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Keywords:

  • heterotrophic bacteria;
  • Legionella;
  • rainwater;
  • storage tank

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Many administrative agencies in Japan are encouraging installation of household rainwater-storage tanks for more effective use of natural rainwater. Water samples were collected periodically from 43 rainwater tanks from 40 households and tested for the presence of Legionella species and the extent of heterotrophic bacteria in Azumino city, Nagano prefecture, Japan. PCR assays indicated the presence of Legionella spp. in 12 (30%) of the 43 tank water samples. Attempts were made to identify correlations between PCR positive samples, topography, pH, chemical oxygen demand (COD), atmospheric temperature and the numbers of heterotrophic bacteria. Between June and October, 2012, the numbers of heterotrophic bacteria in rainwater tanks and the values of COD positively correlated with the presence of Legionella species. In most of the Legionella-positive cases, heterotrophic bacterial cell counts were >104 CFU/mL. Moreover, Legionella species were less frequently detected when the COD value was >5 mg KMnO4/L. Therefore, at least in Azumino, Japan between June and October 2012, both heterotrophic bacterial counts and COD values may be considered index parameters for the presence of Legionella cells in rainwater tanks. Much more accumulation of such data is needed to verify the accuracy of these findings.

List of Abbreviations
BCYE-α

buffered charcoal yeast extract agar with 0.1% α-ketoglutarate

COD

chemical oxygen demand

GVPC

glycine, vancomycin, polymyxin B and cycloheximide

Q-Q

quantile–quantile

WYO-α

Wadowsky–Yee–Okuda agar with 0.1% α-ketoglutarate

σ

standard deviation

Since the first reported occurrence of Legionnaire disease in 1976 [1], infectious diseases caused by Legionella pneumophila have been successively reported worldwide [2-8]. Currently, Legionella species are considered common causes of community-acquired and nosocomial pneumonia [9-11]. Water environments appear to be the natural habitat and to serve as amplifiers for Legionella species, which are facultative intracellular parasites [12, 13]. Roof-harvested rainwater has attracted significant attention as a potential alternative source of potable and nonpotable water, especially in regions where water is scarce [14]. To encourage the use of roof-harvested rainwater for domestic purposes, governmental bodies of many countries, such as Australia [15, 16], Denmark [17], and New Zealand [18], have been providing subsidies to residents. However, there are insufficient data concerning the microbiological quality of roof-harvested rainwater and its potential health risks.

In Japan, there has also been increasing recognition in both urban and rural areas that it is important to utilize rainwater with the aim of effective water reuse and water conservation. Accordingly, many administrative agencies in Japan have also been encouraging installation of rainwater tanks by subsidizing the costs of installation. As a result, many more rainwater tanks have been installed in Azumino city, Nagano prefecture, a rural area of Japan.

The increasing installation of rainwater tanks in urban and/or rural environments necessitates greater understanding of the quality of water they are able to supply. Although there are few published studies on the physico-chemical properties of harvested rainwater [19-23], various factors including topography, climate, atmospheric temperature and surrounding circumstances are all considered to influence the quality of rainwater stored in such tanks.

To obtain current information on the microbiology of tank rainwater in rural area of Japan, we investigated water samples from rainwater tanks for microbiological contamination, focusing on the presence of Legionella species. We simultaneously assessed the pH, COD, atmospheric temperature, climate, number of heterotrophic bacteria and topography, in order to evaluate their mutual relationships.

The objectives of this study were to assess the presence of Legionella and to compare it with various variables concerning harvested rainwater in samples taken during spring, summer and autumn.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Experimental design and collection of water samples

Prior to initiating this study, we asked to participate in a project in which 90 users were to install rainwater tanks with subsidies from Azumino city, Nagano prefecture, Japan. Informed consents for the present study were obtained from the prospective users. In all, 40 users living in four districts of Azumino city, designated as A, B, C and D in Figure 1, were enrolled in the present study; the remaining users did not participate in this study. District A contained nine users including two users who installed two tanks each, district B 12 users, district C 8 users, including one who installed two tanks, and district D 11 users, including one who installed two tanks.

image

Figure 1. Districts A, B, C, and D in Azumino city, in which the 40 rainwater-storage tank users enrolled in the study reside.

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Water samples were collected from 43 rainwater tanks of 40 users three times, that is, in June (spring), August (summer) and October (autumn) of 2012. In addition, four times each month on weekends, rainwater samples were collected from approximately 10 users. In all, 129 rainwater samples comprising 43 in June, 43 in August, and 43 in October in 2012, were collected in 1000 mL sterile polypropylene containers, maintained at 4°C during transport to the laboratory, and tested within 6 hr.

Water quality and associated circumstances

The pH of each sample was recorded with a compact Twin pH meter (model AS-211; AS ONE., Osaka, Japan). Data concerning the topography from which the samples were collected were obtained. The weather, including the amounts of precipitation and atmospheric temperatures, was recorded. COD values were determined with the KMnO4-method using Packtest-COD (WAK-COD, Kyoritsu Chemical-Check, Tokyo, Japan).

Quantitative cultures of Legionella species in rainwater samples

All rainwater samples were filter concentrated in a biological safety cabinet by pouring 500 mL water samples into a sterile 47 mm filter funnel assembly containing Millipore cellulose acetate membrane filters with pore size 0.22 mm (Millipore, Tokyo, Japan), using the vacuum source and side-arm flasks necessary to operate the apparatus. When the 500 mL water samples had passed through the filters, the filters were removed aseptically from the holders with sterile filter forceps, folded outwards and placed in sterilized screw-capped containers with 5 mL of sterile water.

The screw-capped containers were then vortexed for 1 min to free bacteria and organic material from the filter to achieve 100-fold concentrated water samples. If more than one filter was required to concentrate a sample, additional filters were added to the same container. The concentrated water samples were heat-treated by placing in a water bath at 50°C for 20 min. Next, 0.1 mL of the 100-fold filter-concentrated water samples were inoculated onto GVPC or WYO-α (Sysmex–Biomerieux, Tokyo, Japan) media and incubated at 37°C for 5–7 days. All suspected Legionella colonies were spread onto both B-CYE-α (Sysmex–Biomerieux) media and sheep blood agar (Nippon Becton Dickinson, Tokyo, Japan) media and incubated at 37°C. Colonies grown on B-CYE-α media and not on sheep blood agar media were subjected to further PCR examination to confirm the genus.

Detection of the genus Legionella in rainwater samples and discrimination of L. pneumophila from other Legionella species

Concentration of water samples, extraction and preparation of DNAs were performed precisely according to the Manual for the Detection of Pathogenic Microorganisms of National Institute of Infectious Diseases, Japan (http://www.nih.go.jp/niid/ja/labo-manual.html).

After extracting the DNA, PCR assay was carried out with legionellae-specific LEG primers based on 16S-rRNA genes [24]. Against the samples that were positive on PCR assay, two-step PCR analysis was performed to detect L. pneumophila using Lmip primers targeting the mip gene of L. pneumophila [25]. The presence or absence of amplified products was determined following gel electrophoresis and L. pneumophila thus differentiated from non-L. pneumophila species.

Total viable counts of heterotrophic bacteria

Each rainwater sample was immediately diluted with sterile physiological saline solution in a serial 10-fold fashion. Counts of the total viable populations of heterotrophic bacteria were obtained by plating 100 mL of each dilution of water collected from the rainwater tanks on R2A agars (Nippon Becton Dickinson) in triplicate, using sterile glass rods to spread the samples evenly over the agar surfaces. The inoculated plates were incubated at 20°C for 2 weeks. The agar plates containing the dilution yielding the largest number of colonies that were sufficiently distinct to allow accurate counting were selected, and from these the total numbers of heterotrophic bacterial colonies were calculated.

Statistical analyses

The following statistical analyses were all performed using Excel 2010. Logarithmic transformations were utilized in statistical analyses to evaluate the distributions of bacterial cell counts by determining the mean and standard deviation of the distribution. Analyses by null hypothesis with non-parametric Wilcoxon's signed-rank sum test and by means of Pearson's product-moment correlation coefficient were used to characterize which water variables were associated with the occurrence of Legionella. Q-Q plot tests were also used to confirm the log-normal distribution of heterotrophic bacterial cell counts with PCR-positive Legionella.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Detection of the genus Legionella in rainwater samples by quantitative cultures and by PCR

Quantitative cultures on GVPC or WYO-α (Sysmex–Biomerieux) media yielded no growth, except for one sample obtained on 30 June 2012, which was found to be inhabited by 80 CFU/100 mL of Legionella.

In contrast, as shown in Table 1, around 30% of rainwater samples were demonstrated by PCR to be positive for the presence of Legionella species; namely, 12/43 samples (27.9%) in both June and August, and 17/43 (39.5%) in October. As shown in Table 1, two-step PCR analyses to detect L. pneumophila among the Legionella-positive rainwater samples showed an obvious bias by month; that is, 83.3% (10/12) were positive in June, 41.6% (5/12) in August and 23.5% (4/17) in October.

Table 1. Percentages of L. pneumophila-positive samples according to two-step PCR among Legionella-positive samples from roof-harvested rainwater tanks
Date of collectionTotal No. of samples testedNo. of Legionella positive samplesNo. of L. pnemophila positive samplesPercentage (%) of L. pnemophila positive samples
June43121083.3
August4312541.6
October4317423.5
Total12941946.5

Water quality and associated circumstances

Occurrence of Legionella-positive samples according to PCR in relation to the amount of precipitation

Rainwater samples were collected four times a month from about 10 rainwater tanks on each occasion. Table 2 and Figure 2 show findings for samples from 43 different rainwater tanks on days in June 2012, namely days 2, 16, 23 and 30. According to PCR, the percentages of Legionella-positive rainwater samples were 20%, 30%, 10% and 42% on these days, respectively. As clearly indicated in Figure 2, when there had been continuing fine weather (Days 16 and 30) before collecting the rainwater samples from tanks, the positive percentages were high and tended to be lower after rain (Day 23). Regarding this tendency, the findings in August and October were similar.

Table 2. Percentages of Legionella-positive samples according to PCR in relation to the amount of precipitation
Date of collection2 June16 June23 June30 June
Rainfall-amount (mm/week)10.56.079.00
No. of Legionella-positive samples2415
Percentage (%) of Legionella-positive samples20301042
image

Figure 2. Climate of Azumino city in June 2012.

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Determination of COD values in rainwater samples

The COD values ranged between 1 and 10 mg KMnO4/L in June, August and October. Nearly half of the rainwater samples showed COD values of 1 mg KMnO4/L with no bias by month. As shown in Table 3, Legionella-positive percentages in relation to COD values were 45.4% of rainwater samples with COD values of 1 mg KMnO4/L, 27.7% of rainwater samples with COD values of 2 to 4 mg KMnO4/L and 5.5% of rainwater samples with COD values of more than 5 mg KMnO4/L. Increases in COD values tended to be associated with decreases in Legionella-positive percentages by PCR.

Table 3. Detection of Legionella species by PCR in conjunction with COD values
COD (mg KMnO4/L)No. of PCR positive samplesNo. of PCR negative samplesPositive percentage (%) of Legionella
1253045.4
2–4153927.7
5<1175.5
Total418632.3
Total counts of heterotrophic bacterial cells in rainwater samples

Heterotrophic bacterial cell counts ranged between 1.0 × 103/mL and 1.9 × 107/mL (σ = 2.91 × 106) in June, 5.5 × 104/mL and 2.2 × 107/mL (σinline image = 4.20 × 106) in August and 1.0 × 103/mL and 5.2 × 107/mL (σinline image = 1.44 × 106) in October. As shown in the histogram of Figure 3, when the numbers of heterotrophic bacterial cells (CFU/mL) were converted to logarithmic scales, they showed a purely normal distribution. In addition, as shown in Figure 4, when they were plotted on lognormal probability paper, clean linearity was demonstrated, indicating normal distribution. It should be noted that the large majority of Legionella-positive samples by PCR were recovered from rainwater samples that were inhabited by 1.0 × 104/mL of heterotrophic bacteria and that no Legionella-positivity by PCR was detected in samples with less than 103/mL of heterotrophic bacteria.

image

Figure 3. Histogram of Legionella-positive rainwater samples according to heterotrophic cell counts.

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image

Figure 4. Distribution of heterotrophic bacterial cells by lognormal probability plot .

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Legionella species are ubiquitous in freshwater environments and have been isolated from various aquatic habitats, including man-made water distribution systems [10-13]. Legionella species have frequently been isolated in association with amoebae, which may serve as a host for this species. The primary route of infection with Legionella species is considered to be inhalation. Roof-harvested rainwater in household tanks can be used in many aspects of daily life, such as washing family cars, sprinkling gardens with water and watering flowers and plants, all of which may result in the formation of potentially infectious aerosols.

Among the water samples from 43 rainwater tanks of 40 users, we found that only one sample was inhabited by 80 CFU/100 mL of Legionella. Therefore, almost all rainwater samples that were PCR positive for Legionella were below the detection limit for Legionella cells, that is, less than 10 CFU/100 mL; it should be noted that PCR assays detect both living and dead Legionella.

It should be noted that we found that approximately one third of storage tanks were contaminated with Legionella. In detail, among the 40 households investigated in this survey, we demonstrated five to be PCR-positive for Legionella on all three test occasions, seven were positive twice and 11 were PCR-positive for Legionella only once. That is, we found that Legionella species inhabited 23 of 40 household rainwater tanks in Azumino city. As shown in Figure 1, an interesting finding was that samples PCR-positive for Legionella were from households situated in districts A and B, which are both concentrated on the conventional railway Oito line and along national highway Route 147. On the other hand, most households in districts C and D are located in submontane districts and were PCR-negative for Legionella. However, we observed no distinctive differences in heterotrophic bacterial counts and COD values between districts A and B and districts C and D. As demonstrated in Table 2 in conjunction with Figure 2, the PCR-positive percentages for Legionella varied according to the amount of precipitation. Our results suggest that Legionella contamination is related to the amount of precipitation and the location of the rainwater tanks. Indeed, in high-traffic districts, rainwater tanks are apt to be exposed to the spreading of Legionella-contaminated soil or rainwater [26]. Water in transient puddles formed on roads on rainy days might be splashed into the air by moving vehicles on national highway Route 147. During ongoing sunny days, hetrotrophic bacteria multiply profusely. On the other hand, with ongoing rain, heterotrophic bacterial counts decreased, probably because of dilution by continual flow of rainwater. This would lead to degradation of rainwater in tanks allowing habitation by the amoebae required for proliferation of Legionella.

Although not presented in the Results section, pH of the rainwater was distributed unevenly between 3.6 and 7.0, but was approximately constant over the three measurements in each household; we observed no obvious correlations between heterotrophic cell counts and PCR-positivity for Legionella. Variations in pH of rainwater in household storage tanks probably depend on the amount of automobile emissions and the quality of roofs, walls and other building materials.

Legionella species are known to infect the trophozoite form of free-living amoebae and to replicate intracellularly at temperatures over 25°C [27]. In contrast, at temperatures below 20°C, Legionella species are actively digested by amoebae and eliminated from them. The temperatures during the investigation periods of June and August were mostly over 20°C; we measured comparatively higher temperatures in August. Therefore, at least during this period, we consider that atmospheric temperatures probably did not influence detection of Legionella.

Identification of indicator microorganisms or other measureable indicators of Legionella pollution is desirable. We attempted to examine the relationships between our findings and the presence of Legionella. We found that both the number of heterotrophic bacteria in rainwater tanks and COD values appeared to correlate with the presence of Legionella species during the period between June and October.

When we converted the numbers of heterotrophic bacterial cells with PCR-positivity for Legionella to logarithmic scales, almost all heterotrophic bacterial cell counts in rainwater samples that were PCR-positive for Legionella were over 104 CFU/mL, as shown in Figure 3. We proved this finding was significant by means of null hypothesis using non-parametric Wilcoxon's signed-rank sum test with a significance level of 5%. In addition, we demonstrated clean linearity by means of Q–Q plot test on log-normal probability paper, as shown in Figure 4. The log-normal distribution was moreover verified both by means of Kolmogorov–Smirnov and Shapiro–Wilk tests. That is, they were confirmed according to the summary statistics as a normal distribution with an average of 5.2581, normal distribution of standard deviation of 1.0572 from unbiased variance. In this analysis, the position of 4 (104 CFU/mL of heterotrophic cell counts) was standardized by the following formula: z = (4−5.2581)/1.0572 = −1.194. Thus, the probability of distribution of less than 104 CFU/mL became 0.117 of the original value. In other words, 88.3% (1−0.117 = 0.883) of the samples that were PCR-positive for Legionella was distributed in the rainwater samples with heterotrophic bacterial cell counts of more than 104 CFU/mL.

These results were fairly consistent with a recent survey that used multivariate regression to show a relationship between the presence of Legionalla and heterotrophic cell counts of over 1 × 105 CFU/100 mL (i.e., 103 CFU/mL) [28].

Moreover, that the positive percentages for detection of Legionella species were demonstrated to be reduced when COD was more than 5 mg KMnO4/L, as shown in Table 3, is of considerable interest. This finding was statistically investigated by means of Pearson's product-moment correlation coefficient test and found to be significant with a coefficient of correlation value of −0.9970.

As far as we know, there have been few reports concerning index organisms associated with contamination with Legionella cells. Adequate numbers of amoebae would be required to achieve substantial multiplication of intracellular parasitic Legionella. In addition, adequate cell counts of heterotrophic bacteria are needed for substantial proliferation of amoebae. However, the higher the heterotrophic bacterial count, the higher the COD value. An increase in COD values implies deterioration of water conditions; this could be unfavorable for inhabitation by amoebae. Amoebae might have difficulty proliferating in water conditions with COD values of >5 mg KMnO4/L, as shown in Table 3.

Therefore, we consider that a threshold number (less than 104 CFU/mL) of heterotrophic bacteria as well as an amount of COD (more than 5 mg KMnO4/100 mL) might be useful environmental management indicators of the presence of Legionella cells in rainwater tanks during the period between June and October, at least in Azumino, Japan. Much more accumulation of such data is needed to verify the accuracy of our findings.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This study was conducted in co-operation with Azumino city. We would like to express our sincere appreciation to Azumino city for their support and are also grateful for the active support of the local communities of rainwater tank users in Azumino city during the course of this study.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors declare that they have no conflicts of interest.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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