Prevalence and Molecular Characteristics of Waterborne Pathogen Legionella in Industrial Cooling Tower Environments

Cooling towers are a source of Legionnaires’ disease. It is important from a public health perspective to survey industrial cooling towers for the presence of Legionella. Prospective surveillance of the extent of Legionella pollution was conducted at factories in Shijiazhuang, China between March 2011 and September 2012. Overall, 35.7% of 255 industrial cooling tower water samples showed Legionella-positive, and their concentrations ranged from 100 Colony-Forming Units (CFU)/liter to 88,000 CFU/liter, with an average concentration of 9100 CFU/liter. A total of 121 isolates were obtained. All isolates were L. pneumophila, and the isolated serogroups included serogroups 1 (68 isolates, 56.2%), 6 (25, 20.7%), 5 (12, 9.9%), 8 (8, 6.6%), 3 (6, 5.0%) and 9 (2, 1.6%). All 121 isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and 64 different patterns were obtained. All 121 isolates were analyzed sequence-based typing (SBT), a full 7-allele profile was obtained from 117 isolates. One hundred and seventeen isolates were divided into 49 sequence types. Two virulence genes, lvh and rtxA, are analyzed by polymerase chain reaction (PCR). 92.6% (112/121) and 98.3% (119/121) isolates carried lvh and rtxA respectively and 90.9% (110/121) of tested isolates carried both genes. Our results demonstrated high prevalence and genetic polymorphism of L. pneumophila in industrial cooling tower environments in Shijiazhang, China, and the SBT and virulence gene PCR results suggested that the isolates were pathogenic. Improved control and prevention strategies are urgently needed.


Introduction
Legionella, the aetiologic agents of legionellosis, are ubiquitous worldwide in rivers and lakes [1] and man-made water systems such as spas and cooling towers [2]. To date, more than 50 Legionella species have been characterized, and 25 species are known to cause human disease [2,3]. Most human infections are caused by Legionella pneumophila, which is responsible for approximately 90% of the identified clinical cases, and the predominant serogroup is serogroup 1 [2,[4][5][6].
Transmission of bacteria from the environment to humans occurs via inhalation or aspiration of Legionella-containing aerosols [7,8]. Cooling towers that are contaminated by Legionella have been identified as the cause of sporadic cases and outbreaks of legionellosis among people living nearby [9,10]. It is important from a public health perspective to survey industrial cooling towers systems for the presence of Legionella [11,12].
Several outbreaks and a number of cases of Legionella infection that were associated with industrial cooling towers have been reported worldwide [13][14][15]. In China, industrial cooling towers are widely distributed. In our previous study that compared the ability of four methods, culture, polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) and Ethidium monoazide (EMA)-quantitative real-time PCR (EMA-qPCR), to detect Legionella in different types of water systems, we isolated Legionella from cooling towers at a rate of up to 31.0% by quantitative real-time PCR. When using the culture method, the percent-positive rate for Legionella was 26.4%, and the concentrations of Legionella were as high as 1500 Colony-Forming Units (CFU)/liter [16]. These results showed high degree of pollution of Legionella in cooling towers water samples in China. However, there is no surveillance system focusing on the Legionella in the industrial cooling towers.
The industrial cooling towers are usually located in the factories; the arrangement of industrial cooling towers is different to that in other public places. In other public places, such as buildings, hospitals and hotels, the cooling towers are located on the roof, where few people access. But in the factories, the cooling towers are located on the edge of the road, where people often go through. Furthermore, the circulating water in cooling towers can be aerosolized. All these factors increase the risk of Legionella infection. It is important to survey the contamination of Legionella in cooling towers to avoid serious outbreaks of Legionnaires' disease.
To systematically investigate the presence, population structure and infection potential of Legionella in industrial cooling towers water systems, we chose cooling towers in 22 factories in Shijiazhuang City, Hebei province, China as study points for 17 months. The species and serogroups of Legionella isolates were determined. Genetic characteristics were analyzed using pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) methods. Furthermore, two virulence genes, lvh and rtxA, which could be used as indicators of infection potential for L. pneumophila isolates, were examined [17].

Sample Collection
We selected cooling towers in 22 factories in Shijiazhuang City, China, as study points. Sodium hypochlorite, chlorine dioxide, glutaraldehyde and isothiazolin-one were used as disinfection/biocidal treatment of cooling tower water in these factories every week in the summer and every month in other seasons. The samples were taken at the same time before the disinfection/biocidal treatment. Among them cooling towers in one factory (factory A) were selected for sampling in March, April and July in 2011 and every month from October 2011 to September 2012. Cooling towers in other 21 factories (factory B-factory V) were irregular selected for sampling between March 2011 and September 2012. All 255 cooling tower water samples were collected during the study period, and the procedure for collection and pretreatments of the environmental water samples was based on the protocol according to ISO 11731 [18]. Five hundred milliliters of each water sample was collected from a reservoir or condensation pan and placed in a sterile, screw-capped container. Chlorine in the water samples was inactivated by the addition of sodium thiosulfate.

Legionella Detection
To detect Legionella, 500 milliliters of the water samples were filtrated through a membrane with 0.22-μm holes and the membranes were resuspended in 5 mL of distilled water. The resuspension were serially diluted 10-fold with sterile water. Diluted and undiluted samples (100 μL each) were plated onto buffered charcoal yeast extract (BCYE) supplemented with glycine (3 g/L), vancomycin (1 mg/L), polymyxin B (80,000 UI/L) and cycloheximide (80 mg/L) (GVPC) agar (Oxoid, Hampshire, United Kingdom), and the plates were incubated at 37 °C for 10 days. Colonies were identified as Legionella using the Gram staining, L-cysteine requirement test and slide agglutination using polyclonal antisera (Denka Seiken, Tokyo, Japan).

PFGE
One-day, standardized PFGE protocol was used for L. pneumophila subtyping [19]. Cell suspensions were prepared with optical density of 3.8-4.0 using a Densimat photometer (BioMérieux, Lyon, France). A 400-μL aliquot of the adjusted cell suspension was transferred to a 1.5-mL centrifuge tube and 20 μL of proteinase K (20 mg/mL stock; Invitrogen, Carlsbad, CA, USA) was added and mixed gently with the pipette tip. Plugs were made by adding an equal volume (400 μL) of molten 1.0% SeaKem Gold (SKG, Cambrex, Rockland, ME, USA) agarose to each cell suspension and the mixture was immediately dispensed into the wells of a reusable plug mold (Bio-Rad Laboratories, Hercules, CA, USA). The plugs were transferred into 50-mL polypropylene tubes containing 5 mL of cell lysis buffer (50 mM Tris, 50 Mm Ethylene Diamine Tetraacetie Acid (EDTA) (pH 8.0), 1% sarcosine, and 0.5 mg of proteinase K/mL). Lysis of cells in the plugs was performed for 2 h at 54 °C in a water bath with constant agitation (160 rpm). The plugs were washed two times with 15 mL of sterile ultrapure water and four times with 15 mL of TE buffer (10 mM Tris: 1 mM EDTA (pH 8.0)) at 50 °C. Legionella slices were digested using 30 U per slice of AscI (New England Biolabs, Ipswich, MA, USA) for 4 h at 37 °C. Electrophoresis was run with a switch time of 6.8 s to 54.2 s for 19 h in a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA). Images were captured using a Gel Doc 2000 system (Bio-Rad) and converted to TIFF files and analyzed using the BioNumerics version 5.1 software (Applied Maths, Kortrijk, Belgium). Similarity analysis of the PFGE patterns was performed by calculating the Dice coefficients (SD) [20] and clustering was created using the unweighted-pair group method with average linkages (UPGMA).

SBT and Allelic Diversity Analyses
Genotyping was conducted via the standard, sequence-based typing (SBT) method of the European Working Group for Legionella Infections (EWGLI) using 7 genes (flaA, pilE, asd, mip, mompS, proA and neuA) [21,22]. The SBT database that was available on the EWGLI website [23] was used for nucleotide analysis, and the sequences were compared with those in the SBT database, which were also available on the website [24].
The clonal complexes were analyzed using the eBURST V3 software [25,26], and clusters of related sequence types (STs) that descended from a common ancestor were defined as clonal groups (CGs). Single genotypes that corresponded to no CG were defined as singletons, and the BioNumerics software was used to create a minimum spanning tree. In the minimum spanning tree, the founder ST was defined as the ST with the greatest number of single-locus variants. Types are represented by circles, and the size of a circle indicates the number of strains of this particular type. Heavy solid lines connect two types that differ within a single locus; light solid lines connect double-locus variants; heavy dotted lines connect triple-locus variants; light dotted lines connect quadruple-locus variants; and gray circles represent STs that are not part of any clonal complex.

Detection of Virulence Genes
lvh and rtxA genes were examined by PCR. The primers and PCR reaction conditions used to detect lvh and rtxA regions were same as in those described by previous studies [27].

The Degree of Pollution of Legionella in Cooling Tower Water Samples
All 255 cooling tower water samples were tested, and Legionella was cultured from 91 of those samples. Different serogroups were detected from 30 samples. Viable counts for the positive samples (by using the Gram staining, L-cysteine requirement test and slide agglutination using polyclonal antisera) ranged from 100 CFU/liter to 88,000 CFU/liter with an average of 9100 CFU/liter. Overall, the data presented a positive rate of Legionella of 35.7% (91/255) in the cooling tower water samples of this study. Of the positive samples, 71.8% were isolated over 1000 CFU/liter. Twelve of the 22 studied factories were positive for Legionella in at least one sample during the study period.
For factory A, continuous sampling of one-year period every month from October 2011 to September 2012 was carried out (Figure 1). The positive rates of Legionella were higher during the summer and autumn (from May to October) than during the winter (from December to April).

Figure 2.
Clustering results of patterns obtained by pulsed-field gel electrophoresis (PFGE) analysis of 121 Legionella pneumophila strains. These strains were isolated from industrial cooling towers in Shijiazhang, China during the 19-month study period.

PFGE and SBT Analysis of Legionella Isolates
All 121 isolates were analyzed by PFGE, and divided into 64 different patterns (Figure 2). The pattern LPA16.CN0245 was the most frequently occurring pattern and contained 18 isolates that were isolated from three factories over 19 months. Seventeen patterns contained more than one isolate, and among these, 7, 16 and 8 patterns contained isolates from different serogroups, months and factories, respectively. Forty-four patterns contained only one isolate. These results indicated a high genetic polymorphism among these tested isolates.

Discussion
In this study, Legionella were detected in 35.7% of collected industrial cooling towers water samples from 22 factories during the 19-month study period, showing a high degree of pollution of Legionella in industrial cooling towers. The concentration of Legionella was high in this study, with 71.8% of the samples giving a Legionella concentration that exceeded 1000 CFU/liter, which was higher than that was found in cooling tower water samples in Shanghai, China [28] and in spring water samples [29,30]. The above findings revealed a high prevalence and high concentration of Legionella in surveyed cooling towers. Legionella widely and long-standingly inhabits industrial cooling towers water in Shijiazhuang.
The Legionella pollution and concentration were different between seasons in this study. The positive rates were higher during the summer and autumn than that during the winter. The highest concentration was observed in the autumn. Such a seasonal variation may be due to the fact that Legionella multiply faster in the warmer waters of summer and autumn. In Shijiazhuang, the air temperatures were higher in the summer and autumn than other seasons. The seasonal differences of Legionella pollution and concentration in cooling tower water observed in this study were opposite to that in spring water samples as observed in our previous study [30]. Air temperature and utilization rate may explain the differences in the presence of Legionella between seasons. In the study area, air temperature in summer (30 °C-36 °C) and autumn (25 °C-30 °C) is higher than that of other seasons (−5 °C-24 °C) and could impact the water temperature of cooling towers. The cooling tower water temperatures in summer and autumn are up to 32 °C, and in other seasons is no more than 25 °C.
We speculate that air temperature was one factor causing higher Legionella pollution and concentration of cooling towers in summer and autumn. Another factor, utilization rate, may also explain the differences in the presence of Legionella between seasons. Higher utilization rate of cooling towers was observed in summer and autumn that in winter; in contrast, the numbers of passengers visiting hot spring were higher in spring and winter than in summer and autumn. Based on our results of this study and a previous one [30], we suggest that more disinfection measures are needed for spring water systems in spring and winter, and more disinfection measures are needed for cooling tower water systems in summer and autumn.
All the strains isolated in this study were L. pneumophila. This was different to that of cooling tower water in other countries and other types of water samples in China. L. pneumophila accounted for 76.2% of the strains that were isolated from cooling tower water samples in South Korea, and this organism was predominantly found in other types of water samples, such as water from buildings, public baths, hospitals and hotels [31]. In the previous studies of cooling tower and spring water samples, other species, Legionella micdadei and Legionella bozemanii, were also isolated. In this study, serogroup 1 was the most frequently L. pneumophila isolate, which was in agreement with that of previous studies of cooling towers in China and other countries [28,31]; however, L. pneumophila serogroup 1 was not predominant in springs in China as described in previous study [30]. This observation suggested that the serogroups may have adaptability of specific kinds of water environment. The adaptability mechanism of serogroups to specific environment should be studied.
PFGE results showed that PFGE patterns of the tested strains were highly diverse. More than one-third of isolates had a unique PFGE pattern. There were some patterns containing isolates from different serogroups, months and factories. However, the most dominant pattern persisted in the investigated cooling towers throughout the 19-month study period, suggesting that although high genetic polymorphism was displayed, a dominant pattern also existed. However, the PFGE patterns of the tested strains in this study were different to these clinical isolates of persons with diagnosed L. pneumophila pneumonia from China (data not shown).
SBT results showed that the population of STs was highly diverse, suggesting high genetic polymorphism. One hundred and seventeen isolates were divided into 49 STs, of which 34 STs had new allelic profiles. After querying the SBT database, we found five STs (ST1, ST59, ST60, ST345, ST595) that had previously caused Legionnaires' diseases worldwide. In our previous study, we reported a Legionnaires' disease that was caused by a L. pneumophila strain of ST59, which was the second predominant ST in this study [32]. ST1, which is the most prevalent ST worldwide [33][34][35][36][37][38][39], was the most predominant ST in this study. Similar results have been reported in other countries. In Japan, the majority (29%) of environmental isolates, especially from cooling tower waters (74%), were ST1 [33]. In our previous study focusing on the ST distribution of L. pneumophila serogroup 1 strains isolated from different type of water samples in China, results showed that ST1 was the predominant type, accounting for nearly half of the analyzed strains [40]. L. pneumophila serogroup 1 and ST1 are the predominant serogroup and ST caused clinical Legionnaires' disease cases, so the Legionella isolates in cooling towers surveyed in this study have the potential to caused Legionella infections and Legionnaires' disease outbreaks.
lvh and rtxA are two virulence genes of L. pneumophila [41,42]. So, a PCR detection test strategy with lvh and rtxA as pathogenesis markers would be useful for determining the infection potential of an isolate (17). In this study, 90.9% of L. pneumophila isolates, regardless of the serogroups, carried both lvh and rtxA; which further suggests the infection potential of L. pneumophila isolates in cooling tower waters.

Conclusions
Our results demonstrated high prevalence and genetic polymorphism of Legionella in industrial cooling towers. Furthermore, the serogroup, SBT and virulence genes detection results suggested that the Legionella isolates of industrial cooling tower environments may be pathogenic. Improved control and prevention strategies of Legionella, containing routine monitoring of industrial cooling tower water samples, using more detection technologies such as PCR, qPCR and EMA-qPCR, for detection of viable but nonculturable (VBNC) cells and to overcome underestimation by culture methods, as well as monitoring of Legionella-containing aerosols, are urgently needed.