- Email: [email protected]
Molecular detection of Campylobacter spp. in drinking, recreational and environmental water supplies
International Journal of Hygiene and Environmental Health
Int. J. Hyg. Environ. Health 204, 185 ± 189 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Short Communication Molecular detection of Campylobacter spp. in drinking, recreational and environmental water supplies John Moore, Patricia Caldwell, Beverley Millar Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK Received: January 30, 2001 ´ Accepted: July 31, 2001
Abstract A molecular detection assay was performed on 207 samples of drinking, recreational and environmental waters collected in Northern Ireland. The water sources which were PCR positive for Campylobacter spp. included 2/91 (2.2%) drinking water from domestic household taps, 5/57 (8.8%) swimming pool water, 1/23 (4.3%) lake water and 1/1 water from a jacuzzi. Extracted DNA from all water samples was amplified employing a sequencespecific PCR assay based on a 206bp conserved region of the flagellin A-flagellin B (flaA/ flaB) loci for Campylobacter jejuni, C. coli and C. lari. Given the physiological and cultural fragile nature of these species, no waters were cultured using conventional methods due to concern for reversion to non-culturability from time of collection to laboratory analysis. As this genus has been demonstrated to form a `viable but non-culturable' (VBNC) form, failure to culture organisms conventionally from water does not necessarily equate to a negative result, hence molecular detection assays, especially those which can demonstrate cell viability, may be useful in helping to elucidate potential epidemiological sources and reservoirs of this organism, especially where water is suspected as being the vehicle of transmission. Key words: Campylobacter spp. ± PCR ± phenotyping ± water ± flaA
Introduction The past three decades have witnessed the rise of Campylobacter enteritis in humans from virtual obscurity to notoriety, with present isolation rates superceding those of other enteric pathogens such as Salmonella spp. and Shigella spp. in most developed countries (Skirrow, 1990). Unlike the salmonellae and other enteric pathogens, the majority (ca. 99%) of clinical reports concerning Campylobacter are sporadic and Campylobacter enteritis outbreaks are rare (Cowden, 1992). This may be due to discrimi-
natory genotyping schemes not being able to clearly identify outbreaks within the majority of sporadic cases. Additionally, campylobacters do not multiply in foodstuffs and are lethally damaged by drying and exposure to atmospheric oxygen. While it is difficult to determine the sources of individual cases, epidemiological investigations by case-control studies can identify likely sources or exposures. The lack of welldeveloped typing schemes has hindered the epidemiological investigations seeking natural reservoirs of the organism and modes of transmission from these sources to humans. Although campylobacters
Corresponding author: Dr. John E. Moore, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, United Kingdom, Phone: 44 28 9026 3554, Fax: 44(28) 2589 2887, E-mail: [email protected] 1438-4639/01/204-185 $ 15.00/0
186
J. Moore, P. Caldwell, B. Millar
are not completely new to applied bacteriology, they have evaded traditional techniques used for the isolation of pure cultures, apart from single isolations that were free from competing organisms. Until the development of a selective medium by Skirrow (1977), these organisms were known mainly by veterinarians as animal pathogens which were responsible for a wide variety of disorders in cattle, sheep and pigs (Bokkenheuser and Mosenthal 1981). Since the development of more sophisticated isolation techniques (Dekeyser et al. 1972), the true disease potential of these organisms has become apparent and today campylobacteriosis is regarded as a zoonoses, which is capable of being transmitted to humans, mainly through contaminated foodstuffs by a wide range of domestic animals (Prescott and Munroe 1982). Campylobacters can be detected using conventional culture (Bolton et al., 1999), however, they can be difficult to isolate in the laboratory due to their fastidiousness. There have been several reports of outbreak epidemiology implicating water as the vehicle of transmission of this organism to humans, however extended analyses of the water by conventional cultural techniques has not been successful in obtaining a positive culture. There are a number of possible explanations for this failure to detect viable organisms and this may not only be due to VBNC status. At the time of outbreak recognition and investigation, original water specimens may no longer be available and this may be exacerbated by problems associate with the viability of cells due to storage conditions including transport factors, time and temperature, prior to laboratory analyses. Hence it was the aim of this study to employ a molecular detection assay for a diverse variety of drinking, recreational and environmental waters, which could potentially act as a reservoir for human campylobacteriosis.
Materials and methods Collection and processing of water samples All samples (1000 ml) were collected by Environmental Health Officers from the 26 local council authorities within Northern Ireland in sterile plastic disposable containers. The samples were collected as part of a routine sampling programme from various and commercial premises (Table 1). In addition, samples were collected from various leisure facilities, such as public leisure centres and hotel leisure complexes. Surface waters from lakes, reservoirs, springs and wells were also analysed. All samples were transported and maintained at 48C prior to analysis and were processed within 24 ± 72 h following collection. In those cases, where free residual chlorine was
Table 1. Molecular prevalence of Campylobacter spp. in drinking, recreational and environmental water supplies in Northern Ireland. Water Type (i). Drinking waters Tap Spring Bore hole Well Bottled (ii). Recreational waters Swimming pool Jacuzzi Lough River Sea TOTAL
No. of samples examined
No. positive (% positive)
91 6 1 4 1
2 (2.2%) 0 0 0 0
57 1 23 17 6 207
5 (8.8%) 1 1 (4.3%) 0 0 9 (8.7%)
present, i.e. tap water, swimming pools and jacuzzi water, sodium thiosulphate was added at a concentration of 18 mg/l (18 ppm) to the sampling bottles, as previously described (Anon., 1994a), to neutralize up to 5 mg/l (5 ppm) residual chlorine in the sample. Treatment of drinking and recreational waters Drinking water from the tap was treated at local public water treatment works. Raw drinking water was subjected to the following processes, including (i) clarification for the removal of silt, algae, and colour. (ii) filtration for the removal of smaller particles remaining after clarification and also for the removal of iron and manganese from ground water sources. A variety of filters including rapid gravity sand filters were used to allow disinfection to be optimum. (iii) disinfection for the killing of any remaining bacteria in the water following filtration, by the introduction of chlorine to leave free residual chlorine in the finished water supply. Spring, bore-hole and well waters were not treated by any process, except by natural sedimentation. Likewise, for recreational waters, lough, river and sea waters were not treated by any process. Swimming pools were treated by automated systems by a combination of (i) chlorination and ozonation to 1.5 ± 2.0 ppm free residual chlorine and 0.5 ± 1.0 ppm ozone, respectively, (ii) maintenance of pH 7.4 ± 7.6 and (iii) continuous filtration through fine mesh, sand and carbon filters. Jacuzzi water was treated as previously described (Anon., 1994b). Molecular detection of Campylobacter spp. All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet in a room physically separate from that used to set up reaction mixes and also from the `post-PCR' room in order to minimise the production of false-positive results. In all methods tested and where applicable, molecular grade water was employed (Biowhittaker Inc, Maryland, USA, LAL Grade cat no: W50100) to reduce contamination. The type strain C. jejuni NCTC 11351 and molecular grade water were employed as positive and negative extraction controls respectively.
Campylobacter in water Water samples (400 ml) were initially filtered through a sterile polycarbonate membrane filter (43 mm diameter; pore size 0.20 mm) (Whatman Ltd., England) employing a sterile Millipore Water Filtration system (Millipore Inc., USA). Filters containing filtrate were removed and placed in sterile 450 ml Tris-HCl [10 mM Tris HCl pH 8.0] and were sonicated in a 1.5 ml microfuge tube undergoing two cycles of sonication for 30 sec each (MSE 150-W ultrasonic disintegrator, model Mk2, minimal power) and cooled in an ice bath. The cells and particles remaining after lysis were removed by centrifugation (Minimix, 14,000 g) for a 15 sec burst and all genomic DNA in the supernatent was then extracted employing the Roche High Purity PCR Template Kit (Roche Ltd., England), in accordance with the manufacturer's instructions. Extracted DNA was transferred to a sterile tube and stored at ÿ 808C. All water samples were examined by PCR amplification of the flagellin A-flagellin B (flaA/flaB) gene as previously described (Wegmüller et al., 1993). Sensitivity assays were performed with known cell densities, as previously described (Kirk and Rowe, 1997).
Results Four water categories were positive for Campylobacter spp. by PCR as shown in Table 1, demonstrating a low prevalence of this organism in waters. The sensitivity of the PCR assay equated to between 10 ± 20 colony forming units per ml of water. No inhibition of PCR amplification was noted throughout demonstrating the lack of PCR inhibitors in all water samples examined.
Discussion The aim of this study was to examine the prevalence of Campylobacter spp. in a variety of waters, using a PCR-based assay as the method of detection. No waters were cultured using conventional methods due to concern for the potential of reversion to nonculturability from time of collection to laboratory analysis. With culture, the absence of a positive result does not equate to the water source being free of campylobacters. Therefore employment of a molecular based assay in such circumstances may be a more useful method of determining the potential of infectivity to humans from contaminated water supplies. In this study 2.2% of tap waters were positive. Normal water processing regimes allow initially for the physical filtration of drinking water through filters, including slow sand filtration, followed by disinfection (chlorination) of water supplies through
187
the introduction of chlorine. Campylobacters are sensitive to free residual levels of chlorine as found in water. Previously, Megraud and Serceau (1990) described the cultural isolation of atypical campylobacters from untreated water in Bordeaux, France, and these workers suggested that such campylobacters were sensitive to chlorination. Hence, it is unlikely that final processed tap water should be a source of these organisms, provided that the proper chlorination and quality control measures are in place to eliminate these pathogens. Therefore, in the present study, those PCR positive samples from tap water may represent water which was previously positive, i.e. possibly due to faecal contamination at the reservoir by birds and/or animals. A slightly higher proportion of recreational waters were positive, particularly water taken from municipal swimming pools. In this case although appropriate controls were in place including filtration and chlorination the events of faecal accidents in the pool especially with young children highlights the potential problem and risks associated with this recreational activity. Likewise, for whirlpools/spas and jacuzzies, these amenities traditionally have been associated with a high risk of acquiring an infection, especially Pseudomonas aeruginosa related infections, i.e. including otitis externa and folliculitis, as previously described (Berrouane et al. 2000). Previously, there have been several studies employing PCR techniques to detect campylobacters from waters (Oyofo and Rollins, 1993; Hernandez et al. 1995; Jackson et al. 1996). In a more recent publication, Waage et al. (1999) described a sensitive method based on PCR amplification of the intergenic sequence between the flagellin gene loci (flaA and flaB). Although the method was able to detect 3 to 15 colony forming units per 100 ml water of C. jejuni, there was a necessity to use an enrichment stage. However, by enriching the membrane filter in 10 ml Rosef broth, cells which are present in low numbers and also those which are non-culturable would not be detected, due to both the dilution factor of the enrichment broth and the potential PCR inhibitors introduced by the broth (Wilson, 1997). For all PCR positive samples, the absence of a positive culture would still not have equated to the sample being safe for human consumption or recreational use. Therefore all PCR positive samples should be further examined for viable campylobacters and hence infectivity by employing either appropriate animal models or further molecular characterisation such as RT-PCR. Regardless of this, all such PCR positive waters should therefore be queried as being potentially infectious due to the presence of the
188
J. Moore, P. Caldwell, B. Millar
Table 2. Advantages of the use of PCR-based techniques over cultural techniques in the detection of thermophilic campylobacters from waters. Assay type Test parameter
PCR-based assays
Culture-based assays
Filtration of test water Need for culture/selective enrichment of organisms Affect of antibiotics on assay Ability to detect non-culturable types Time to detection Specificity Cost Ability for rapid screening of large numbers of samples
yes no none yes 8h good moderate high
yes yes inhibition of certain sensitive strains no 48 ± 72 h moderate moderate low
viable but non-culturable form of this organism in water. Therefore employment of PCR-based techniques for the detection of campylobacters from waters, offers several advantages as outlined in Table 2, particularly the ability to screen large numbers of environmental samples without the need to culture, whereby only PCR-positive samples are processed further by conventional culture techniques. The presence of the non-culturable forms has significant importance to the water industry. One difficulty in elucidating the potential hazard of the viable but non-culturable cells is the inability to detect such cells in finished water by employing routine bacteriological methods. Furthermore any laboratory detection method that is employed must be capable of detecting low numbers (102) of campylobacters against a large background flora and other food materials, as campylobacters have a relatively low infective dose (Robinson 1981). As Campylobacter spp. are rapidly inactivated by chlorine, the use of untreated water, the failure of disinfection and poor water system maintenance are important causes of outbreaks. Bathing in surface water or pools has up to now not been reported, as a cause of human campylobacteriosis outbreaks, although it has been reported at a number of freshwater bathing sites (Obiri-Danso and Jones, 1999). In conclusion, untreated surface waters may represent a source of contamination with campylobacters in Northern Ireland, where they have a recreational involvement or are used as a drinking water source by either human or agricultural livestock. Therefore consideration of waterborne campylobacteriosis should be given to patients presenting with acute enteritis and a history of participation in water sports/activities. As faecal coliform organisms have been previously shown to be poor markers of water quality, especially for Campylobacter spp, new criteria should be established to assess the risk
from Campylobacter and to evaluate and monitor the quality of water used for recreational purposes. Acknowledgements. The authors wish to thank the staff of the Food Hygiene Laboratory, Northern Ireland Public Health Laboratory and the Environmental Health Officers from the 26 local district council regions in Northern Ireland, for their help and assistance with the collection of samples. This work was supported financially by the Department of Health and Social Services (Northern Ireland) and the European Social Fund.
References Anon. The microbiology of water. Part 1 ± drinking water. pp 58. Report on public health and medical subjects no. 71. HMSO, London (1994a). Anon. Hygiene for spa pools. Public Health Laboratory Service. Blackmore Press, Shaftsbury, England. pp 20 ± 30 (1994b). Berrouane, Y. F., McNutt, L. A., Buschelman, B. J., Rhomberg, P. R., Sanford, M. D., Hollis, R. J., Pfaller, M. A., Herwaldt, L. A. Outbreak of severe Pseudomonas aeruginosa infections caused by a contaminated drain in a whirlpool bathtub. Clin, Infect. Dis.31, 1331 ± 1337 (2000). Bokkenheuser, V. D., Mosenthal, A. C. Campylobacteriosis: A foodborne disease. J. Food Safe. 3, 127 ± 143 (1981). Bolton, F. J., Surman, S. B., Martin, K., Wareing, D. R., Humphrey, T. J. Presence of Campylobacter and Salmonella in sand from bathing beaches. Epidemiol. Infect. 122, 7 ± 13 (1999). Cowden, J.: Campylobacter: epidemiological paradoxes. Brit. Med. J. 305, 132 ± 133 (1992). Dekeyser, P., Gossuin-Detrain, M., Butzler, J. P. Sternon, J.: Acute enteritis due to related Vibrio: First positive stool cultures. J. Infect. Dis. 125, 390 ± 392 (1972). Hernandez, J., Alonso, J. L., Fayos, A., Amoros, I., Owen, R. J. Development of a PCR assay combined with a short enrichment culture for detection of
Campylobacter in water Campylobacter jejuni in estuarine surface waters. FEMS Microbiol. Lett. 127, 201 ± 206 (1995). Jackson, C. J., Fox, A. J., Jones, D. M. A. A novel polymerase chain reaction assay for the detection and speciation of thermophilic Campylobacter spp. J. Appl. Bacteriol. 81, 467 ± 473 (1996). Kirk, R., Rowe, MT. A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water. Lett Appl Microbiol. 19, 301 ± 303 (1994). Megraud, F., Serceau, R. Search for Campylobacter species in the public water supply of a large urban community. Zentralbl. Hyg. Umweltmed. 189, 536 ± 542 (1990). Obiri-Danso, K., Jones, K. Distribution and seasonality of microbial indicators and thermophilic campylobacters in two freshwater bathing sites on the River Lune in northwest England. J Appl Microbiol. 87, 822 ± 832 (1999). Oyofo, B. A., Rollins, D. M. Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction. Appl. Environ. Microbiol. 59, 4090 ± 4095 (1993).
189
Prescott, J. L., Munroe, D. L.: Campylobacter jejuni enterititis in human and domestic animals. J. Am. Vet. Med. Assoc. 12, 1524 ± 1530 (1982). Robinson, D. A.: Infective dose of Campylobacter jejuni in milk. Brit. Med. J. 282, 1584 (1981). Skirrow, M. B.: Campylobacter enteritis: a `new' disease. Brit. Med. J. 2, 9 ± 11 (1977). Skirrow, M. B.: Campylobacter. The Lancet 336, 921 ± 923 (1990). Vogt, R. L., Sours, H. E., Barrett, T., Feldman, R. A., Dickinson, R. J., Witherell, L. Campylobacter enteritis associated with contaminated water. Ann. of Intern. Med. 96, 292 ± 296 (1982). Waage, A. S., Vardund, T., Lund, V., Kapperud, G. Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay. Appl. Environ. Microbiol. 65, 1636 ± 1643 (1999). Wegmüller B, Luthy J, Candrian U.: Direct PCR detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products. Appl. Environ. Microbiol. 59: 2161 ± 2165 (1993). Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741 ± 3751 (1997).
Int. J. Hyg. Environ. Health 204, 185 ± 189 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Short Communication Molecular detection of Campylobacter spp. in drinking, recreational and environmental water supplies John Moore, Patricia Caldwell, Beverley Millar Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK Received: January 30, 2001 ´ Accepted: July 31, 2001
Abstract A molecular detection assay was performed on 207 samples of drinking, recreational and environmental waters collected in Northern Ireland. The water sources which were PCR positive for Campylobacter spp. included 2/91 (2.2%) drinking water from domestic household taps, 5/57 (8.8%) swimming pool water, 1/23 (4.3%) lake water and 1/1 water from a jacuzzi. Extracted DNA from all water samples was amplified employing a sequencespecific PCR assay based on a 206bp conserved region of the flagellin A-flagellin B (flaA/ flaB) loci for Campylobacter jejuni, C. coli and C. lari. Given the physiological and cultural fragile nature of these species, no waters were cultured using conventional methods due to concern for reversion to non-culturability from time of collection to laboratory analysis. As this genus has been demonstrated to form a `viable but non-culturable' (VBNC) form, failure to culture organisms conventionally from water does not necessarily equate to a negative result, hence molecular detection assays, especially those which can demonstrate cell viability, may be useful in helping to elucidate potential epidemiological sources and reservoirs of this organism, especially where water is suspected as being the vehicle of transmission. Key words: Campylobacter spp. ± PCR ± phenotyping ± water ± flaA
Introduction The past three decades have witnessed the rise of Campylobacter enteritis in humans from virtual obscurity to notoriety, with present isolation rates superceding those of other enteric pathogens such as Salmonella spp. and Shigella spp. in most developed countries (Skirrow, 1990). Unlike the salmonellae and other enteric pathogens, the majority (ca. 99%) of clinical reports concerning Campylobacter are sporadic and Campylobacter enteritis outbreaks are rare (Cowden, 1992). This may be due to discrimi-
natory genotyping schemes not being able to clearly identify outbreaks within the majority of sporadic cases. Additionally, campylobacters do not multiply in foodstuffs and are lethally damaged by drying and exposure to atmospheric oxygen. While it is difficult to determine the sources of individual cases, epidemiological investigations by case-control studies can identify likely sources or exposures. The lack of welldeveloped typing schemes has hindered the epidemiological investigations seeking natural reservoirs of the organism and modes of transmission from these sources to humans. Although campylobacters
Corresponding author: Dr. John E. Moore, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, United Kingdom, Phone: 44 28 9026 3554, Fax: 44(28) 2589 2887, E-mail: [email protected] 1438-4639/01/204-185 $ 15.00/0
186
J. Moore, P. Caldwell, B. Millar
are not completely new to applied bacteriology, they have evaded traditional techniques used for the isolation of pure cultures, apart from single isolations that were free from competing organisms. Until the development of a selective medium by Skirrow (1977), these organisms were known mainly by veterinarians as animal pathogens which were responsible for a wide variety of disorders in cattle, sheep and pigs (Bokkenheuser and Mosenthal 1981). Since the development of more sophisticated isolation techniques (Dekeyser et al. 1972), the true disease potential of these organisms has become apparent and today campylobacteriosis is regarded as a zoonoses, which is capable of being transmitted to humans, mainly through contaminated foodstuffs by a wide range of domestic animals (Prescott and Munroe 1982). Campylobacters can be detected using conventional culture (Bolton et al., 1999), however, they can be difficult to isolate in the laboratory due to their fastidiousness. There have been several reports of outbreak epidemiology implicating water as the vehicle of transmission of this organism to humans, however extended analyses of the water by conventional cultural techniques has not been successful in obtaining a positive culture. There are a number of possible explanations for this failure to detect viable organisms and this may not only be due to VBNC status. At the time of outbreak recognition and investigation, original water specimens may no longer be available and this may be exacerbated by problems associate with the viability of cells due to storage conditions including transport factors, time and temperature, prior to laboratory analyses. Hence it was the aim of this study to employ a molecular detection assay for a diverse variety of drinking, recreational and environmental waters, which could potentially act as a reservoir for human campylobacteriosis.
Materials and methods Collection and processing of water samples All samples (1000 ml) were collected by Environmental Health Officers from the 26 local council authorities within Northern Ireland in sterile plastic disposable containers. The samples were collected as part of a routine sampling programme from various and commercial premises (Table 1). In addition, samples were collected from various leisure facilities, such as public leisure centres and hotel leisure complexes. Surface waters from lakes, reservoirs, springs and wells were also analysed. All samples were transported and maintained at 48C prior to analysis and were processed within 24 ± 72 h following collection. In those cases, where free residual chlorine was
Table 1. Molecular prevalence of Campylobacter spp. in drinking, recreational and environmental water supplies in Northern Ireland. Water Type (i). Drinking waters Tap Spring Bore hole Well Bottled (ii). Recreational waters Swimming pool Jacuzzi Lough River Sea TOTAL
No. of samples examined
No. positive (% positive)
91 6 1 4 1
2 (2.2%) 0 0 0 0
57 1 23 17 6 207
5 (8.8%) 1 1 (4.3%) 0 0 9 (8.7%)
present, i.e. tap water, swimming pools and jacuzzi water, sodium thiosulphate was added at a concentration of 18 mg/l (18 ppm) to the sampling bottles, as previously described (Anon., 1994a), to neutralize up to 5 mg/l (5 ppm) residual chlorine in the sample. Treatment of drinking and recreational waters Drinking water from the tap was treated at local public water treatment works. Raw drinking water was subjected to the following processes, including (i) clarification for the removal of silt, algae, and colour. (ii) filtration for the removal of smaller particles remaining after clarification and also for the removal of iron and manganese from ground water sources. A variety of filters including rapid gravity sand filters were used to allow disinfection to be optimum. (iii) disinfection for the killing of any remaining bacteria in the water following filtration, by the introduction of chlorine to leave free residual chlorine in the finished water supply. Spring, bore-hole and well waters were not treated by any process, except by natural sedimentation. Likewise, for recreational waters, lough, river and sea waters were not treated by any process. Swimming pools were treated by automated systems by a combination of (i) chlorination and ozonation to 1.5 ± 2.0 ppm free residual chlorine and 0.5 ± 1.0 ppm ozone, respectively, (ii) maintenance of pH 7.4 ± 7.6 and (iii) continuous filtration through fine mesh, sand and carbon filters. Jacuzzi water was treated as previously described (Anon., 1994b). Molecular detection of Campylobacter spp. All DNA isolation procedures were carried out in a Class II Biological Safety Cabinet in a room physically separate from that used to set up reaction mixes and also from the `post-PCR' room in order to minimise the production of false-positive results. In all methods tested and where applicable, molecular grade water was employed (Biowhittaker Inc, Maryland, USA, LAL Grade cat no: W50100) to reduce contamination. The type strain C. jejuni NCTC 11351 and molecular grade water were employed as positive and negative extraction controls respectively.
Campylobacter in water Water samples (400 ml) were initially filtered through a sterile polycarbonate membrane filter (43 mm diameter; pore size 0.20 mm) (Whatman Ltd., England) employing a sterile Millipore Water Filtration system (Millipore Inc., USA). Filters containing filtrate were removed and placed in sterile 450 ml Tris-HCl [10 mM Tris HCl pH 8.0] and were sonicated in a 1.5 ml microfuge tube undergoing two cycles of sonication for 30 sec each (MSE 150-W ultrasonic disintegrator, model Mk2, minimal power) and cooled in an ice bath. The cells and particles remaining after lysis were removed by centrifugation (Minimix, 14,000 g) for a 15 sec burst and all genomic DNA in the supernatent was then extracted employing the Roche High Purity PCR Template Kit (Roche Ltd., England), in accordance with the manufacturer's instructions. Extracted DNA was transferred to a sterile tube and stored at ÿ 808C. All water samples were examined by PCR amplification of the flagellin A-flagellin B (flaA/flaB) gene as previously described (Wegmüller et al., 1993). Sensitivity assays were performed with known cell densities, as previously described (Kirk and Rowe, 1997).
Results Four water categories were positive for Campylobacter spp. by PCR as shown in Table 1, demonstrating a low prevalence of this organism in waters. The sensitivity of the PCR assay equated to between 10 ± 20 colony forming units per ml of water. No inhibition of PCR amplification was noted throughout demonstrating the lack of PCR inhibitors in all water samples examined.
Discussion The aim of this study was to examine the prevalence of Campylobacter spp. in a variety of waters, using a PCR-based assay as the method of detection. No waters were cultured using conventional methods due to concern for the potential of reversion to nonculturability from time of collection to laboratory analysis. With culture, the absence of a positive result does not equate to the water source being free of campylobacters. Therefore employment of a molecular based assay in such circumstances may be a more useful method of determining the potential of infectivity to humans from contaminated water supplies. In this study 2.2% of tap waters were positive. Normal water processing regimes allow initially for the physical filtration of drinking water through filters, including slow sand filtration, followed by disinfection (chlorination) of water supplies through
187
the introduction of chlorine. Campylobacters are sensitive to free residual levels of chlorine as found in water. Previously, Megraud and Serceau (1990) described the cultural isolation of atypical campylobacters from untreated water in Bordeaux, France, and these workers suggested that such campylobacters were sensitive to chlorination. Hence, it is unlikely that final processed tap water should be a source of these organisms, provided that the proper chlorination and quality control measures are in place to eliminate these pathogens. Therefore, in the present study, those PCR positive samples from tap water may represent water which was previously positive, i.e. possibly due to faecal contamination at the reservoir by birds and/or animals. A slightly higher proportion of recreational waters were positive, particularly water taken from municipal swimming pools. In this case although appropriate controls were in place including filtration and chlorination the events of faecal accidents in the pool especially with young children highlights the potential problem and risks associated with this recreational activity. Likewise, for whirlpools/spas and jacuzzies, these amenities traditionally have been associated with a high risk of acquiring an infection, especially Pseudomonas aeruginosa related infections, i.e. including otitis externa and folliculitis, as previously described (Berrouane et al. 2000). Previously, there have been several studies employing PCR techniques to detect campylobacters from waters (Oyofo and Rollins, 1993; Hernandez et al. 1995; Jackson et al. 1996). In a more recent publication, Waage et al. (1999) described a sensitive method based on PCR amplification of the intergenic sequence between the flagellin gene loci (flaA and flaB). Although the method was able to detect 3 to 15 colony forming units per 100 ml water of C. jejuni, there was a necessity to use an enrichment stage. However, by enriching the membrane filter in 10 ml Rosef broth, cells which are present in low numbers and also those which are non-culturable would not be detected, due to both the dilution factor of the enrichment broth and the potential PCR inhibitors introduced by the broth (Wilson, 1997). For all PCR positive samples, the absence of a positive culture would still not have equated to the sample being safe for human consumption or recreational use. Therefore all PCR positive samples should be further examined for viable campylobacters and hence infectivity by employing either appropriate animal models or further molecular characterisation such as RT-PCR. Regardless of this, all such PCR positive waters should therefore be queried as being potentially infectious due to the presence of the
188
J. Moore, P. Caldwell, B. Millar
Table 2. Advantages of the use of PCR-based techniques over cultural techniques in the detection of thermophilic campylobacters from waters. Assay type Test parameter
PCR-based assays
Culture-based assays
Filtration of test water Need for culture/selective enrichment of organisms Affect of antibiotics on assay Ability to detect non-culturable types Time to detection Specificity Cost Ability for rapid screening of large numbers of samples
yes no none yes 8h good moderate high
yes yes inhibition of certain sensitive strains no 48 ± 72 h moderate moderate low
viable but non-culturable form of this organism in water. Therefore employment of PCR-based techniques for the detection of campylobacters from waters, offers several advantages as outlined in Table 2, particularly the ability to screen large numbers of environmental samples without the need to culture, whereby only PCR-positive samples are processed further by conventional culture techniques. The presence of the non-culturable forms has significant importance to the water industry. One difficulty in elucidating the potential hazard of the viable but non-culturable cells is the inability to detect such cells in finished water by employing routine bacteriological methods. Furthermore any laboratory detection method that is employed must be capable of detecting low numbers (102) of campylobacters against a large background flora and other food materials, as campylobacters have a relatively low infective dose (Robinson 1981). As Campylobacter spp. are rapidly inactivated by chlorine, the use of untreated water, the failure of disinfection and poor water system maintenance are important causes of outbreaks. Bathing in surface water or pools has up to now not been reported, as a cause of human campylobacteriosis outbreaks, although it has been reported at a number of freshwater bathing sites (Obiri-Danso and Jones, 1999). In conclusion, untreated surface waters may represent a source of contamination with campylobacters in Northern Ireland, where they have a recreational involvement or are used as a drinking water source by either human or agricultural livestock. Therefore consideration of waterborne campylobacteriosis should be given to patients presenting with acute enteritis and a history of participation in water sports/activities. As faecal coliform organisms have been previously shown to be poor markers of water quality, especially for Campylobacter spp, new criteria should be established to assess the risk
from Campylobacter and to evaluate and monitor the quality of water used for recreational purposes. Acknowledgements. The authors wish to thank the staff of the Food Hygiene Laboratory, Northern Ireland Public Health Laboratory and the Environmental Health Officers from the 26 local district council regions in Northern Ireland, for their help and assistance with the collection of samples. This work was supported financially by the Department of Health and Social Services (Northern Ireland) and the European Social Fund.
References Anon. The microbiology of water. Part 1 ± drinking water. pp 58. Report on public health and medical subjects no. 71. HMSO, London (1994a). Anon. Hygiene for spa pools. Public Health Laboratory Service. Blackmore Press, Shaftsbury, England. pp 20 ± 30 (1994b). Berrouane, Y. F., McNutt, L. A., Buschelman, B. J., Rhomberg, P. R., Sanford, M. D., Hollis, R. J., Pfaller, M. A., Herwaldt, L. A. Outbreak of severe Pseudomonas aeruginosa infections caused by a contaminated drain in a whirlpool bathtub. Clin, Infect. Dis.31, 1331 ± 1337 (2000). Bokkenheuser, V. D., Mosenthal, A. C. Campylobacteriosis: A foodborne disease. J. Food Safe. 3, 127 ± 143 (1981). Bolton, F. J., Surman, S. B., Martin, K., Wareing, D. R., Humphrey, T. J. Presence of Campylobacter and Salmonella in sand from bathing beaches. Epidemiol. Infect. 122, 7 ± 13 (1999). Cowden, J.: Campylobacter: epidemiological paradoxes. Brit. Med. J. 305, 132 ± 133 (1992). Dekeyser, P., Gossuin-Detrain, M., Butzler, J. P. Sternon, J.: Acute enteritis due to related Vibrio: First positive stool cultures. J. Infect. Dis. 125, 390 ± 392 (1972). Hernandez, J., Alonso, J. L., Fayos, A., Amoros, I., Owen, R. J. Development of a PCR assay combined with a short enrichment culture for detection of
Campylobacter in water Campylobacter jejuni in estuarine surface waters. FEMS Microbiol. Lett. 127, 201 ± 206 (1995). Jackson, C. J., Fox, A. J., Jones, D. M. A. A novel polymerase chain reaction assay for the detection and speciation of thermophilic Campylobacter spp. J. Appl. Bacteriol. 81, 467 ± 473 (1996). Kirk, R., Rowe, MT. A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water. Lett Appl Microbiol. 19, 301 ± 303 (1994). Megraud, F., Serceau, R. Search for Campylobacter species in the public water supply of a large urban community. Zentralbl. Hyg. Umweltmed. 189, 536 ± 542 (1990). Obiri-Danso, K., Jones, K. Distribution and seasonality of microbial indicators and thermophilic campylobacters in two freshwater bathing sites on the River Lune in northwest England. J Appl Microbiol. 87, 822 ± 832 (1999). Oyofo, B. A., Rollins, D. M. Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction. Appl. Environ. Microbiol. 59, 4090 ± 4095 (1993).
189
Prescott, J. L., Munroe, D. L.: Campylobacter jejuni enterititis in human and domestic animals. J. Am. Vet. Med. Assoc. 12, 1524 ± 1530 (1982). Robinson, D. A.: Infective dose of Campylobacter jejuni in milk. Brit. Med. J. 282, 1584 (1981). Skirrow, M. B.: Campylobacter enteritis: a `new' disease. Brit. Med. J. 2, 9 ± 11 (1977). Skirrow, M. B.: Campylobacter. The Lancet 336, 921 ± 923 (1990). Vogt, R. L., Sours, H. E., Barrett, T., Feldman, R. A., Dickinson, R. J., Witherell, L. Campylobacter enteritis associated with contaminated water. Ann. of Intern. Med. 96, 292 ± 296 (1982). Waage, A. S., Vardund, T., Lund, V., Kapperud, G. Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay. Appl. Environ. Microbiol. 65, 1636 ± 1643 (1999). Wegmüller B, Luthy J, Candrian U.: Direct PCR detection of Campylobacter jejuni and Campylobacter coli in raw milk and dairy products. Appl. Environ. Microbiol. 59: 2161 ± 2165 (1993). Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741 ± 3751 (1997).