DETECTION OF LEGIONELLA PNEUMOPHILA IN ENVIRONMENTAL AEROSOLS OF COOLING TOWERS BY IMPACT SAMPLING METHODS

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1 DETECTION OF LEGIONELLA PNEUMOPHILA IN ENVIRONMENTAL AEROSOLS OF COOLING TOWERS BY IMPACT SAMPLING METHODS L Bordenave, S Dubrou, A Mouilleseaux, V Bex 1, D Carlier and F Squinazi Hygiene Laboratory of Paris, Paris, France ABSTRACT To evaluate the efficiency of classic air sampling methods in case of suspicious bioaerosols of Legionella, we tested 3 biocollectors in outdoor conditions : a six-stage microbial air sampler (S6, Andersen sampler), a single-stage multiple-hole impactor (ATΩ) and a liquid impinger sampler (AGI 30). The 3 biocollectors were used simultaneously in 7 assays on-site near cooling towers. The selected sites were located in Paris city and the samples were obtained close to the installations in aerosols discharged from the towers. Each of the 3 sampling methods allowed the detection of airborne Legionella pneumophila (2 to 4 CFU /m 3 ) but was not related to the concentration of bacteria in water. The background flora present in the water of cooling towers could inhibit the growth of Legionella on culture media. The results showed that the impact sampling methods are useful in heterogeneous and variable conditions. INDEX TERMS Bioaerosols, Legionella pneumophila, Measurement methods, Infectious diseases, Cooling tower INTRODUCTION Legionella pneumophila is an hydrotelluric bacteria potentially responsible of severe pneumonia when inhaled from environmental contaminated aerosols. Usually, the agent is detected from natural or manmade sites where it is able to develop in favourable conditions. Among the different sources known to produce these contaminated bioaerosols, the cooling towers are often mentioned. In France, as no community outbreak had been related to these devices until the latest years, the risk linked to these equipments had been neglected (Dubrou et al, 2002). However, since the governmental guidelines of 1997, a revaluation of the health risk associated to the cooling systems has been noted. Three outbreaks of legionnaire s diseases have been reported, 2 in Paris area and 1 in Rennes city, respectively in June 1998, August 1999 and between July and November 2000, where cooling towers had been offending as sources of the contamination. (Decludt et al, 1999 ; BEH n 41/99 ; Schvoerer et al, 2001). The strains of legionellae supposed to be responsible of the outbreaks were identified in the water of the cooling systems. However, these results can not be directly related to human risk because Legionella is an airborne pathogen. In these 3 outbreaks as in a more preventive investigation, the failure of proof of air discharge contamination is evident. Actually, no method for outdoor air sampling in the research of Legionella had been recognized. The extreme variability of the environmental conditions, the significant microbial flora generally present in the water of the cooling systems, may result in difficulties to validate reproductive sampling and analysis methods. Recently, two reports deal with the 1 Contact author valerie.bex@mairie-paris.fr 411

2 detection of Legionella in outdoor air (Pascual et al, 2001 ; Ishimatsu et al, 2001). Using modified techniques, the authors showed the need to evaluate the performance of the biocollectors in real conditions and elaborated an adapted method for the detection of Legionella from air. This study was performed to develop a specific sampling air strategy to detect Legionella near different cooling towers (independent of outbreaks of legionnaire s diseases). The multiplication of the sampling campaigns on different sites has been chosen to compare 3 different biocollectors in real conditions. METHODS Sampling sites The study was performed between May and November 2000, i.e. during the period of cooling systems running. Samples were taken from 4 towers (TA, TB, TC and TD) located in different sites in Paris city. The cooling equipments were situated on the terrace of buildings (TA, TB, TC) or on ground floor (TD). All of the sites were chosen according to (i) the accessibility of the cooling tower (space around the tower, possibility of plugging electrical appliances), (ii) the proximity of office/home-buildings (TA, TB, TC) or passageway (TD). The concentration of Legionella in cooling water was also considered. In the case of TC and TD, campaigns at different periods of the seasons were performed. The air samples were collected in the plume of the towers, close to the air discharge when it was possible (top of the TC and TD) and downwind from TA (3, 7 and 11 m), TB (2 m), TC (1, 2m), TD (3 and 10m). Sampling methods The water samples were obtained from the cooling pond just before the air sampling. One-liter sample was collected in a sterile plastic bottle and analyzed on the same day. For air sampling methods, 3 biocollectors has been tested. All glass impinger (AGI 30) sampler containing 20 ml of PBS was used. The sampler was connected to vacuum pump and flow meters. It was calibrated with a flow rate of 12 L/min (Lin et al, 1997). The sixstage microbial air sampler (S6) consists of a serie of six metal plates with perforations of smaller diameter in each succeeding plate (Andersen, 1958). The method allows the separation of the particles by size ranges and collects them on glass Petri dishes containing 27 ml of GVPC medium placed on each stage. The sampling was performed with a calibrated flow rate of 28 L/min. The single-stage multiple-hole impactor (ATΩ) with GVPC plates was used to collect Legionella. ATΩ was battery operated, equipped with a screen of 265 perforations and was given to collect particles with a diameter ranged from 1 µm to >10 µm. The flow rate of the sampler was 100 L/min and can sample up to 1000L each cycle. Sampling strategy As the outdoor conditions are very changing the 3 biocollectors were used simultaneously, except for the campaign N TA, TB and TD1. The flow rate sampler was different for each biocollector, so we collected in parallel 3 different volumes of air and during the same time. With this strategy, we considered the simultaneous samples to be more homogenous and representative of the airborne contamination. Several volumes of air have been tested. When the sampling time exceeded 10 minutes, the number of samples obtained with ATΩ was proportional to the number of the period necessary to sample during the same period with S6 and AGI (Ex : 30 minutes with S6 = 3 x 10 minutes with ATΩ). During air sampling, temperature and humidity were measured with a portable instrument (Testo 615) equipped with appropriate calibrated probes. 412

3 Laboratory investigation Legionella and Legionella pneumophila were determinated in accordance with the French standard procedure XP T For the water samples, the method consists of filtration of 1- liter sample through 0.4-µm-pore-size polycarbonate membrane, decontamination by heat or acid, inoculation onto a GVPC medium, and incubation at 37 C for 10 days. A combined heat and acid decontamination procedure was added to eliminate competing bacteria, often heavily present in such samples. The detection level was 10 2 CFU/L. For the air samples, plastic and glass Petri dishes were incubated at 37 C for 10 days. The samples obtained with the impinger method were filtered through a 0.4-µm-pore-size membrane, then the decontamination procedure was performed before the incubation. The number of colony-forming units (CFU) recovered from each solid impacted sample was corrected using a corresponding conversion table (Andersen or ATΩ table). The result was reported in CFU/m 3. RESULTS The first campaigns on-site was made to test the feasibility of the air biocollection in outdoor environmental conditions (TA, TB and TD1). For technical and/or consenting reasons, no further campaign was performed on the site of TA and TB. Sampling strategy assays Different air volumes have been tested with the 3 biocollectors (table1). For 3 campaigns (TB, TC1 and TD3) positive results were obtained and corresponded to 280 to 840 liters of sampled air. For these 3 assays, replicated samples were performed and positive results represented 50%, 14%, 20% and 33% of the samples obtained with a volume of 280, 360, 500 Table 1 : Detection of L.pneumophila from water samples and specimens of air discharge from 4 cooling towers Water Air Tower N (1) Water biocollector L.pneumophila CFU/l Air volume tested (l) L.pneumophila positive air volume Total number of samples Air total flora (2) CFU/m 3 A ATΩ 100, 200, 400 no B 1 <10 2 ATΩ 100, 200, 400, 500, 1000 no S6 140, 280, ATΩ 200, 400, 500, S6 280, 420, 560, C AGI ATΩ 500, 1000 no <10 2 S6 840 no AGI 360 no ATΩ 50,100, 200 no ATΩ 500, 1000 no <10 2 S6 840 no D AGI 360 no ATΩ 500, S AGI 360 no (1) campaign number, (2) bacteria (including Legionella spp.) and/or fungi growing on GVPC media and 840 liters, respectively. The collection duration was 5 minutes to 30 minutes. 413

4 The count of Legionella pneumophila in air (2 to 4 CFU/m 3 ) was not proportional to the water contamination (<10 2 to CFU/L). Note that an associated flora was heavily present in cooling towers water which was confirmed by the significant contamination of the GVPC media impacted by airborne particles. When the concentration of Legionella in water was inferior to the detection level, the contaminant was not detected, except in the TB assays. For this last site, inhibition due to background flora could explain the negative result obtained in cooling water. Another campaign on this site would have been helpful for interpretation. The 3 positive assays were obtained with the biocollectors located near the tower (<2 meters), at the fan of the tower (TC1 and TD3) or downwind the exhaust vent of TB. The samples obtained on further distance where often contaminated by airborne flora. For example, 10 of the 16 samples performed with ATΩ were overgrown with fungi in TD1 assays. Comparison of biocollectors Table 2 : Comparison of the 3 biocollectors for L.pneumophila positive detection Biocollector S6 ATΩ AGI Temp. Hum. number of CFU / plate Tower Flora C % CFU/m 3 CFU/m 3 CFU/m 3 L.pneumophila (b) B 27,0 nd Bacteria (a) _ Fungi (a) _ L.pneumophila C 24,9 42 Bacteria (a) Fungi (a) L.pneumophila (c) 0 (d) D 9,0 nd Bacteria (a) (c) 13 (d) Fungi (a) (c) 0 (d) (a) bacteria (except Legionella spp.) or fungi collected on GVPC media, (b) not done, (c) sample obtained in parallel with S6, (d) sample obtained in parallel with ATΩ Among the 3 positive assays, S6 sampler allowed regularly detection of Legionella (once with 280L, twice with 840L), ATΩ got positive results with a 500 liters volume of air and AGI permitted positive result with 360L. The results expressed in CFU per m 3 of air showed in table 2 were comparable. The S6 collector shows that the size of the collected particles associated with L.peumophila was in a range of 1.1 to 3.3 µm (stage 4 and 5), which confirm the respirable potentiality of these contaminated aerosols. In these S6 assays, we noted that the positive plates were not contaminated with bacteria or fungi. DISCUSSION Using different air sampling methods to detect Legionella pneumophila at the air discharge from cooling towers, we tested classics methods in outdoor environmental conditions. Different water contaminations were observed among the 7 assays on-site which could help to determinate the limits of sampling methods. To estimate the more efficient air volume to detect L. pneumophila, we performed several samples with each of the biocollectors. We detected the airborne contaminant in each case of heavily amount of L. peumophila in water (> CFU/L) when particles were collected during long period of time with the Andersen 414

5 microbial sampler. When shorter period were chosen (with ATΩ), the percent of detection was lower which mean that one needs more samples with this method. The results showed also that different air collection time should be tested for each sampling equipment more than specific volumes of air. To counterbalance the air turbulences induced by the exhaust vent from the tower and by wind around the site, longer sampling periods should be applied. In the case of positive result obtained near one cooling tower by Ishimatsu et al, 2001, a modified impinger AGI30 was used during 120 minutes at a flow rate of 5L/min. Concerning the amount of airborne Legionella detectable in our study, the results were similar to that obtained with a modified single stage sampler in waterfalls of cooling tower (4 CFU/m3) by Pascual and al, The 3 sampling methods allowed detection of Legionella but the more efficient method was S6 sampler, certainly because of the repartition of airborne microorganisms on different stages. The repartition prevent inhibition of legionellae s growth which can occur when air is sampled on an unique plate. This point should be take into account with attention when samples of aerosols coming from high contaminated water are performed and analysis methods are cultural techniques. All our positive results were obtained close to the tower (<2 meters), downwind or at the fan of the towers, although all the further samples were negative. Even if sites outside a tower can be contaminated over distance of up to 200 meters (Bornstein, 1990), our results confirm the extreme variations of the air contamination few meters (3 to 11 meters) from the outlet of air. It should egg us on sample closer to the tower to increase the probability of Legionella detection if one research for sources of airborne contamination linked to such device. As positive results were obtained near an installation located on passageway (TD), we showed also that a cooling tower can be a non-negligible direct source of contaminated aerosols. With the six stage microbial sampler Andersen, we showed that small particles (<3,3 µm) were concerned. These particles are susceptible to penetrate into the lung, in particular the alveoli and then can be considered as potential infectious aerosols. That confirms the suspicions about cooling systems in the case of community outbreaks. However, the results are effective in 3 of the 4 towers we chose and can not be extrapolated to other installation as the amount and the size of the emitted droplets are closely dependant of the structure and functioning of each cooling system. CONCLUSION AND IMPLICATIONS The ability of impact methods in outdoor conditions has been study for the detection of Legionella near cooling towers. Although the variable conditions involved adapted methods, it has been able to apply a specific strategy increasing the probability to detect Legionella in aerosols discharge from outdoor installations. An appropriate strategy would be a sufficient number of large air samples, obtained with a low flow rate sampler, the nearest of the tower exhaust vent. This strategy could be helpful in an investigation during an outbreak of legionnaire s disease. In an another way, this study allows serious hopes in the application of its strategy in more homogenous indoor conditions. ACKNOWLEDGEMENTS We thank Olivier Challemel, Florence Pigeon and Martine Adnane for technical assistance and advice. 415

6 REFERENCES Andersen AA New sampler for the collection, sizing, and enumeration of viable airborne particles. J. Bacteriol. Vol. 76, pp BEH n 41/99, Cas groupés de Légionelloses dans le 15 e arrondissement de Paris, août Bornstein N Dispersion and survival of Legionella in air. Aerobiologia. Vol. 6, pp Decludt B, Guillotin L, Van Gastel B, et al Epidemic cluster of legionnaires disease : Paris, June Eurosurveillance. Vol. 4 (11), p 115. Dubrou S, Guillotin L, Cabon S, et al Cooling towers and Legionellosis : a large urban area experience. In Legionella, Reinhard and al, eds. Washington DC : ASM Press, pp Ishimatsu S, Miyamoto H, Hori H, et al Sampling and detection of Legionella pneumophila aerosols generated from an industrial cooling tower. Ann. Occup. Hyg.. Vol. 45 (6), pp Lin X, Willeke K, Ulevicius V, et al Effect of sampling time on the collection efficiency of All-Glass Impingers. Am. Ind. Hyg. Assoc. J. Vol. 58, pp Pascual L, Pérez-Luz S, Amo A, et al Detection of Legionella pneumophila in bioaerosols by polymerase chain reaction. Can. J. Microbiol. Vol. 47 (4), pp Schvoerer C, Campese C, Chichizola MN, et al. 2001, Epidémie communautaire de légionellose, Rennes, France, 2000, Journées Scientifiques de l InVS novembre Saint-Maurice. 416