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Detection of Legionella bacteria in sewage by polymerase chain reaction and standard culture method
E)
Pergamon 0273-1223(95)00302-9
Wal. Sci. Tech. Vol. 31, No. 5-6, pp. 409-416,1995. Copyright@ 1995 IAWQ Printed in Great Bntaio. All rights reserved. 0273-1223/95 $9'50 + 0'00
DETECTION OF LEGIONELLA BACTERIA IN SEWAGE BY POLYMERASE CHAIN REACTION AND STANDARD CULTURE METHOD Bruce M. Roll and Roger S. Fujioka Water Resources Research Center and Department ofMicrobiology, University of Hawaii, Honolulu. H/96822, USA
Abstract
Legionella bacteria are ubiquitous in environmental waters. Only a few species of Legionella, especially, L. pneumophila are pathogenic to humans and cause a sometimes fatal Legionnnaires disease as well as a less fatal disease called Pontiac fever. The presence of
Legionella in sewage and aerosolized sewage is the subject of this investigation because reuse
of sewage may involve the exposure of people to aerosolization, the mode of transmission of
Legionella bacteria. The objective of this study was to determine the prevalence of Legionella species and L. pneumophila in wastewater and their fate after various stages of treatment. The polymerase chain reaction (PCR) and standard culture method were utilized to detect
Legionella species and L. pneumophila. PCR results indicated that Legionella species were 3
present at levels > 10 cells / ml during all phases of sewage treatment including chlorinated effluents. Culture results indicatedlevels at least one log lower than seen with PCR. Legionella species were also recovered from air samples collected from secondary aeration basins at levels 3 < 10 cells/ml. PCR was shown to be the most rapid and sensitive method for detecting Legionella in sewage. KEYWORDS
Legionella, detection, polymerase chain reaction (PCR). culture method. wastewater, seweage, aerosolized sewage
Introduction In 1976 at a Legionnaires' convention in Philadelphia, an epidemic occurred that led to the discovery of Legionella pneumophila (Fraser et aI., 1977). Since its discovery, the family Legionellae has been responsible for numerous outbreaks of Legionnaires disease and non• pneumonic Pontiac fever. To date, 48 species of Legionella have been identified with eighteen of these species being associated with either Legionnaire's disease or Pontiac fever. According to the Centers for Disease Control (CDC), the vast majority (85%) of all Legionella pneumonias are caused by the species Legionella pneumophila (Reingold et aI., 1984). The major vehicle of transmission for these diseases is the inhalation of aerosols containing 409
B. M. ROLL and R. S. FUJIOKA
410
Legionel/a. Although Legionel/a appears to be a normal inhabitant of most aquatic environments, under certain condition their numbers may increase. This is of particular concern when virulent strains of Legionel/a are able to multiply to high concentrations in aquatic environments. Cooling towers (Addiss et aI., 1989), whirl pool baths (Mangione, et aI., 1982), and showers (Bollin et aI., 1985) have been shown to be sources for transmission of Le~ionella. As more wastewaters are directed toward reuse, it is critical that we know the disease potential of these waters. Research conducted in a temperate climate (palmer et aI.,1993) demonstrated that sewage is a potential reservoir for Legionel/a. To date, however, no studies have surveyed the prevalence of Legionel/a in sewage found in tropical climates. In addition it is critical that we know the disease potential of this organism for sewage treatment plant, workers who may be exposed to aerosols created during the sewage treatment process. The purpose of this study was to determine the prevalence of Legionel/a in the various stages of sewage treatment at a wastewater treatment plant in a tropical climate. This study utilized the polymerase chain reaction (PCR) and standard culture methods to examine various stages of sewage treatment for the presence of Legionella pneumophila and other Legionella species. In addition, air samples were collected from secondary aeration basins and analyzed for the presence of Legionel/a. Materials and Methods Water Sampling. Wastewater samples were collected from the Wahiawa sewage treatment facility located on the island of Oahu in the State of Hawaii. This treatment plant utilizes settling tanks, aeration basins (activated sludge) and chlorination to treat sewage. Samples of raw, primary, secondary treated and chlorinated wastewater were collected in sterile 500 ml nalgene plastic bottles containing sodium thiosulfate (40mg). transported at
40 C
All samples were
and analyzed within 4 hours of collection.
Air Sampling. Air samples were collected from aerated activated sludge basins using a portable vacuum pump (Nelson model 921 Berkeley, California) connected to the side port of a WOO ml erlenmeyer vacuum flask. The flask contained an aquarium bubbling stone and was connected to a glass funnel that served as an air collector. The apparatus was sterilized by autoclaving and filled with 800 ml of sterile phosphate buffered water. The flask was then placed in an ice cooler and maintained at 40C during the sampling period. The collection funnel was suspended four feet above the water surface in the secondary aeration basin. Samples were collected over a four hour period at an air flow rate of 283 liters per hour.
Detection of Ltgionella bacteria in sewage
411
During this time approximately 1133 liters of air was bubbled through the phosphate buffer. These air treated buffer samples were transported at 40 C and analyzed within 4 hours of collection. Detection of Legionella Using PCR. For genetic detection of Legionella, the EnviroAMP~ kit developed by Perkin Elmer Roche (Alameda, CAl
was used as per the
manufacturer's instructions. Many samples required 1:10 or 1:100 dilutions to eliminate interference caused by undefmed subsJances in sewage. Detection of Legionella species and L.
pnewnophi/a was accomplished by filtering 10 ml of the sewage sample and using PCR to amplify DNA sequences in the conserved regions of the 5S ribosomal RNA gene (Chumkov et
aI., 1986, MacDonell and Caldwell. 1987, Wolters and Erdmann, I988)andsequences from the miD. (macrophage infectivity potentiator) gene, unique to L. pnewnophila (Cianciotto et aI., 1990, Engleberg et aI., 1989). PCR products were detected by a reverse dot blot technique using
Legionella specific probes immobilized on nylon membrane strips. Biotinylated primers were used and the products which reacted with the probes were detected as colored dots resulting from a streptavidin-horseradish peroxidase conjugate reaction. Formation of a blue "L" dot on the strip indicated the presence one or more of twenty Legionella species. Formation of a blue "P" dot indicated the presence of L. pnewnophi/a. Positive and negative controls are built into the strip. The test is semi-quantitative since the intensity of the blue positive control dot is 3
equivalent to 10 cells per mI. When the "L" or " P" dots were darker than the control dot, the sample was considered to contain
> lif cells per ml.
When the "L" or "P" dots were lighter
than the positive control dot the sample was considered to contain
<
103 cells per mI.
Culture Isolation. Ten mls of sewage was filtered through a 0.45 l1m HLVP Millipore filter(Bedford, MA) and pretreated with acid(Bopp etal., 198 I) to inactivate competing organisms. Following acid treatment 0.1 ml samples were spread plated onto buffered charcoal-yeast extract agar containing a-ketogluterate
and glycine (3 gIl) and supplemented with
cyclohexamide (80mg/I), vancomycin (5 mg/I) and polymyxin B (lOOUlml). The plates were then incubated at 37"C in a humid 4 % CO2 environment for 5 to 20 days. Typical Legionella colonies appeared light blue to gray-white.
412
B. M. ROLL and R. S. FUJIOKA
Confinnation of Legionella Colonies. Suspected Legionella colonies were confirmed using direct fluorescent antibody staining with FITC labelled Legionella polyclonal antibody (Scimedix, Denville, N. J.) that detected both L. pneumophila and other Legionella species.
Results Recovery of Legionella From Sewage. Using PCR, Legionella gene sequences were 3
detected in all phases of sewage treatment and at levels> 10 cells per ml (Table 1). Of the 3
18 raw sewage samples collected, 89 % demonstrated levels above 10 cells/ml. Legionella 3
species were detected in excess of 10 cell/ml in 100 % of the samples collected from primary effluent, secondary effluent and even chlorinated effluent containing an average of 1.20 mg/l of free chlorine. Of the 54 samples collected throughout the treatment process, L. pneumophila was detected in only one sample (secondary effluent). Plate culture did not detect L.
pneumophila but detected the presence of Legionella species in 67 % of raw sewage, in 64 % of primary effluent, in 38 % secondary effluent, and in 10 % of chlorinated effluent (Table 2). The recovered concentration of Legionella ranged from 190-267 colony forming units per ml which was at least one log below the numbers detected using PCR. Table 1. Recovery of Legionella from Sewage using PCR
L. pneumophila # positive 1 # samples
Cells/ml
Legionella species
Cell sImi
Raw sewage
0118 (0%)
0
16/18 (89%)
> 10
10 Effluent
0/14 (0%)
0
14/14 (100 %)
> 10
20 Effluent
1/12 (8%)
< 103
12/12 (100 %)
>10
20
0110 (0 %)
0
10/10 (100%)
>10
Source
+ Ch Effluent
3 3 3 3
Detection of Ltgionella bacteria in sewage
413
Table 2. Recovery of ugionel/a from Sewage using Colony Culture
Legionella species # positive / # samples
Average Colony Forming Units (CFU) / ml
Raw sewage
12/18 (67%)
203
10 Effluent
9/14 (64%)
267
20 Effluent
5/13 (38%)
190
20
1110 (10%)
225
Source
+ CI 2 Effluent
Recovery of Legionel/a from air samples. A total of nine air samples was collected from secondary aeration basins. An average of 1133 liters of air were processed for each sample. Legionella species were detected by PCR in 7/9 (77%) of the air samples collected from secondary aeration basins at levels
< 103
cells/ml (Table 3). No L. pneumophila was
detected in any of these samples. Culture results recovered Legionella species in 3/9 (33 %) of the air samples at
< l
which was at least 1 log lower than the PCR results. No L.
pneumophila was detected in air samples by either peR or culture. Table 3. Recovery of ugioneUa from Air Samples using PeR and Colony Culture
L. pneumophila
Legionella spp.
# positive / # samples
# positive I # samples
Cell Concentration
PeR
0/9 (0%)
7/9 (77%)
< 10) Cells! ml
Culture
0/9 (0%)
3/9 (33%)
< 102 Cells! ml
Discussion This study demonstrated that Legionella species were present during all stages of a sewage treatment plant in a tropical climate indicating the prevalence of Legionella in tropical environmental waters. PCR results indicated no reduction in the levels of Legionella gene sequences throughout the treatment process including chlorination. These results indicate that
414
B. M. ROLL and R S. fUJIOKA
Legionella may be a natural inhabitant of sewage. These finding could also be explained by the fact that Legionella are able to survive in protozoans (Fields, 1993) which could protect
Legionella throughout the treatment process. In addition, Legionella may be associated with biofilms found in sewage pipes and various structures within the treatment process. In chlorinated effluent with average free chlorine levels of 1.2 mg/l, 100% of the samples was positive for Legionella species by PCR while only 10% of the samples was positive by culture.These findings could be explained by the fact that PCR does not distinguish between live and dead cells. Chlorination is known to disinfect Legionella. In addition, chlorination may also induce stressed or viable but non-culturable Legionella which would not be detected by culture. Finally, acid treatment of samples may be detrimental to stressed
Legionella. Sewage treatment plant workers are exposed to aerosols created during wastewater treatment, particularly aerosols created during secondary treatment. Air samples indicate that
Legionella species can be found in air samples collected near secondary aeration basins. The volume sampled was estimated to be equal to a two hour exposure to these aerosols. These results indicate that sewage treatment plant workers may be at risk to Legionella infections. However, the levels of Legionella recovered by PCR and culture methods were below the estimated infectious dose for Legionella. Moreover, L. pneumophila was not detected in the air samples by either PCR or culture indicating tllat environmental strains of Legionella, which are less pathogenic, predominate in sewage. In summary, PCR detected Legionella species in 95 % of the samples collected while culture method detected Legionella species in 50% of these samples. Lower recovery rates by culture are due to the inefficiency of cultural methods such as competing organisms that were not suppressed by acid treatment or antibiotics. In addition, acid treatment may have detrimental effects upon culture of Legionella. Finally only viable Legionella are detected by culture method.
Detection of LegioneUa bacteria in sewage
415
Higher recovery rates by PCR are due to the sensitivity of the method and the fact that dead and live cells are detected by PCR. One major advantage in the use of PCR is that results can be obtained in less than one day as compared to the time-consuming culture methods that can take up to three weeks. On the one occasion that L. pneumophila was detected by PCR, we were unable to detect L. pneumophila by culture.
This study
demonstrates that PCR is a rapid and sensitive means for detecting Legionella in wastewater. Further research is needed to determine the persistence of Legionella genetic material in sewage and especially aerosolized sewage so that we may better understand the issues related to viability and risks to humans when the peR technique is used. References Addiss, D. G., Davis, 1. P., laVenture, M., Wand, P. J. Hutchinson, M. A., and McKinney, R. M. 1989. Community-acquired Legionnaires' disease associated with a cooling tower: evidence for longer distance transport of Legionella pneumophila. Am. J.
Epidemio/. 130:557-568.
Bollin, G. E., Plouffe, J. F., Para, M. F., and Hackman, 8. 1985. Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets. Appl. Environ. Microbiol. SO: 1128-113 I. Bopp, C. A., Sumner, J. W., Morris, G. K. and Wells, J. G. 1981. Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J. C/in. Microbiol. 13:714. Chumakov, K. M., Tartakovsky, I. S., Ogarkova, O. A., and Krozorovsky, S. V. 1986. Use of the 5S ribosomal RNA nucleotide sequence analysis for the study of the phylogeny of the genus Legionella. Mo/ekuliaraia Genelika. Mikrobi%gia, I Viros%g, 7:38-40. Fields, B. S. 1993. Legionella and protozoa: Interaction of pathogen and its natural host. In Barbee, J. M. Breiman, R. F., and Dufour, A. P. (Eds.) Legionella: current status and emerging perspectives. p.129. American Society for Microbiology, Washington, D. C. Fraser, D. W., Tasi, T. R., Orenstein, W., et aI., 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl J. Med. 297: 1189·1197.
416
B. M. ROLL and R S. FUJIOKA
MacDonell, M. T., and Colwell, R. R. 1987. The nucleotide sequence of the 5S rRNA from Legionel/a pneumophila. Nuc. Acids Res. 15: 1335. Mangione, E. J., Remis, R. S., Tait, K. A., McGee, W. B., Gorman, G. W., Wentworth, B. B., Baron, P. A., Hightower, A. W., Barbaree, J. M., and Bromme, C. V. 1982. An outbreak of Pontiac fever related to whirlpool use, Michigan. lAMA 253: 535-539. Palmer, C. J., Tasi, Y. L., Paszko-Kolva, C., Mayer, C., and Sangermano, L. R. 1993. Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods. Appl. Environ. Microbiol. 59:3618-3624. Reingold, A. L., Thomason, B. M., Brake, B. J., Thacker, L., Wilkinson, H. W. and Kuritsky, J. N.1984.Legionel/a pneumonia in the United States: the distribution of serogroups and species causing human illness, l. Infecl. Dis. 149:819. Wolters, J., and Erdmann, V. A. 1988. Compilation of 5S rRNA and 5S rRNA gene sequences.Nuc. Acids. Res 16 (Supplement) rl-r70.
Pergamon 0273-1223(95)00302-9
Wal. Sci. Tech. Vol. 31, No. 5-6, pp. 409-416,1995. Copyright@ 1995 IAWQ Printed in Great Bntaio. All rights reserved. 0273-1223/95 $9'50 + 0'00
DETECTION OF LEGIONELLA BACTERIA IN SEWAGE BY POLYMERASE CHAIN REACTION AND STANDARD CULTURE METHOD Bruce M. Roll and Roger S. Fujioka Water Resources Research Center and Department ofMicrobiology, University of Hawaii, Honolulu. H/96822, USA
Abstract
Legionella bacteria are ubiquitous in environmental waters. Only a few species of Legionella, especially, L. pneumophila are pathogenic to humans and cause a sometimes fatal Legionnnaires disease as well as a less fatal disease called Pontiac fever. The presence of
Legionella in sewage and aerosolized sewage is the subject of this investigation because reuse
of sewage may involve the exposure of people to aerosolization, the mode of transmission of
Legionella bacteria. The objective of this study was to determine the prevalence of Legionella species and L. pneumophila in wastewater and their fate after various stages of treatment. The polymerase chain reaction (PCR) and standard culture method were utilized to detect
Legionella species and L. pneumophila. PCR results indicated that Legionella species were 3
present at levels > 10 cells / ml during all phases of sewage treatment including chlorinated effluents. Culture results indicatedlevels at least one log lower than seen with PCR. Legionella species were also recovered from air samples collected from secondary aeration basins at levels 3 < 10 cells/ml. PCR was shown to be the most rapid and sensitive method for detecting Legionella in sewage. KEYWORDS
Legionella, detection, polymerase chain reaction (PCR). culture method. wastewater, seweage, aerosolized sewage
Introduction In 1976 at a Legionnaires' convention in Philadelphia, an epidemic occurred that led to the discovery of Legionella pneumophila (Fraser et aI., 1977). Since its discovery, the family Legionellae has been responsible for numerous outbreaks of Legionnaires disease and non• pneumonic Pontiac fever. To date, 48 species of Legionella have been identified with eighteen of these species being associated with either Legionnaire's disease or Pontiac fever. According to the Centers for Disease Control (CDC), the vast majority (85%) of all Legionella pneumonias are caused by the species Legionella pneumophila (Reingold et aI., 1984). The major vehicle of transmission for these diseases is the inhalation of aerosols containing 409
B. M. ROLL and R. S. FUJIOKA
410
Legionel/a. Although Legionel/a appears to be a normal inhabitant of most aquatic environments, under certain condition their numbers may increase. This is of particular concern when virulent strains of Legionel/a are able to multiply to high concentrations in aquatic environments. Cooling towers (Addiss et aI., 1989), whirl pool baths (Mangione, et aI., 1982), and showers (Bollin et aI., 1985) have been shown to be sources for transmission of Le~ionella. As more wastewaters are directed toward reuse, it is critical that we know the disease potential of these waters. Research conducted in a temperate climate (palmer et aI.,1993) demonstrated that sewage is a potential reservoir for Legionel/a. To date, however, no studies have surveyed the prevalence of Legionel/a in sewage found in tropical climates. In addition it is critical that we know the disease potential of this organism for sewage treatment plant, workers who may be exposed to aerosols created during the sewage treatment process. The purpose of this study was to determine the prevalence of Legionel/a in the various stages of sewage treatment at a wastewater treatment plant in a tropical climate. This study utilized the polymerase chain reaction (PCR) and standard culture methods to examine various stages of sewage treatment for the presence of Legionella pneumophila and other Legionella species. In addition, air samples were collected from secondary aeration basins and analyzed for the presence of Legionel/a. Materials and Methods Water Sampling. Wastewater samples were collected from the Wahiawa sewage treatment facility located on the island of Oahu in the State of Hawaii. This treatment plant utilizes settling tanks, aeration basins (activated sludge) and chlorination to treat sewage. Samples of raw, primary, secondary treated and chlorinated wastewater were collected in sterile 500 ml nalgene plastic bottles containing sodium thiosulfate (40mg). transported at
40 C
All samples were
and analyzed within 4 hours of collection.
Air Sampling. Air samples were collected from aerated activated sludge basins using a portable vacuum pump (Nelson model 921 Berkeley, California) connected to the side port of a WOO ml erlenmeyer vacuum flask. The flask contained an aquarium bubbling stone and was connected to a glass funnel that served as an air collector. The apparatus was sterilized by autoclaving and filled with 800 ml of sterile phosphate buffered water. The flask was then placed in an ice cooler and maintained at 40C during the sampling period. The collection funnel was suspended four feet above the water surface in the secondary aeration basin. Samples were collected over a four hour period at an air flow rate of 283 liters per hour.
Detection of Ltgionella bacteria in sewage
411
During this time approximately 1133 liters of air was bubbled through the phosphate buffer. These air treated buffer samples were transported at 40 C and analyzed within 4 hours of collection. Detection of Legionella Using PCR. For genetic detection of Legionella, the EnviroAMP~ kit developed by Perkin Elmer Roche (Alameda, CAl
was used as per the
manufacturer's instructions. Many samples required 1:10 or 1:100 dilutions to eliminate interference caused by undefmed subsJances in sewage. Detection of Legionella species and L.
pnewnophi/a was accomplished by filtering 10 ml of the sewage sample and using PCR to amplify DNA sequences in the conserved regions of the 5S ribosomal RNA gene (Chumkov et
aI., 1986, MacDonell and Caldwell. 1987, Wolters and Erdmann, I988)andsequences from the miD. (macrophage infectivity potentiator) gene, unique to L. pnewnophila (Cianciotto et aI., 1990, Engleberg et aI., 1989). PCR products were detected by a reverse dot blot technique using
Legionella specific probes immobilized on nylon membrane strips. Biotinylated primers were used and the products which reacted with the probes were detected as colored dots resulting from a streptavidin-horseradish peroxidase conjugate reaction. Formation of a blue "L" dot on the strip indicated the presence one or more of twenty Legionella species. Formation of a blue "P" dot indicated the presence of L. pnewnophi/a. Positive and negative controls are built into the strip. The test is semi-quantitative since the intensity of the blue positive control dot is 3
equivalent to 10 cells per mI. When the "L" or " P" dots were darker than the control dot, the sample was considered to contain
> lif cells per ml.
When the "L" or "P" dots were lighter
than the positive control dot the sample was considered to contain
<
103 cells per mI.
Culture Isolation. Ten mls of sewage was filtered through a 0.45 l1m HLVP Millipore filter(Bedford, MA) and pretreated with acid(Bopp etal., 198 I) to inactivate competing organisms. Following acid treatment 0.1 ml samples were spread plated onto buffered charcoal-yeast extract agar containing a-ketogluterate
and glycine (3 gIl) and supplemented with
cyclohexamide (80mg/I), vancomycin (5 mg/I) and polymyxin B (lOOUlml). The plates were then incubated at 37"C in a humid 4 % CO2 environment for 5 to 20 days. Typical Legionella colonies appeared light blue to gray-white.
412
B. M. ROLL and R. S. FUJIOKA
Confinnation of Legionella Colonies. Suspected Legionella colonies were confirmed using direct fluorescent antibody staining with FITC labelled Legionella polyclonal antibody (Scimedix, Denville, N. J.) that detected both L. pneumophila and other Legionella species.
Results Recovery of Legionella From Sewage. Using PCR, Legionella gene sequences were 3
detected in all phases of sewage treatment and at levels> 10 cells per ml (Table 1). Of the 3
18 raw sewage samples collected, 89 % demonstrated levels above 10 cells/ml. Legionella 3
species were detected in excess of 10 cell/ml in 100 % of the samples collected from primary effluent, secondary effluent and even chlorinated effluent containing an average of 1.20 mg/l of free chlorine. Of the 54 samples collected throughout the treatment process, L. pneumophila was detected in only one sample (secondary effluent). Plate culture did not detect L.
pneumophila but detected the presence of Legionella species in 67 % of raw sewage, in 64 % of primary effluent, in 38 % secondary effluent, and in 10 % of chlorinated effluent (Table 2). The recovered concentration of Legionella ranged from 190-267 colony forming units per ml which was at least one log below the numbers detected using PCR. Table 1. Recovery of Legionella from Sewage using PCR
L. pneumophila # positive 1 # samples
Cells/ml
Legionella species
Cell sImi
Raw sewage
0118 (0%)
0
16/18 (89%)
> 10
10 Effluent
0/14 (0%)
0
14/14 (100 %)
> 10
20 Effluent
1/12 (8%)
< 103
12/12 (100 %)
>10
20
0110 (0 %)
0
10/10 (100%)
>10
Source
+ Ch Effluent
3 3 3 3
Detection of Ltgionella bacteria in sewage
413
Table 2. Recovery of ugionel/a from Sewage using Colony Culture
Legionella species # positive / # samples
Average Colony Forming Units (CFU) / ml
Raw sewage
12/18 (67%)
203
10 Effluent
9/14 (64%)
267
20 Effluent
5/13 (38%)
190
20
1110 (10%)
225
Source
+ CI 2 Effluent
Recovery of Legionel/a from air samples. A total of nine air samples was collected from secondary aeration basins. An average of 1133 liters of air were processed for each sample. Legionella species were detected by PCR in 7/9 (77%) of the air samples collected from secondary aeration basins at levels
< 103
cells/ml (Table 3). No L. pneumophila was
detected in any of these samples. Culture results recovered Legionella species in 3/9 (33 %) of the air samples at
< l
which was at least 1 log lower than the PCR results. No L.
pneumophila was detected in air samples by either peR or culture. Table 3. Recovery of ugioneUa from Air Samples using PeR and Colony Culture
L. pneumophila
Legionella spp.
# positive / # samples
# positive I # samples
Cell Concentration
PeR
0/9 (0%)
7/9 (77%)
< 10) Cells! ml
Culture
0/9 (0%)
3/9 (33%)
< 102 Cells! ml
Discussion This study demonstrated that Legionella species were present during all stages of a sewage treatment plant in a tropical climate indicating the prevalence of Legionella in tropical environmental waters. PCR results indicated no reduction in the levels of Legionella gene sequences throughout the treatment process including chlorination. These results indicate that
414
B. M. ROLL and R S. fUJIOKA
Legionella may be a natural inhabitant of sewage. These finding could also be explained by the fact that Legionella are able to survive in protozoans (Fields, 1993) which could protect
Legionella throughout the treatment process. In addition, Legionella may be associated with biofilms found in sewage pipes and various structures within the treatment process. In chlorinated effluent with average free chlorine levels of 1.2 mg/l, 100% of the samples was positive for Legionella species by PCR while only 10% of the samples was positive by culture.These findings could be explained by the fact that PCR does not distinguish between live and dead cells. Chlorination is known to disinfect Legionella. In addition, chlorination may also induce stressed or viable but non-culturable Legionella which would not be detected by culture. Finally, acid treatment of samples may be detrimental to stressed
Legionella. Sewage treatment plant workers are exposed to aerosols created during wastewater treatment, particularly aerosols created during secondary treatment. Air samples indicate that
Legionella species can be found in air samples collected near secondary aeration basins. The volume sampled was estimated to be equal to a two hour exposure to these aerosols. These results indicate that sewage treatment plant workers may be at risk to Legionella infections. However, the levels of Legionella recovered by PCR and culture methods were below the estimated infectious dose for Legionella. Moreover, L. pneumophila was not detected in the air samples by either PCR or culture indicating tllat environmental strains of Legionella, which are less pathogenic, predominate in sewage. In summary, PCR detected Legionella species in 95 % of the samples collected while culture method detected Legionella species in 50% of these samples. Lower recovery rates by culture are due to the inefficiency of cultural methods such as competing organisms that were not suppressed by acid treatment or antibiotics. In addition, acid treatment may have detrimental effects upon culture of Legionella. Finally only viable Legionella are detected by culture method.
Detection of LegioneUa bacteria in sewage
415
Higher recovery rates by PCR are due to the sensitivity of the method and the fact that dead and live cells are detected by PCR. One major advantage in the use of PCR is that results can be obtained in less than one day as compared to the time-consuming culture methods that can take up to three weeks. On the one occasion that L. pneumophila was detected by PCR, we were unable to detect L. pneumophila by culture.
This study
demonstrates that PCR is a rapid and sensitive means for detecting Legionella in wastewater. Further research is needed to determine the persistence of Legionella genetic material in sewage and especially aerosolized sewage so that we may better understand the issues related to viability and risks to humans when the peR technique is used. References Addiss, D. G., Davis, 1. P., laVenture, M., Wand, P. J. Hutchinson, M. A., and McKinney, R. M. 1989. Community-acquired Legionnaires' disease associated with a cooling tower: evidence for longer distance transport of Legionella pneumophila. Am. J.
Epidemio/. 130:557-568.
Bollin, G. E., Plouffe, J. F., Para, M. F., and Hackman, 8. 1985. Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets. Appl. Environ. Microbiol. SO: 1128-113 I. Bopp, C. A., Sumner, J. W., Morris, G. K. and Wells, J. G. 1981. Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. J. C/in. Microbiol. 13:714. Chumakov, K. M., Tartakovsky, I. S., Ogarkova, O. A., and Krozorovsky, S. V. 1986. Use of the 5S ribosomal RNA nucleotide sequence analysis for the study of the phylogeny of the genus Legionella. Mo/ekuliaraia Genelika. Mikrobi%gia, I Viros%g, 7:38-40. Fields, B. S. 1993. Legionella and protozoa: Interaction of pathogen and its natural host. In Barbee, J. M. Breiman, R. F., and Dufour, A. P. (Eds.) Legionella: current status and emerging perspectives. p.129. American Society for Microbiology, Washington, D. C. Fraser, D. W., Tasi, T. R., Orenstein, W., et aI., 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl J. Med. 297: 1189·1197.
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B. M. ROLL and R S. FUJIOKA
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