- Email: [email protected]
Rapid enumeration of Salmonella in environmental waters and wastewater
PII: S0043-1354(99)00401-7
Wat. Res. Vol. 34, No. 8, pp. 2397±2399, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
TECHNICAL NOTE RAPID ENUMERATION OF SALMONELLA IN ENVIRONMENTAL WATERS AND WASTEWATER BELLA S. W. HO1M and T.-Y. TAM2*M 1
Environmental Microbiology Section, Waste Policy and Services Group, Environmental Protection Department, Government of Hong Kong, Revenue Tower, 5 Gloucester Road, Wanchai, Hong Kong and 2Local Control Oce (Territory West), Environmental Protection Department, Government of Hong Kong, 7/F Chinachem Tsuen Wan Plaza, 455±457 Castle Peak Road, New Territories, Hong Kong (First received 1 April 1999; accepted in revised form 1 September 1999) AbstractÐWe have incorporated an enzyme-linked ¯uorescent immunoassay technique into the conventional multiple-tube analytical procedure to rapidly enumerate Salmonella in environmental waters and wastewater. Fifty-two environmental water and wastewater samples from diverse sources were used in the present study. The concentrations of Salmonella present ranged from 1 to 4.6 103 per l, of which 22 were merely 1 per l. The technique rapidly screened all positive samples (i.e. samples with Salmonella ), hence, shortened the enumeration of Salmonella in environmental waters and wastewater from ®ve days to three. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐSalmonella, screening, enumeration, most-probable-number, enzyme-linked ¯uorescent immunoassay, environmental waters, wastewater
INTRODUCTION
Salmonella is a bacterial pathogen that may be excreted in faeces and may end up in environmental waters through sewage pollution (Ashbolt, 1996). The determination of level of Salmonella in environmental waters and wastewater is required in assessing the related health risk. However, only tests with high sensitivity can be used as the level of Salmonella in environmental waters is often very low. Conventionally, the level of Salmonella in environmental waters and wastewater is determined by the multiple-tube method (APHA, 1992). The concentration in terms of most-probable-number (MPN) is estimated by reference to a probability table. The whole process of detection and enumeration takes about ®ve days to complete. The process is both time-consuming and labour-intensive. Furthermore, Salmonella was often absent in a large proportion of the environmental water samples we had examined so far; thus it would be ideal to have a rapid screening test for subsequent enumeration by the multiple-tube method. It would also be desirable to
*Author to whom all correspondence should be addressed. Tel.: +852-2411-9604; fax: +852-2611-9149; e-mail: [email protected]
develop a procedure to shorten the duration for the whole enumeration process. The widespread use of any enzyme-linked ¯uorescent immunoassay technique capable of rapid detection of Salmonella in food is further enhanced by the availability of a relevant immunoanalyzer commercially. In the present study, we assessed the suitability of incorporating a similar technique and instrumentation into the conventional multiple-tube analytical procedure for routine enumeration of Salmonella in environmental waters and wastewater. We found that the technique allowed rapid screening, thus shortening the whole duration for enumeration of Salmonella from ®ve days to three. MATERIALS AND METHODS
Environmental waters and wastewater Environmental water and wastewater samples were collected from dierent environmental sources for the present study. These included a total of 52 water samples comprising seven river water, 27 bathing beach water, ®ve marine coastal water, one well water, eight stream water and four sewage samples. They were enumerated for Salmonella using both the conventional multiple-tube method alone and in conjunction with the immunoassay technique. Conventional multiple-tube method
2397
A three-tube approach was used for the conventional
2398
Bella S. W. Ho and T.-Y. Tam
multiple-tube method, i.e. three tubes of each dilution were prepared and ®ltered. For water samples from sources with less sewage contamination, triplicates of 500, 100 and 10 ml were ®ltered; whereas for sewage samples, higher dilutions were made before ®ltration. Each membrane ®lter was then inoculated into a bottle of Buered Peptone Water (BPW) (Oxoid, CM509) for pre-enrichment 378C for 24 h. An aliquot of 0.2 ml was subsequently enriched in 20 ml of Rappaport Vassiliadis (RV) broth (Oxoid, CM669) at 428C for 48 h. To examine for Salmonella-suspected colonies, each RV broth culture was plated onto Xylose Lysine Desoxycholate (XLD) agar (Oxoid, CM469) and incubated at 378C for 242 h. Suspected colonies were identi®ed using the VITEK (bioMerieux Inc., USA) automatic microbial identi®cation system; this ®nal con®rmation step took about 24 h to complete. The number of Salmonella-positive tubes in each dilution was then recorded and the MPN of Salmonella present in the sample was back-calculated by reading from the MPN table. Procedure for Salmonella immunoassay technique After pre-enrichment for 24 h in BPW as described in the conventional multiple-tube method above, aliquots of 0.1 and 1 ml pre-enriched BPW broth (from each tube of the lowest dilution) were subcultured into 10 ml of RV broth and 10 ml of Selenite Cystine (SC) broth [4 g per l sodium biselenite (Oxoid L121) and 19 g per l SC broth base (Oxoid CM699)], respectively. Both RV and SC broths were incubated for 6±8 h at respective temperatures of 42 and 378C. Subsequently, aliquots of 1 ml from RV and SC broths were each subcultured by inoculation into a 10-ml M broth (bioMerieux Inc., USA). After incubation for 18 h at 428C, 1 ml from each RV- and SCinoculated M broths were mixed together thoroughly and heated for 15 min at 1008C. An aliquot of 500 ml of this 2 boiled mixture was added onto a VIDAS Salmonella reagent-strip impregnated with a speci®c substrate for ¯uorescence production in the presence of Salmonella. Positive tubes could therefore be identi®ed and recorded and the number of Salmonella present in each sample was then back-calculated. 2 The VIDAS immunoanalyzer (bioMerieux Inc., USA) system was used for the detection of ¯uorescence production. Essentially, the process involved the antigens of Salmonella present reacted with the Salmonella-speci®c antibodies on the pre-coated wall of the solid phase receptacle inside the analyzer system. The antigen-antibody complex thus formed in turn reacted with a second anti-
body conjugated with an enzyme to form a large complex. This larger complex further reacted with a speci®c sub2 strate impregnated on a VIDAS Salmonella reagent-strip to produce a ¯uorescent endproduct which could be easily detected by the system. Sensitivity and speci®city of the detection system were ®rst checked using a pure culture of Salmonella. An overnight broth culture of Salmonella (containing approximately 108 cells per ml) was serially 10-fold diluted with Ringer's solution to give a range of cell concentrations between 0.1 to 105 per 100 ml. A volume of 250 ml was prepared for each dilution and each was then enumerated using the same immunoassay protocol as described above. The concentrations of Salmonella in the serial dilutions were all further con®rmed with plate-counting on Trypticase Soy Agar. RESULTS
Among the 52 environmental water samples tested, 34 were shown to be positive for Salmonella by the conventional multiple-tube method. Salmonella was not detected in the remaining 18 samples (Table 1). The level of Salmonella found in the positive samples ranged from 1 to 4.6 103 per l and among them 65% (i.e. 22 samples) contained a very low Salmonella level of less than 10 per l. The screening results obtained using the technique were also in good agreement with those obtained using the conventional multiple-tube method (Table 1). Hence, the speci®city of Salmonella immunoassay technique for screening Salmonella in environmental waters and wastewater was 100% on a per sample basis. When an ``individual'' result was obtained from each tube (i.e. on a per tube basis) of the lowest dilution based on the conventional multiple-tube method was used to compare with that based on the method incorporating the Salmonella immunoassay technique, the sensitivity and speci®city of the technique was 97.7 and 94.3%, respectively (Table 2). Four of the tubes were found immunoassay-positive but culture-negative; of which three did contain Salmonella, whereas in two water samples
Table 1. Comparison of Salmonella screening results on a per sample basis Concentration of Salmonella per l
Type
Number of samples tested
Number of positive samples Conventional MPN method
Immunoassay technique
0
Bathing beach water Well water
17 1
0
0
1±10
Bathing beach water Marine coastal water River water Stream water
10 4 6 2
22
22
11±100
Stream water Marine coastal water
4 1
5
5
101±1000
River water Stream water Sewage
1 1 1
3
3
> 1000
Stream water Sewage
1 3
4
4
Salmonella in environmental waters and wastewater
2399
Table 2. Comparison of Salmonella screening results on a per tube basis Number of tubes
Immunoassay-positive Immunoassay-negative
Conventional MPN culture-positive
Conventional MPN culture-negative
86 2
4 66
with very low concentrations (1 per l), there were two tubes both of which did contain Salmonella and were found immunoassay-negative but culturepositive; thus a slightly lower than ideal (100%) sensitivity for an individual tube containing such a low level of Salmonella screened by the immunoassay technique was observed. DISCUSSION
Environmental samples of a dierent nature have been tested in the present study. The sample types tested covered almost all the types that might be encountered in a water quality analytical laboratory. Our results indicated that, even for samples with a low Salmonella concentration (i.e. 1 per l), the immunoassay technique was able to give a clear positive signal with high accuracy. Even with samples containing high concentrations of mixed bacterial ¯ora, e.g. sewage samples, the immunoassay technique would still detect the presence of Salmonella. In the present study, we found that the Salmonella immunoassay technique was capable of detecting a very low level of Salmonella (1 per l). The immunoassay technique could therefore reliably be used as a rapid screening tool for detecting Salmonella in environmental waters and wastewater. When the multiple-tube method is performed in conjunction with the immunoassay technique, the enumeration of Salmonella in an environmental sample could be easily accomplished within three
days instead of the usual ®ve-day period when using the conventional multiple-tube method alone. The present results have shown the practical application of the immunoassay technique routinely screening for Salmonella in environmental waters and wastewater in conjunction with using the conventional multiple-tube method. Our study also suggested that, besides Salmonella, a similar immunoassay technique (simply by replacing the reagent strips with those impregnated with appropriate substrates) could be applicable to screening and enumeration of other bacterial pathogens such as Campylobacter, E. coli O157, etc. AcknowledgementsÐThe expert technical assistance of Mr Jonathan Tse, Misses Florence Ho and Niki Ho is gratefully acknowledged. We thank Drs David Ha and Malcolm Broom for perusal of this manuscript and the Director of Environmental Protection for permission to publish this paper. The views in this paper are those of the authors; and do not necessarily represent the view of the Government of Hong Kong SAR. REFERENCES
Ashbolt N. J. (1996) Human health risk from microorganisms in the Australian marine environment. Technical Annex 2 of the ``State of the Marine Environment Report for Australia. 16 pp. APHA (1992) Quantitative Salmonella procedures. In: Standard Methods for the Examination of Water and Wastewater, Part 9260D. Greenberg AE, Clesceri LS, Eaton AD (eds), 18th edn. American Publication Health Association, Washington, DC, USA. pp. 91±92.
Wat. Res. Vol. 34, No. 8, pp. 2397±2399, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
TECHNICAL NOTE RAPID ENUMERATION OF SALMONELLA IN ENVIRONMENTAL WATERS AND WASTEWATER BELLA S. W. HO1M and T.-Y. TAM2*M 1
Environmental Microbiology Section, Waste Policy and Services Group, Environmental Protection Department, Government of Hong Kong, Revenue Tower, 5 Gloucester Road, Wanchai, Hong Kong and 2Local Control Oce (Territory West), Environmental Protection Department, Government of Hong Kong, 7/F Chinachem Tsuen Wan Plaza, 455±457 Castle Peak Road, New Territories, Hong Kong (First received 1 April 1999; accepted in revised form 1 September 1999) AbstractÐWe have incorporated an enzyme-linked ¯uorescent immunoassay technique into the conventional multiple-tube analytical procedure to rapidly enumerate Salmonella in environmental waters and wastewater. Fifty-two environmental water and wastewater samples from diverse sources were used in the present study. The concentrations of Salmonella present ranged from 1 to 4.6 103 per l, of which 22 were merely 1 per l. The technique rapidly screened all positive samples (i.e. samples with Salmonella ), hence, shortened the enumeration of Salmonella in environmental waters and wastewater from ®ve days to three. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐSalmonella, screening, enumeration, most-probable-number, enzyme-linked ¯uorescent immunoassay, environmental waters, wastewater
INTRODUCTION
Salmonella is a bacterial pathogen that may be excreted in faeces and may end up in environmental waters through sewage pollution (Ashbolt, 1996). The determination of level of Salmonella in environmental waters and wastewater is required in assessing the related health risk. However, only tests with high sensitivity can be used as the level of Salmonella in environmental waters is often very low. Conventionally, the level of Salmonella in environmental waters and wastewater is determined by the multiple-tube method (APHA, 1992). The concentration in terms of most-probable-number (MPN) is estimated by reference to a probability table. The whole process of detection and enumeration takes about ®ve days to complete. The process is both time-consuming and labour-intensive. Furthermore, Salmonella was often absent in a large proportion of the environmental water samples we had examined so far; thus it would be ideal to have a rapid screening test for subsequent enumeration by the multiple-tube method. It would also be desirable to
*Author to whom all correspondence should be addressed. Tel.: +852-2411-9604; fax: +852-2611-9149; e-mail: [email protected]
develop a procedure to shorten the duration for the whole enumeration process. The widespread use of any enzyme-linked ¯uorescent immunoassay technique capable of rapid detection of Salmonella in food is further enhanced by the availability of a relevant immunoanalyzer commercially. In the present study, we assessed the suitability of incorporating a similar technique and instrumentation into the conventional multiple-tube analytical procedure for routine enumeration of Salmonella in environmental waters and wastewater. We found that the technique allowed rapid screening, thus shortening the whole duration for enumeration of Salmonella from ®ve days to three. MATERIALS AND METHODS
Environmental waters and wastewater Environmental water and wastewater samples were collected from dierent environmental sources for the present study. These included a total of 52 water samples comprising seven river water, 27 bathing beach water, ®ve marine coastal water, one well water, eight stream water and four sewage samples. They were enumerated for Salmonella using both the conventional multiple-tube method alone and in conjunction with the immunoassay technique. Conventional multiple-tube method
2397
A three-tube approach was used for the conventional
2398
Bella S. W. Ho and T.-Y. Tam
multiple-tube method, i.e. three tubes of each dilution were prepared and ®ltered. For water samples from sources with less sewage contamination, triplicates of 500, 100 and 10 ml were ®ltered; whereas for sewage samples, higher dilutions were made before ®ltration. Each membrane ®lter was then inoculated into a bottle of Buered Peptone Water (BPW) (Oxoid, CM509) for pre-enrichment 378C for 24 h. An aliquot of 0.2 ml was subsequently enriched in 20 ml of Rappaport Vassiliadis (RV) broth (Oxoid, CM669) at 428C for 48 h. To examine for Salmonella-suspected colonies, each RV broth culture was plated onto Xylose Lysine Desoxycholate (XLD) agar (Oxoid, CM469) and incubated at 378C for 242 h. Suspected colonies were identi®ed using the VITEK (bioMerieux Inc., USA) automatic microbial identi®cation system; this ®nal con®rmation step took about 24 h to complete. The number of Salmonella-positive tubes in each dilution was then recorded and the MPN of Salmonella present in the sample was back-calculated by reading from the MPN table. Procedure for Salmonella immunoassay technique After pre-enrichment for 24 h in BPW as described in the conventional multiple-tube method above, aliquots of 0.1 and 1 ml pre-enriched BPW broth (from each tube of the lowest dilution) were subcultured into 10 ml of RV broth and 10 ml of Selenite Cystine (SC) broth [4 g per l sodium biselenite (Oxoid L121) and 19 g per l SC broth base (Oxoid CM699)], respectively. Both RV and SC broths were incubated for 6±8 h at respective temperatures of 42 and 378C. Subsequently, aliquots of 1 ml from RV and SC broths were each subcultured by inoculation into a 10-ml M broth (bioMerieux Inc., USA). After incubation for 18 h at 428C, 1 ml from each RV- and SCinoculated M broths were mixed together thoroughly and heated for 15 min at 1008C. An aliquot of 500 ml of this 2 boiled mixture was added onto a VIDAS Salmonella reagent-strip impregnated with a speci®c substrate for ¯uorescence production in the presence of Salmonella. Positive tubes could therefore be identi®ed and recorded and the number of Salmonella present in each sample was then back-calculated. 2 The VIDAS immunoanalyzer (bioMerieux Inc., USA) system was used for the detection of ¯uorescence production. Essentially, the process involved the antigens of Salmonella present reacted with the Salmonella-speci®c antibodies on the pre-coated wall of the solid phase receptacle inside the analyzer system. The antigen-antibody complex thus formed in turn reacted with a second anti-
body conjugated with an enzyme to form a large complex. This larger complex further reacted with a speci®c sub2 strate impregnated on a VIDAS Salmonella reagent-strip to produce a ¯uorescent endproduct which could be easily detected by the system. Sensitivity and speci®city of the detection system were ®rst checked using a pure culture of Salmonella. An overnight broth culture of Salmonella (containing approximately 108 cells per ml) was serially 10-fold diluted with Ringer's solution to give a range of cell concentrations between 0.1 to 105 per 100 ml. A volume of 250 ml was prepared for each dilution and each was then enumerated using the same immunoassay protocol as described above. The concentrations of Salmonella in the serial dilutions were all further con®rmed with plate-counting on Trypticase Soy Agar. RESULTS
Among the 52 environmental water samples tested, 34 were shown to be positive for Salmonella by the conventional multiple-tube method. Salmonella was not detected in the remaining 18 samples (Table 1). The level of Salmonella found in the positive samples ranged from 1 to 4.6 103 per l and among them 65% (i.e. 22 samples) contained a very low Salmonella level of less than 10 per l. The screening results obtained using the technique were also in good agreement with those obtained using the conventional multiple-tube method (Table 1). Hence, the speci®city of Salmonella immunoassay technique for screening Salmonella in environmental waters and wastewater was 100% on a per sample basis. When an ``individual'' result was obtained from each tube (i.e. on a per tube basis) of the lowest dilution based on the conventional multiple-tube method was used to compare with that based on the method incorporating the Salmonella immunoassay technique, the sensitivity and speci®city of the technique was 97.7 and 94.3%, respectively (Table 2). Four of the tubes were found immunoassay-positive but culture-negative; of which three did contain Salmonella, whereas in two water samples
Table 1. Comparison of Salmonella screening results on a per sample basis Concentration of Salmonella per l
Type
Number of samples tested
Number of positive samples Conventional MPN method
Immunoassay technique
0
Bathing beach water Well water
17 1
0
0
1±10
Bathing beach water Marine coastal water River water Stream water
10 4 6 2
22
22
11±100
Stream water Marine coastal water
4 1
5
5
101±1000
River water Stream water Sewage
1 1 1
3
3
> 1000
Stream water Sewage
1 3
4
4
Salmonella in environmental waters and wastewater
2399
Table 2. Comparison of Salmonella screening results on a per tube basis Number of tubes
Immunoassay-positive Immunoassay-negative
Conventional MPN culture-positive
Conventional MPN culture-negative
86 2
4 66
with very low concentrations (1 per l), there were two tubes both of which did contain Salmonella and were found immunoassay-negative but culturepositive; thus a slightly lower than ideal (100%) sensitivity for an individual tube containing such a low level of Salmonella screened by the immunoassay technique was observed. DISCUSSION
Environmental samples of a dierent nature have been tested in the present study. The sample types tested covered almost all the types that might be encountered in a water quality analytical laboratory. Our results indicated that, even for samples with a low Salmonella concentration (i.e. 1 per l), the immunoassay technique was able to give a clear positive signal with high accuracy. Even with samples containing high concentrations of mixed bacterial ¯ora, e.g. sewage samples, the immunoassay technique would still detect the presence of Salmonella. In the present study, we found that the Salmonella immunoassay technique was capable of detecting a very low level of Salmonella (1 per l). The immunoassay technique could therefore reliably be used as a rapid screening tool for detecting Salmonella in environmental waters and wastewater. When the multiple-tube method is performed in conjunction with the immunoassay technique, the enumeration of Salmonella in an environmental sample could be easily accomplished within three
days instead of the usual ®ve-day period when using the conventional multiple-tube method alone. The present results have shown the practical application of the immunoassay technique routinely screening for Salmonella in environmental waters and wastewater in conjunction with using the conventional multiple-tube method. Our study also suggested that, besides Salmonella, a similar immunoassay technique (simply by replacing the reagent strips with those impregnated with appropriate substrates) could be applicable to screening and enumeration of other bacterial pathogens such as Campylobacter, E. coli O157, etc. AcknowledgementsÐThe expert technical assistance of Mr Jonathan Tse, Misses Florence Ho and Niki Ho is gratefully acknowledged. We thank Drs David Ha and Malcolm Broom for perusal of this manuscript and the Director of Environmental Protection for permission to publish this paper. The views in this paper are those of the authors; and do not necessarily represent the view of the Government of Hong Kong SAR. REFERENCES
Ashbolt N. J. (1996) Human health risk from microorganisms in the Australian marine environment. Technical Annex 2 of the ``State of the Marine Environment Report for Australia. 16 pp. APHA (1992) Quantitative Salmonella procedures. In: Standard Methods for the Examination of Water and Wastewater, Part 9260D. Greenberg AE, Clesceri LS, Eaton AD (eds), 18th edn. American Publication Health Association, Washington, DC, USA. pp. 91±92.