs found in treated water samples. The concentration of six haloacetic acids (HAA6) exceeded the concentrations of trihalomethanes (THMs). Chlorinated drinking waters (DWs) originating from surface waters contained the highest concentration of HAA6 and THMs: 108 and 26 microg/l, respectively. The lowest concentrations of DBPs were measured from ozonated and/or activated carbon filtrated and chloraminated DWs. Higher concentrations of HAA6, THMs, and adsorbable organic halogens were measured in summer compared to winter. The levels of chlorinated acetic acids, chloroform, and bromodichloromethane correlated positively with mutagenicity. Past mutagenicity levels of DWs were examined. A major reduction in the use of prechlorination, increased use of chloramine disinfection, and better removal of organic carbon were the most important reasons for the 69% decrease in mutagenicity from 1985 to 1994.

Molecular size distribution of natural organic matter in raw and drinking waters

The purpose of this study was to compare the molecular size distribution (MSD) of natural organic matter (NOM) in raw waters (RW) and drinking waters (DW), and to find out the differences between MSD after different water treatment processes. The MSD of NOM of 34 RW and DW of Finnish waterworks were determined with high-performance size-exclusion chromatography (HPSEC). Six distinct fractions were generally separated from water samples with the TSK G3000SW column, using sodium acetate at pH 7 as an eluent. Large and intermediate humic fractions were the most dominant fractions in surface waters (lakes and rivers), while in artificially recharged groundwaters and natural groundwaters intermediate and small fractions predominated. Water treatment processes removed the two largest fractions almost completely shifting the MSD towards smaller molecular size in DW. Granular activated carbon (GAC) filtration, ozonation, and their combination reduced all humic fractions compared to the conventional treatment. Humic fractions correlated with total organic carbon (TOC) content and chemical oxygen demand, this being especially true in RW. The results demonstrate that the HPSEC method can be applied for a qualitative and also for rough estimate quantitative analyzes of NOM directly from RW and DW samples without sample pretreatment.

Changes in content of microbially available phosphorus, assimilable organic carbon and microbial growth potential during drinking water treatment processes

There are regions where microbial growth in drinking water is limited by phosphorus instead of organic carbon. In phosphorus limited waters small changes in phosphorus concentration significantly affect microbial growth. We studied how water treatment processes in waterworks affect the availability of microbial nutrients and microbial growth potential in drinking water. The nutrients studied were assimilable organic carbon (AOCpotential) and microbially available phosphorus (MAP) which both were quantified by bioassays. Chemical coagulation, commonly used in surfacewater works, effectively removed AOCpotential and MAP. In contrast to activated carbon filtration, ozonation increased the concentrations of AOCpotential and MAP, and also microbial growth potential. In most of the drinking waters, microbial growth was limited by phosphorus, and microbial growth potential correlated with the MAP concentration. Microbial growth potential was lowest in drinking waters produced from surface waters with efficient treatment technique and highest in less treated ground waters.

Microbially available organic carbon, phosphorus, and microbial growth in ozonated drinking water

Ozonation is a disinfection technique commonly used in the treatment of drinking water. It destroys harmful microbes, but it also degrades organic matter in water, increasing the bioavailability of organic matter. Recently, it was found that not only organic carbon but also phosphorus can limit the microbial growth in drinking water, which contains high amount of organic matter. We used a bioassay to analyze whether ozone could also increase the microbially available phosphorus (MAP) in drinking water, and whether MAP in ozone-treated water was associated with the growth of heterotrophic microbes. We found that both assimilable organic carbon and MAP concentrations were increased by ozone treatment. In ozonated water, microbial growth was mainly limited by phosphorus, and even minor changes in MAP concentration dramatically increased the growth potential of heterotrophic microbes. In this study, ozonation increased the MAP by 0.08-0.73 microgram P/l, resulting in an increase of 80,000-730,000 CFU/ml in water samples. In contrast to MAP, the content of assimilable organic carbon (AOCpotential) did not correlate with microbial growth. The results show that in water treatment not only AOCpotential but also MAP should be considered as an important factor that can limit microbial growth in drinking water.

Determination of assimilable organic carbon in humus-rich drinking waters

Abstract The potential for microbial growth in drinking waters is considered to be associated with the content of easily assimilable organic carbon (AOC). The bacterial test for the determination of the AOC concentration presumes that microbial growth in the test is limited by the availability of organic carbon. However, in the northern latitudes inorganic nutrients, particularly phosphorus, may limit the microbial growth in drinking waters. In our studies, addition of an inorganic nutrient mixture (N, P, K, S, Ca, Mg) to soft, humus-rich drinking waters increased the AOC yield in most of the drinking waters. The assimilable organic carbon determined with the addition of nutrients describes that pool of organic carbon potentially available for microbial growth (AOC potential ). The AOC yield when there is no without addition of inorganic nutrients describes that part of organic carbon (AOC native ) which bacteria can utilize in the prevailing conditions and it is composed of the availability of both organic substrates and inorganic nutrients. We found that the bacterial strains, Pseudomonas fluorescens P 17 and Aquaspirillum NOX , used in the standard AOC method, can also be applied in humus-rich drinking waters.

The microbial community structure of drinking water biofilms can be affected by phosphorus availability.

ABSTRACT Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter−1 and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7c and 18:1ω7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.