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Quantification of thermotolerant Campylobacter in Swiss watertreatment plants, by real-time quantitative polymerase chain

reaction

Rinsoz, T; Hilfiker, S; Oppliger, A

Rinsoz, T; Hilfiker, S; Oppliger, A (2009). Quantification of thermotolerant Campylobacter in Swiss watertreatment plants, by real-time quantitative polymerase chain reaction. Water environment research, 81(9):929-933.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Water environment research 2009, 81(9):929-933.

Rinsoz, T; Hilfiker, S; Oppliger, A (2009). Quantification of thermotolerant Campylobacter in Swiss watertreatment plants, by real-time quantitative polymerase chain reaction. Water environment research, 81(9):929-933.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Water environment research 2009, 81(9):929-933.

Quantification of Thermotolerant Campylobacter in Swiss Water Treatment

Plants, by Real-Time Quantitative Polymerase Chain Reaction

Thomas Rinsoz 1, Silvia Hilfiker2 , Anne Oppliger h

ABSTRACT: A total of 49 wastewater samples from 23 different wastewater treatment plants (WWTPs) were analyzed using real-time quantitative polymerase chain reaction for the presence and quantity of thermotolerant campylobacters. Thermotolerant campylobacters were detected in 87.5% (21124) and 64% (16125) of untreated and treated wastewater samples, respectively. Their concentration was sufficiently high to be quantified in 20.4% (10/49) of the samples. In these samples, the concentration ranged from 68 000 to 2292 000 cellslL in untreated wastewater and from 10 800 to 28 000 cellslL in treated water. We conclude that thennotolerant campylobacters present a health hazard for workers at WWTPs in Switzerland. Water Environ. Res., 81, 929 (2009).

KEYWORDS: Campylobacter spp., occupational health, quantitative polymerase chain reaction, wastewater treatment plant.

doi:lO.2175/106143009X407429

Introduction Wastewater is recognized as a significant vehicle for the

transmission of several viral and bacterial diseases. The type and quantity of microorganisms present in wastewater reflect both clinical and subclinical infections prevalent in the human population (Vaidya et al., 2002). In the genus Campylobaeter, the main human pathogens are the thermotolerant species C. jejuni, C. coli, and C. lari. These are the most common human enteric pathogens and cause acute diarrhea (Frost, 2001). Some infections also have been associated with chronic, debilitating sequelae, such as Guillain-Barre syndrome and Fischer Miller syndrome (Nachamkin et al., 1998). Studies carried out in several countries have shown that these bacteria are ubiquitous in wastewater and that human and animal waste from slaughter­houses and animal livestock are the major sources of human contamination (Corry and Atabay, 2001; Jones, 2001).

A quantitative exposure assessment is necessary to estimate the risk caused by pathogenic bacteria to wastewater treatment plant

1 Institut Universitaire Romand de Sante au Travail (Institute for Work and Health), University of Lausanne and Geneva, Lausanne, Switzerland.

2 Abteilung fur Arbeits- und Umweltmedizin (Occupational and Environ­mental Health Unit), Med. Poliklinik USZ, Zurich, Switzerland.

* Institut Universitaire Romand de Sante au Travail (Institute for Work and Health), University of Lausanne and Geneva, rue du Bugnon 21, CH-lOl1 Lausanne, Switzerland; e-mail: Anne.Oppliger@hospvd.ch.

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(WWTP) workers. Traditional methods available to quantify bacteria use culture-dependent protocols. However, Campy[abae­ter spp. have the ability to persist in aquatic environments in viable but nonculturable form (Cellini et aI.. 1994; Rollins and Colwell, 1986; Shahamat et aI., 1993; West et aI., 1990). Similar phenomena also have been reported for milk and foodbome bacteria (Oliver et al., 2005). Moreover, it is now widely accepted that our knowledge of microbial diversity is severely limited by the fact that the vast majority (>90%) of naturally occurring microorganisms cannot be cultivated using standard techniques (Amann et aL, 1995; DeLong and Pace, 2001). Therefore, attempts to quantify pathogens in wastewater may benefit from the development of a culture-independent method. Polymerase chain reaction (PCR) has been used successfully to detect Campylobaeter spp. (Moreno, Botella, Alonso, Ferrus, Hernandez, and Hernandez, 2003) in wastewater. However, that study did not provide quantitative levels of the pathogens in wastewater. Real­time quantitative peR (Q-PCR) is a non-culture-dependent method that has the potential to quantify bacterial load. The Q­PCR method has been used in many environmental and clinical microbiological studies. For example, it has been used previously to quantify campylobacters in cattle feces (Inglis and Kalischuk. 2004), Helieobacter pylori in clinical samples (Rinttilii et al., 2004), and Giardia and Cryptosporidiurn in wastewater (Guy et aI., 2003). To date, the Q-PCR techniques have not been used to quantify thermotolerant campylobacters.

The purpose of this study was to develop a real-time Q-PCR protocol able to detect and quantify thermo tolerant campylobac­ters in wastewater to assess the following:

(1) Potential exposure of WWTP workers to those pathogens, and (2) Efficacy of the wastewater treatment process in removal of

those bacteria from water to be recycled for human consumption.

Methodology Bacterial Strain. We chose Campy/ohaeter ji~illlli (ATCC

33291) as our standard strain for serial dilution and PCR calibration, because it is recognized as one of the most frequent etiological agents of campylobacteriosis (Tauxe, 1992; \Vaage et al., 1999). This strain was grown on Blood Agar Base (Oxoid, Pratteln, Switzerland) at 37)C under microaerophilic condi­tions.

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Table 1-Quantifieation of thermotholerant eampylo­baeters in wastewater where eampylobaeters were quantifiable.*

Quantification WWTP Origin ( campylobacters/L)

F F G G J J J J K K K N

Influent 69 000 Effluent Detected Influent 85 000 Effluent Detected Influent (before decantation) Detected Influent (after decantation) 96 000 Effluent (before chlorination) 18600 Effluent (after chlorination) Detected Influent Detected Effluent (physico-chemical treatment) Detected Effluent (biological treatment) 10 800 Influent Detected Effluent 28 000 Influent 2292 000 Effluent 23500 Influent 99 000 Effluent Not detected Influent 68 000 Effluent Detected

* F = Zurich region (urban), G = Zurich region (rural), J and K = Lausanne region (urban), and N through W = Lausanne region (rural).

Environmental Sampling. A total of 23 WWTPs were sampled in Switzerland, in the Zurich and Lausanne areas; 11 WWTPs were sitw;lted in "urban" areas, and 12 were situated in "rural" areas (see detail in Table 1). For one WWTP (J), untreated wastewater was sampled before and after decantation, and treated wastewater was sampled before and after chlorination. In WWTP "K", biological and physico-chemical methods were used as wastewater treatments. Samples were taken following each of the treatments. Each sample corresponded to 1 L of wastewater collected with an automatic flow sampler used continuously (1 mL/minute) over 24 hours. The sampling scheme yielded a total of 24 untreated and 25 treated wastewater samples to be analyzed by Q-PCR.

Preparation of Wastewater Samples for Polymerase ~hain Reaction. Samples of untreated and treated wastewater {ere filtered through a 37-mm diameter and 0.22-l1m pore

polycarbonate filter (Millipore, Billerica, Massachusetts). This type of filter has a smooth, glass-like surface recommended for bacterial collection. To avoid any risk of saturating the filters, while also optimizing the chances of detecting the lower levels of campylobacter in treated water, slightly smaller volumes were filtered from the untreated wastewater (approximately 50 mL) than from the treated wastewater (approximately 100 mL), as the organic matter concentration was expected to be higher in the untreated water. The data for each sample were subsequently normalized to reflect the amount of cells detected per liter of water. DNA was extracted directly from the filter with the FastDNA SPIN Kit for Soil and the Fastprep Instrument (Bio101, Carlsbad, California), which performs a mechanical lysis of the bacterial wall. The extraction was done in accordance with the manufacturer's instructions, with the filter replacing the soil sample. DNA was eluted in 200 I1L EB buffer (Qiagen, Basel, Switzerland).

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Real-Time Quantitative Polymerase Chain Reaction. Each PCR reaction was performed in a total volume of 20 J.LL using the qPCR Core kit No ROX (Eurogentec, Seraing, Belgium). Two I1L of diluted DNA samples were added to the PCR mix. To avoid a potential negative effect of inhibitors, samples of untreated wastewater were diluted 10- and 40-fold, and samples of treated wastewater were diluted 1- and 5-fold. No-template controls with Qiagen EB buffer instead of template DNA were included in each run. All PCR assays were run on a RotorGene 3000 (Corbett Research, Sydney, Australia). A 287-bp fragment of the 16S ribosomal gene of thermotolerant campylobacters, including C. jejuni, C. coli, and C. lari, was quantified with a primer-probe system (Josefsen et al., 2004; Lubeck et al., 2003). The PCR was perfonned with the following concentrations: 2.5 mM MgCI2,

0.4 mM dNTPs mix, 0.44 11M forward primer (5'- CTG CTT AAC ACA AGT TGA GTA GG -3'), 0.48 11M reverse primer (5'­TTC CTT AGG TAC CGT CAG AA -3'), 0.02 11M probe (5'­FAM- TGT CAT CCT CCA CGC GGC GTT GCT GC -Tamra-3'), and 0.625 U/reaction of HotGoldStar polymerase (Qiagen, Basel, Switzrerland). The amplification was carried out under the following conditions: 95°C for 10 minutes and 50 cycles of 95°C for 15 seconds and 58°C for 60 seconds, with acquisition of the fluorescence on the FAM channel (excitation at 485 nm) at the end of the elongation step at 58°C.

Standard Curves and Statistical Analysis. Serial dilutions of C. jejuni genomic DNA (ATCC 33291) were used as standards for determining bacterial number by real-time PCR. The number of copies of the Q-PCR standards was calculated using the following equations:

Genome weight (Dalton) W

= [%GC x total length] x 618.4 / 100 (1)

+ [(100 - %GC) x total length] x 617.4 / 100 + 36

Where %GC and total length = 30.6% and 1.64 X 106 bp for C. jejuni (Parkhill et al., 2000).

Genome copies per nanogram of DNA = (NL/W) x 10-9 (2)

Where NL = Avogadro constant (6.02 X 1023 molecules per mol). The calibration curve was generated with the RotorGene

software, version 6.0.16 (Corbett Research). Using the calibration curve, the initial number of target copies in the PCR reaction was calculated by the RotorGene software. For example, the number of campylobacters per liter was calculated in the following way for the influent of WWTP "P": serial 5-time dilutions of C. jejuni genomic DNA ranging from 5.05 to 0.0404 pglreaction were used as a standard. The raw result from the Q-PCR indicated a mean of 0.965 pglreaction, with this amount corresponding to 1146 Campylobacters/reaction. As 1/20 L of wastewater was filtered and 11100 of the extracted DNA was used for the PCR reaction, the concentration in the influent of WWTP "P" was estimated as 1146 X 20 X 100 = 2292 000 cellslL. The Pearson Chi-square test was used to evaluate whether the differences in Campylo­bacter numbers between the different water sources were statistically meaningful.

Results The results for the detection/quantification of thermotolerant

campylobacters DNA in wastewater by real-time Q-PCR are summarized in Tables 1 and 2. DNA was detected by Q-PCR in

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Table 2-Detection of thermotholerant campylobacters in wastewater.

Type of WWTP

Urban Rural Total (number of (number of (number of

Influent Effluent WWTPs) WWTPs) WWTPs)

Yes Yes 8 4 12 Yes No 3 5 8 No No 0 1 1 No Yes 0 2 2 Total 11 12 23

87.5% (21124) and 64% (16/25) of untreated and treated wastewater samples, respectively (Table 2). The difference in campylobacter detection between influent and effluent water is statistically significant (Pearson Chi-square test: X2= 4.059, df = 1, and P = 0.044). Thennotolerant camp1y10bacters could be quantified by Q-PCR in 20.4% (10/49) of the samp1es-25% (6/ 24) for untreated wastewater samples and 16% (4/25) for treated wastewater (Table 1). In samples for which a quantification was possible, concentrations ranged from 68 000 to 2292 000 cellslL of untreated wastewater (mean = 451 500, standard deviation = 901 751) and 10 800 to 28 000 cellslL (mean= 20 225, standard deviation = 7363) of treated water. In WWTPs "T" and "W", DNA was detected in treated wastewater, but not in untreated wastewater. We found no statistically meaningful difference in the presence of thennotolerant campylobacters in comparisons between urban and rural influents or effluents (influents: Yates corrected Chi-square test: X2= 1.342, df = 1, and P = 0.247; effluents: Pearson Chi­square test: X2= 1.245, df = 1, and P = 0.265). In the single WWTP (P), where quantification of Campylobaeter was possible both for influent and effluent water, we found that treatment eliminated 99% of bacteria originally present.

Discussion Quantification of Bacteria. Our results show that the

occurrence of detectable levels of thermotolerant campylobacters in Swiss wastewater is high in untreated wastewater (87.5%) and remains significant in treated wastewater (64%), therefore indicating that the current treatment process does not constitute a fully effective barrier to these bacteria. These results confirm observations made by others researchers in different countries (Arimi et aI., 1988; Betaieb and Jones, 1990; Moreno, Botella, Alonso, Ferrus, Hernandez, and Hernandez, 2003; Sahlstrom et al., 2004; Stampi et al., 1993).

Previous studies that attempted to quantify Campylobaeter spp. in wastewater used culture methods in combination with the most probable number method (Arimi et aI., 1988; Betaieb and Jones, 1990; Stampi et aI., 1993). This method is based on culture after serial dilutions of the sample and allows an estimation of the bacterial concentration in the sample. In a study carried out in England, thennophilic campylobacters were quantified every hour, from 6:00 a.m. to 8:00 p.m., over a 5-day period, in wastewater of a single WWTP. The maximum concentration of bacteria observed in this study was 180 000 cellslL in untreated wastewater. This concentration was reduced by 99.9% after treatment (Arimi et aI., 1988). In a study carried out in Libya, thermophilic campylobacters were quantified on two different

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dates, in each of two WWTPs (Betaieb and Jones, 1990). In the first WWTP, the numbers of campylobacters in incoming water were 82 000 and 58 670 cellslL. Campylobacters were not detectable after the water had been treated. In the second WWTP, the numbers of campylobacters per liter were 82 000 and 59 670 in incoming water and 16 330 and 17 670 in treated water, respectively. Two separate studies in a single Italian WWTP showed average concentrations between 16 300 and 17 200 cellslL of incoming water (Stampi et al., 1992, 1993). These concentra­tions were reduced by more than 99% in treated water. All these studies used culture methods to quantify campylobacters in wastewater. Because Campylobacter spp. have the ability to survive in viable but nonculturable forms (Rollins and Colwell, 1986), such methods tend to underestimate their concentration in environmental samples.

In the only previous study of wastewater contamination by campylobacters where Q-PCR was used, C. jejuni/coli DNA was not detected in untreated or treated wastewater in two WWTPs in Germany (Alexandrino et al., 2004). Our study of Swiss wastewater plants is based on more WWTPs, but less replicates per WWTP, than the studies cited above. Its results are in general accordance with those of other studies on these pathogens, in terms of occurrence, concentration, and efficacy of removal during wastewater treatment.

Our results show that the occurrence and quantity of thermotolerant campylobacters is much higher in untreated wastewater than in treated wastewater. Combined with the fact that the detection limit is lower in treated wastewater, it indicates that there is a significant reduction of the quantity of bacteria by the wastewater treatment process. The lower detection limit observed in treated water can be explained by the fact that untreated wastewater is rich in PCR inhibitors, such as humic acid, polysaccharides, and metal ions, which are eliminated partially by the wastewater treatment. Thus, samples coming from treated wastewater need to be less diluted before the Q-PCR reaction than samples of untreated wastewater. This difference in the detection limit for incoming and outgoing water probably explains why, in two WWTPs (T and W), similar to the study of Moreno, Ferrus, Alonso, Jimenez, and Hernandez (2003), DNA was detected only in treated wastewater.

Risk for Wastewater Workers. Wastewater treatment work­ers have been shown to be at a higher risk of developing a large variety of work-related symptoms compared with the general population, including respiratory, neurological, and gastrointesti­nal (e.g., diarrhea) effects (Douwes et al., 2001; Rylander, 1999; Thorn et aI., 2002; Thorn and Beijer, 2004). However, specific causal agents have not been identified clearly (Douwes et al., 2001). As symptoms of campylobacter infection range from watery diarrhea to dysentery, often accompanied by fever and abdominal cramps, campylobacters could be responsible for some of the gastrointestinal work-related symptoms of wastewater treatment workers. Campylobacters also are associated with significant sequelae, such as Guillain-Barre syndrome and Fischer Miller syndrome (Nachamkin et al., 1998); however, the prevalence of those syndromes in wastewater treatment workers has not been documented. We have shown that the concentration of thermotolerant Campylobaeter in wastewater can be as high as 2 millions cellslL in untreated wastewater (WWTP "P"), whereas the infectivity dose of this pathogen is known to be as low as 500 to 800 cells (Black et al., 1988; van Vliet and Ketley, 2001). With

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the caveat that our study makes no distinction between pathogenic and non-pathogenic or between viable and non-viable Campylo­baeter sp., we suggest that the potential risk for wastewater workers of exposure to pathogenic Campylobaeter infections is sufficiently high to justify the imposition of stronger precautions to prevent direct contact with wastewater. Campylobaeter spp. are recognized as a major source of gastroenteritis throughout the world. Published estimates suggest that, in the United Kingdom and in the United States, approximately 1 % of the population is affected annually by Campylabaeter infection, resulting in substantial financial loss, as a result of clinical costs and lost working hours (Ketley, 1997; On et aI., 1998). It is presumed that such infection mainly occurs through the consumption of contaminated food, but occupational infection also is likely to occur. Campylobaeter spp. are present in work environments, such as the poultry industry (Haas et al., 2005; Rivoal et aI., 2005), where infection could occur both by hand-to-mouth contact and by direct airborne transmission via inhalation (Wilson, 2004). The Q-PCR could be applied in poultry work environments, including poultry houses and slaughterhouses, to assess airborne exposure of workers to Campylobaeter spp. or other pathogenic bacteria.

Conclusion The use of the real-time Q-PCR method to quantify thermo­

tolerant Campylabaeter in wastewater suggests that the levels of such species in untreated wastewater could be higher than has been appreciated previously; untreated wastewater samples from 6 of the 23 WWTP tested showed Campylobaeter levels of 68 000 cells/mL or higher. Our study provides evidence that current wastewater treatment procedures can greatly reduce the concen­trations of such species; however, it also establishes that traces of Campylobaeter are still detectable in more than one-half of the investigated effluent samples from water treatment plants (at sufficiently high levels to be quantified in two cases). The high concentrations of thermotolerant campylobacters found in waste­water suggest that exposure to these bacteria could present an occupational risk for wastewater workers in Switzerland. We recommend that workers in WWTPs be informed of the potential risk of infection by pathogenic bacteria and the preventive hygiene measures that must be taken.

Credits Thanks to Moira Cockell (Alwrite, Savigny, Switzerland) for

helpful comments and suggestions on the draft. We greatly appreciate the support and cooperation of owners and staff of the WWTPs that were involved in this study. This work was supported by a Swiss National Foundation (Bern, Switzerland) grant 3200BO-I04246 to Anne Oppliger.

Submitted for publication August 28, 2008; revised manuscript submitted January 7,2009; accepted for publication Janum) 29, 2009.

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