Combining Environmental Investigation and a Dual-Analytical Strategy to Isolate the Legionella longbeachae Strain Linked to Two Occupational Cases of Legionellosis

Combining Environmental Investigation and a Dual-Analytical Strategy to Isolate the Legionella... Abstract Legionella has a global distribution, mainly in aquatic and man-made environments. Under the right conditions, this bacterium is a notorious human pathogen responsible for severe pulmonary illnesses. Legionellosis outbreaks are reported around the world, and exposure to water droplet aerosols containing Legionella pneumophila is usually the mechanism of its transmission. Even if L. pneumophila causes most outbreaks, Legionella longbeachae also accounts for some cases. Unlike most other Legionella strains, L. longbeachae is typically found in soil. Given the wide diversity and high concentration of microorganisms found in soil, isolating L. longbeachae by culture can be challenging. Because the chances of successfully isolating the strain are low, it is often not even attempted. This study reports the strategies used to successfully isolate L. longbeachae strain that was responsible of the two occupational legionellosis in Quebec. Fifteen random samples were collected from the soil of the metal recycling plant where the diagnosed workers were employed, covering 1.5% of the accessible surface of the plant. All samples were analyzed with both the quantitative polymerase chain reaction (qPCR) and culture methods. Four qPCR detection systems targeting Legionella spp, L. pneumophila, L. pneumophila serogroup 1, and L. longbeachae were used. Acid, heat, and acid/heat treatments were used for the culture method. For the qPCR method, all samples were positives for Legionella spp but only four were positives for L. longbeachae. For the culture method, only one isolate could be confirmed to be L. longbeachae. However, that strain proves to be the same one that caused the occupational legionellosis. Detecting the presence of L. longbeachae using the qPCR method made it possible to target the right samples to enable the cultivable strain of L. longbeachae to be isolated from the soil of the metal recycling plant. The complementarity of the two methods was established. This paper demonstrated the advantages of selecting the proper sampling and analytical strategies to achieve the isolation of the strain responsible for the infections. It also highlights for the first time in Quebec the potential occupational risks associated with L. longbeachae from soil and should motivate questioning soil exposures when all sources of water contamination have been eliminated from the causal analysis of legionellosis. bacterial strain isolation, culture method, environmental investigation, Legionella longbeachae, Legionellosis, qPCR detection, soil samples Introduction Legionella bacteria have a global distribution and are mainly found in natural and man-made aquatic environments (Friedman et al., 1987; Yu et al., 2002). More than 50 species of Legionella have been described and almost half of them are recognized as potential pathogens. Legionellaceae may be responsible for two different types of pathology. While Pontiac fever is a mild respiratory infection, Legionnaires’ disease is a severe pneumonia with fatality rates of between 10 and 20% (Bartram, 2007). Legionellosis outbreaks are reported all over the globe every year. Usually, exposure to water droplet aerosols from cooling towers, fountains, and spas are the identified source of Legionellosis. In Europe and North America, Legionella pneumophila is known to be responsible for 90 to 95% of cases (Association of Water Technologies, 2003; Bartram, 2007; Whiley and Bentham, 2011). In Australia and New Zealand, Legionella longbeachae is the infectious bacterium responsible for more than 50% of cases (Whiley and Bentham, 2011; Murdoch et al., 2013). Most Legionella species live in aquatic environments (Lawrence et al., 2016), but L. longbeachae is an exception. L. longbeachae is not often isolated from field samples (Koide et al., 2001) and is usually associated with compost, manufactured potting mixes, and gardening soil. It has rarely been found in natural soil and almost never in aquatic environments (Joly et al., 1984; Marrie et al., 1994; Fields, 2007; Whiley and Bentham, 2011; Currie and Beattie, 2015; Isenman et al., 2016). To our knowledge, in Canada, L. longbeachae has only been isolated from field samples twice, and contrary to what has been reported elsewhere, on both occasions, they were detected in water samples (Joly et al., 1984; Marrie et al., 1994) and not from soil. Legionella longbeachae was isolated for the first time in 1980, from the lungs of a patient with pneumonia (McKinney et al., 1981). Even though its occurrence is low, there are now reports from around the world that L. longbeachae is a cause of legionellosis (Okazaki et al., 1998; Centers for Disease Control and Prevention-CDC, 2000; Phares et al., 2007; Whiley and Bentham, 2011; Lindsay et al., 2012; Currie and Beattie, 2015; Picard-Masson et al., 2016). In Canada, human infection from L. longbeachae has rarely been described (Wright et al., 2012; Picard-Masson et al., 2016). Because of the wide diversity and high concentration of microorganisms found in soil, the isolation of a specific bacterium by culture is not only laborious, but often impossible. Isolating a specific species present in very low concentrations in soil samples compared to other species is often an extremely complicated undertaking. During the summer of 2015, two cases of occupational Legionellosis were linked to L. longbeachae in a metal recycling plant (Picard-Masson et al., 2016). L. longbeachae has never been isolated from soil in Canada, much less from poor natural soil such as that found on the work site. In order to determine the possible source of those infections, occupational and environmental investigations were carried out. This study reports the strategies used for environmental sampling and combined analytical approach using quantitative polymerase chain reaction (qPCR) and culture methods to successfully isolate the L. longbeachae strain from one soil samples and connect it to the workers’ disease. Materials and methods Soil sampling The metal-working plant is about 120000 ft2 in area, of which 50000 ft2 are covered by a roof. The shredder and conveyor occupying 10000 ft2, leaving 40000 ft2 accessible. Most of the site is on bare ground. Soil samples were taken from that area since there was greater likelihood of finding L. longbeachae contamination in the soil there (i.e., greater humidity, less exposure to wind and ultraviolet rays from the sun, closer to soil-contaminated metal, and wrecked cars being delivered). The roof covered area was divided into 43 ft2 lots and 15 of them were randomly selected. Each lot was marked, and care was taken not to step on the sample surface. In each lot, 12 soil sub-samples were taken and pooled together in a 1-liter sterile bottle (Nalgene, Rochester, NY, USA). A control sample was also taken from an area of undisturbed soil close to the entrance of the plant. Proper sterilization of the sampling equipment was ensured by using 99% isopropanol, between each lot. Sample preparation Soil suspension and culture inoculation were done the same day as the sampling. DNA extraction and qPCR amplifications were performed the next days. First, all samples were manually homogenized, by stirring the soil and other particles, when present (e.g., stone, plastic debris, etc.), with a sterile glass rod, in the 1-liter plastic container (Fisher Scientific, Ottawa, ON, Canada). Ensuring that stones and pieces of wood were not included, 5.0 ± 0.2g of the pre-mixed soil was weighed and placed in 50 ml Falcon conical centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA); then 30 ml of PCR grade sterile phosphate buffer solution (PBS) (Thermo Fisher Scientific) was added to the soil. The suspensions were shaken on a multi-tube maxi-vortex (VWR International, Ville Mont-Royal, QC, Canada) at 2200 rpm for 15 min. After agitation, the soil suspensions were left at room temperature for at least 15 minutes to allow time for the larger particles to settle out. The suspensions were then separated into four aliquots to perform the qPCR and the culture analysis. Unused fractions of the samples were kept at 4°C (Centers for Disease Control and Prevention-CDC, 2005). PCR detection method Seven ml of the soil in the suspension were concentrated down to a final volume of 1.5 to 2 ml by centrifugation at 14800 rpm for 5 min in an IEC Micro CL 17 centrifuge (Thermo Fisher Scientific). The total genomic DNA was extracted from concentrates using ZR Soil Microbe DNA MiniPrep™ (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instruction manual. To determine whether L. pneumophila could also be present in the natural soil samples, a total of four Taqman detection systems were used for this study. The L. longbeachae system kit was acquired from QIAGEN® (microbial DNA qPCR assay kit BPID00555A, Qiagen, Toronto, ON, Canada). The other detection systems, namely, JFP/JRP: LegLC for Legionella spp (Jonas et al., 1995; Wellinghausen et al., 2001), PT69-PT70: LpneuFL for Legionella pneumophila (Macioszek et al., 1999), and P66/P65: Sg1 for L. pneumophila serotype 1 (Mérault et al., 2011), had previously been thoroughly validated and utilized in our lab, mainly on water samples from manufacturing processes and cooling towers (Marchand and Lacombe, 2015). The probes and primers came from Integrated DNA Technologies (Coralville, IO, USA) and their sequences can be found in Table 1. Target DNA from L. pneumophila ATCC 33152 and L. longbeachae 33462 were used as positive controls, and sterile PCR water as the negative control of each PCR plate. The TaqMan® Exogenous Internal Control from Applied Biosystems (Life Technologies, Austin, TX, USA) was added to the master mix to assess for possible inhibition. All qPCR were performed in a final volume of 25 µl with 2 µl of template DNA. Amplification was completed in an Eppendorf Mastercycle Realplex 2 thermocycler (Thermo Fisher Scientific). The DNA amplification programs for L. longbeachae started at 95°C for 15 min, followed by 40 cycles of 94°C for 15 s and 60°C 1 min, and then a final step of 72°C for 10 s. For the other Legionella, the program was 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 60.5°C for 30 s and 72°C for 15 s, with a final step of 72°C for 10 s. Table 1. Primes and probes sequences of the detection systems for Legionella sp, L. pneumophila, and L. pneumophila serogroup 1. Systems  Sequences  Legionella sp: JFP/JRP: LegLC   Primes  JFP  5-AGG GTT GAT AGG TTA AGA GC-3  JRP  5-CCA ACA GCT AGT TGA CAT CG-3   Probe  LegLC  5′-/56-FAM/TAC TGA CAC/ZEN/TGA GGC ACG AAA GCG T/3IABkFQ/-3  Legionella pneumophila: PT69-PT70: LpneuFL   Primes  PT69  5-GCA TTG GTG CCG ATT TGG-3  PT70  5-GCT TTG CCA TCA AAT CTT TCT GAA-3   Probe  LpneuFL  5′-/56-FAM/CCA CTC ATA/ZEN/GCG TCT TGC ATG CCT TTA/3IABkFQ/-3′  Legionella pneumophila sérogroupe 1: P66/P65: LegSg1   Primes  P66  5-CAA ACA CCC CAA CCG TAA TCA-3  P65  5-CAA AGG GCG TTA CAG TCA AAC C-3   Probe  LegSg1  5′-/56-FAM/TCC TGG GAT/ZEN/TGG GTT GGG TTA TTT TAA CTC CT/3IABkFQ/-3′  Systems  Sequences  Legionella sp: JFP/JRP: LegLC   Primes  JFP  5-AGG GTT GAT AGG TTA AGA GC-3  JRP  5-CCA ACA GCT AGT TGA CAT CG-3   Probe  LegLC  5′-/56-FAM/TAC TGA CAC/ZEN/TGA GGC ACG AAA GCG T/3IABkFQ/-3  Legionella pneumophila: PT69-PT70: LpneuFL   Primes  PT69  5-GCA TTG GTG CCG ATT TGG-3  PT70  5-GCT TTG CCA TCA AAT CTT TCT GAA-3   Probe  LpneuFL  5′-/56-FAM/CCA CTC ATA/ZEN/GCG TCT TGC ATG CCT TTA/3IABkFQ/-3′  Legionella pneumophila sérogroupe 1: P66/P65: LegSg1   Primes  P66  5-CAA ACA CCC CAA CCG TAA TCA-3  P65  5-CAA AGG GCG TTA CAG TCA AAC C-3   Probe  LegSg1  5′-/56-FAM/TCC TGG GAT/ZEN/TGG GTT GGG TTA TTT TAA CTC CT/3IABkFQ/-3′  View Large Culture method Pre-treatment and inoculation To enhance the odds of isolating L. longbeachae in natural soil, three treatments (acid, heat, and acid/heat) were applied to all samples in suspension before plating them on buffer charcoal yeast extracts (BCYE) and BMPA (Oxoid Inc, Nepean, ON, Canada). Heat treatment was performed on 15 ml of extracted soil, which was deposited in a water bath at 50 ± 2°C for 30 ± 2 minutes. Directly afterward, suspensions were diluted (1:10 and 1:100) and plated on both culture media, except for 1 ml that was reprocessed with the acid treatment to obtain a combined acid/heat effect. The acid treatment was performed by adding 1 ml of the sample extract to 1 ml of HCL/KCl pH 2.2 ± 0.2 solution, and after 10 min, the samples were diluted in PBS and 200 µl of the suspensions and 1:10 and 1:100 dilutions were immediately inoculated. The 1:100 sample extract was plated in triplicate, 1:10 in duplicate and the direct suspension was inoculated once on each media. Finally, for each sample, a total of 48 petri dishes were plated. A fresh suspension of L. pneumophila ATCC 33152 was processed in each treatment as a positive control, with sterile water as the negative control. Confirmation of L. longbeachae in culture After 4 days of incubation at 35°C with the addition of 5% CO2, all petri dishes were observed under a Nikon SMZ18 stereomicroscope at magnifications of 10X to 135X (Nikon Instruments Inc., Melville, NY, USA). All colonies that appeared to be Legionella were isolated on BCYE and TSA blood agar (Oxoid Inc.). In the first trial, a total of 127 colonies were isolated for prospective confirmation. The colonies came from all the dilutions, both media and the three treatments. Following the positive PCR results, a second plating trial was performed only on the four positive samples. 1:1000 and 1:10000 dilutions of the suspension, maintained for 12 days at 4°C were re-plated on BMPA and BCYE. After incubation, this second plating resulted in 38 new prospective isolated colonies. All isolate colonies that did not grow on TSA with blood agar were analyzed using the Sherlock fatty acid methyl ester method (GC-FAME) (version 6.1) (MIDI Inc., Newark, NY, USA) following the manufacturer’s instructions and using the IBA1 database for the profiles matching the Legionella species. Dendrogram The dendrogram was used to compare the fatty acid methyl ester (GC-FAME) profiles of the strains and determine the degree of relatedness between the isolates. The seven strains analyzed by the MIDI Sherlock MIS (MIDI Inc.) were the strains of the two patients, isolated from their lung sputum, the soil strain came from sample 183, one reference strain of L. longbeachae (ATCC-33462), and three other ATCC non-L. longbeachae strains of Legionella. Isolated strains linked at a Euclidean distance of 10 units generally belong to the same microbial species; isolates linked at a Euclidean distance of 6 units generally belong to the same subspecies; and isolates linked at a Euclidean distance between 2 and 4 generally are considered to be the same strain (Leonard et al., 1995; Smith and Siegel, 1996; Sasser, 2001; Hinton et al., 2004). Results To our surprise, when analyzed with the qPCR method, all 16 samples were positive for Legionella spp (Table 2). Also surprising was that qPCR could detect L. longbeachae in four of them, but only one had a concentration above the analytical quantification limit (6000 GU/g). After retesting those four qPCR positive samples with the culture method, Legionella spp was finally recovered in one sample. Table 2. Concentration of Legionella spp, L. pneumophila, and L. longbeachae (GU/g) measured by qPCR in natural soil samples collected from the metal recycling plant.   Legionella spp  L. pneumophila  L. pneumophila serogroup 1  L. longbeachae  Samples  GU/g  GU/g  GU/g  GU/g  176  8200  120  <6000  <6000  178  2600  <6000  <6000  <6000  179  9500  <6000  <6000  <6000  180  73000  <6000  <6000  <6000  181  31000  600  <6000  <6000  182  47000  <6000  <6000  <6000  183  690000  500  65  10000  190  370000  <6000  <6000  <6000  192  57000  <6000  <6000  <6000  193  83000  <6000  <6000  <6000  195  360000  <6000  <6000  <6000  196  58000  700  <6000  <6000  197  58000  <6000  <6000  <6000  198  27000  250  <6000  <6000  199  75000  500  65  <6000  200  36000  700  140  <6000    Legionella spp  L. pneumophila  L. pneumophila serogroup 1  L. longbeachae  Samples  GU/g  GU/g  GU/g  GU/g  176  8200  120  <6000  <6000  178  2600  <6000  <6000  <6000  179  9500  <6000  <6000  <6000  180  73000  <6000  <6000  <6000  181  31000  600  <6000  <6000  182  47000  <6000  <6000  <6000  183  690000  500  65  10000  190  370000  <6000  <6000  <6000  192  57000  <6000  <6000  <6000  193  83000  <6000  <6000  <6000  195  360000  <6000  <6000  <6000  196  58000  700  <6000  <6000  197  58000  <6000  <6000  <6000  198  27000  250  <6000  <6000  199  75000  500  65  <6000  200  36000  700  140  <6000  View Large The cultivable Legionella spp was confirmed in one sample at a concentration of ~2.0 × 105 CFU/g. The extremely high concentrations of non-Legionella colonies on the culture media, even with the combined acid/heat treatment, render isolation of the Legionella spp difficult. Of the total 165 colonies isolated in pure culture on the BCYE media, only one isolate strain could be confirmed to be L. longbeachae with the GC-FAME and the qPCR methods. The relatedness between the patients (strains 26 and 46) and the environmental strain (183-17) is shown in Fig. 1. The Euclidean distance calculated for all strains of L. longbeachae tested (reference, patient and soil) is below 5 units. Figure 1. View largeDownload slide Dendrogram displaying the Euclidean distance obtained from the fatty acid profile produced by GC-FAME analysis of the strains isolated from the occupational environment and the lungs of the two patients. Figure 1. View largeDownload slide Dendrogram displaying the Euclidean distance obtained from the fatty acid profile produced by GC-FAME analysis of the strains isolated from the occupational environment and the lungs of the two patients. Discussion For the great majority of sporadic legionellosis cases, the source of infection remains unknown (Den Boer et al., 2008; van Heijnsbergen et al., 2014; Currie and Beattie, 2015). Usually, it is because no environmental investigation has been attempted. When cases are related, sampling may be considered but not attempted due to known deficiencies in sampling or analytical protocol but another reason could be that the pathogenic strain goes undetected because of in the sampling and or analytical protocols used. Obtaining a culture of L. longbeachae from the soil in the work environment was a major coup in this occupational investigation. O’Connor et al. (2007) point out that the presence of Legionella spp in the media does not necessarily indicate that people handling it will become infected. Obtaining a Legionella strain from a culture made it possible to compare the clinical strains obtained from the two workers’ lungs with the environmental soil strain found in the recycling plant. Although most Legionella species are reported in aquatic environments, their presence has also been described in compost, humus, and soil by many authors (Hughes and Steele, 1994; Koide et al., 2001; Casati et al., 2009, 2010; Travis et al., 2012; van Heijnsbergen et al., 2014). In Australia, Steele et al. (1990) reported that Legionella spp could be isolated from as much as 73% of the potting soil produced. In the Netherlands, van Heijnsbergen et al. (2014) found that 30% (6/30) of the natural soil analyzed was positive for Legionella spp. Travis et al. (2012) reported isolation of Legionella spp from 56% of damp soil samples from Thailand, but they did not recover any that tested positive for L. longbeachae. In fact, with the exception of Koide et al. (2001), who reported that 37% of the samples tested positive for L. longbeachae in compost and potting soils without distinction, other authors reported in soil samples none to very sparse occurrences (4.3%) of L. longbeachae. Successful isolation of a cultivable L. longbeachae strain from the soil was considered to be a challenge by most people involved in the investigation and most did not encourage the environmental investigation and microbial analysis, in part due to the unreported presence of L. longbeachae in Canadian soil, and to the poor nature of the soil at the investigation site. This reluctance to gather environmental samples and to perform analyses may possibly be part of the explanation for the failures reported in identifying the source of sporadic cases of legionellosis. The combined approach applied in this study undoubtedly improves the likelihood of finding the environmental source of the strain. The qPCR analysis was used effectively to target samples containing L. longbeachae DNA. Once the samples possibly containing L. longbeachae were identified, our chances of recovering the strain from the soil of the work environment was somewhat improved. From more than 150 colonies isolated, only one was identified as Legionella spp by the GC-FAME method. The identification of this positive strain, obtained by culture, was confirmed with the qPCR using markers specific to L. longbeachae. This study also demonstrates that a dendrogram performed on the GC-FAME profiles obtained from the clinical and the environmental strains can provide preliminary appreciation of the relatedness between isolates. The Euclidean distances calculated between the four Legionella species included in this study are 10, 14, and 16 units, those distances correspond well to other inter-species distances reported in the literature (Sasser, 2001; Hinton et al., 2004). However, the distance found between the 4 L. longbeachae strains is smaller than that reported by Diogo et al. (1999). In fact, the Euclidean distance they reported among the six strains of L. longbeachae tested was 13 units, compared to only 5 units in this study for all tested strains (reference, patients and soil). Furthermore, the isolated strains of L. longbeachae from this study, and a Euclidean distance of only 2.4 units showed a very high relatedness among those three strains. As reported by Picard-Masson (2016), the strains were subsequently analyzed by pulsed-field gel electrophoresis, which revealed that they had the same genotype. Detecting the presence of L. longbeachae using the qPCR method made it possible to target the right samples to enable the cultivable strain of L. longbeachea to be isolated from the soil of the metal recycling plant. The complementarity of the two methods was established. This paper demonstrates the significance of making the proper analytical choices in order to successfully link microorganisms in the workplace to infectious illness; it also highlights the potential occupational risks associated with L. longbeachae in soil. Declaration Genevieve Marchand, Carole Pépin, and Nancy Lacombe are employed by the Institut de recherche Robert Sauvé en santé et en sécurité du travail (IRSST) who provided funding for this study. The authors designed and executed the study and have sole responsibility for the writing and content of the manuscript. Judith Lord is employed by the Direction de la santé public, CISSS de la Montérégie-Centre who provided funding for this study. The authors designed and executed the study and have sole responsibility for the writing and content of the manuscript. Conflict of Interest The authors declare no conflict of interest relating to the material presented in this article. References Association of Water Technologies. ( 2003) Legionella 2003- an update and statement by the association of water technologies (AWT) . Rockville, MD: Association of Water Technologies. Bartram J (ed.). ( 2007) Legionella and the prevention of legionellosis. World Health Organization . Casati S, Conza L, Bruin Jet al.  . ( 2010) Compost facilities as a reservoir of Legionella pneumophila and other Legionella species. Clin Microbiol Infect ; 16: 945– 7. 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( 2002) Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis ; 186: 127– 8. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Occupational Hygiene Society. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Work Exposures and Health (formerly Annals Of Occupational Hygiene) Oxford University Press

Combining Environmental Investigation and a Dual-Analytical Strategy to Isolate the Legionella longbeachae Strain Linked to Two Occupational Cases of Legionellosis

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British Occupational Hygiene Society
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.
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2398-7308
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2398-7316
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10.1093/annweh/wxx109
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Abstract

Abstract Legionella has a global distribution, mainly in aquatic and man-made environments. Under the right conditions, this bacterium is a notorious human pathogen responsible for severe pulmonary illnesses. Legionellosis outbreaks are reported around the world, and exposure to water droplet aerosols containing Legionella pneumophila is usually the mechanism of its transmission. Even if L. pneumophila causes most outbreaks, Legionella longbeachae also accounts for some cases. Unlike most other Legionella strains, L. longbeachae is typically found in soil. Given the wide diversity and high concentration of microorganisms found in soil, isolating L. longbeachae by culture can be challenging. Because the chances of successfully isolating the strain are low, it is often not even attempted. This study reports the strategies used to successfully isolate L. longbeachae strain that was responsible of the two occupational legionellosis in Quebec. Fifteen random samples were collected from the soil of the metal recycling plant where the diagnosed workers were employed, covering 1.5% of the accessible surface of the plant. All samples were analyzed with both the quantitative polymerase chain reaction (qPCR) and culture methods. Four qPCR detection systems targeting Legionella spp, L. pneumophila, L. pneumophila serogroup 1, and L. longbeachae were used. Acid, heat, and acid/heat treatments were used for the culture method. For the qPCR method, all samples were positives for Legionella spp but only four were positives for L. longbeachae. For the culture method, only one isolate could be confirmed to be L. longbeachae. However, that strain proves to be the same one that caused the occupational legionellosis. Detecting the presence of L. longbeachae using the qPCR method made it possible to target the right samples to enable the cultivable strain of L. longbeachae to be isolated from the soil of the metal recycling plant. The complementarity of the two methods was established. This paper demonstrated the advantages of selecting the proper sampling and analytical strategies to achieve the isolation of the strain responsible for the infections. It also highlights for the first time in Quebec the potential occupational risks associated with L. longbeachae from soil and should motivate questioning soil exposures when all sources of water contamination have been eliminated from the causal analysis of legionellosis. bacterial strain isolation, culture method, environmental investigation, Legionella longbeachae, Legionellosis, qPCR detection, soil samples Introduction Legionella bacteria have a global distribution and are mainly found in natural and man-made aquatic environments (Friedman et al., 1987; Yu et al., 2002). More than 50 species of Legionella have been described and almost half of them are recognized as potential pathogens. Legionellaceae may be responsible for two different types of pathology. While Pontiac fever is a mild respiratory infection, Legionnaires’ disease is a severe pneumonia with fatality rates of between 10 and 20% (Bartram, 2007). Legionellosis outbreaks are reported all over the globe every year. Usually, exposure to water droplet aerosols from cooling towers, fountains, and spas are the identified source of Legionellosis. In Europe and North America, Legionella pneumophila is known to be responsible for 90 to 95% of cases (Association of Water Technologies, 2003; Bartram, 2007; Whiley and Bentham, 2011). In Australia and New Zealand, Legionella longbeachae is the infectious bacterium responsible for more than 50% of cases (Whiley and Bentham, 2011; Murdoch et al., 2013). Most Legionella species live in aquatic environments (Lawrence et al., 2016), but L. longbeachae is an exception. L. longbeachae is not often isolated from field samples (Koide et al., 2001) and is usually associated with compost, manufactured potting mixes, and gardening soil. It has rarely been found in natural soil and almost never in aquatic environments (Joly et al., 1984; Marrie et al., 1994; Fields, 2007; Whiley and Bentham, 2011; Currie and Beattie, 2015; Isenman et al., 2016). To our knowledge, in Canada, L. longbeachae has only been isolated from field samples twice, and contrary to what has been reported elsewhere, on both occasions, they were detected in water samples (Joly et al., 1984; Marrie et al., 1994) and not from soil. Legionella longbeachae was isolated for the first time in 1980, from the lungs of a patient with pneumonia (McKinney et al., 1981). Even though its occurrence is low, there are now reports from around the world that L. longbeachae is a cause of legionellosis (Okazaki et al., 1998; Centers for Disease Control and Prevention-CDC, 2000; Phares et al., 2007; Whiley and Bentham, 2011; Lindsay et al., 2012; Currie and Beattie, 2015; Picard-Masson et al., 2016). In Canada, human infection from L. longbeachae has rarely been described (Wright et al., 2012; Picard-Masson et al., 2016). Because of the wide diversity and high concentration of microorganisms found in soil, the isolation of a specific bacterium by culture is not only laborious, but often impossible. Isolating a specific species present in very low concentrations in soil samples compared to other species is often an extremely complicated undertaking. During the summer of 2015, two cases of occupational Legionellosis were linked to L. longbeachae in a metal recycling plant (Picard-Masson et al., 2016). L. longbeachae has never been isolated from soil in Canada, much less from poor natural soil such as that found on the work site. In order to determine the possible source of those infections, occupational and environmental investigations were carried out. This study reports the strategies used for environmental sampling and combined analytical approach using quantitative polymerase chain reaction (qPCR) and culture methods to successfully isolate the L. longbeachae strain from one soil samples and connect it to the workers’ disease. Materials and methods Soil sampling The metal-working plant is about 120000 ft2 in area, of which 50000 ft2 are covered by a roof. The shredder and conveyor occupying 10000 ft2, leaving 40000 ft2 accessible. Most of the site is on bare ground. Soil samples were taken from that area since there was greater likelihood of finding L. longbeachae contamination in the soil there (i.e., greater humidity, less exposure to wind and ultraviolet rays from the sun, closer to soil-contaminated metal, and wrecked cars being delivered). The roof covered area was divided into 43 ft2 lots and 15 of them were randomly selected. Each lot was marked, and care was taken not to step on the sample surface. In each lot, 12 soil sub-samples were taken and pooled together in a 1-liter sterile bottle (Nalgene, Rochester, NY, USA). A control sample was also taken from an area of undisturbed soil close to the entrance of the plant. Proper sterilization of the sampling equipment was ensured by using 99% isopropanol, between each lot. Sample preparation Soil suspension and culture inoculation were done the same day as the sampling. DNA extraction and qPCR amplifications were performed the next days. First, all samples were manually homogenized, by stirring the soil and other particles, when present (e.g., stone, plastic debris, etc.), with a sterile glass rod, in the 1-liter plastic container (Fisher Scientific, Ottawa, ON, Canada). Ensuring that stones and pieces of wood were not included, 5.0 ± 0.2g of the pre-mixed soil was weighed and placed in 50 ml Falcon conical centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA); then 30 ml of PCR grade sterile phosphate buffer solution (PBS) (Thermo Fisher Scientific) was added to the soil. The suspensions were shaken on a multi-tube maxi-vortex (VWR International, Ville Mont-Royal, QC, Canada) at 2200 rpm for 15 min. After agitation, the soil suspensions were left at room temperature for at least 15 minutes to allow time for the larger particles to settle out. The suspensions were then separated into four aliquots to perform the qPCR and the culture analysis. Unused fractions of the samples were kept at 4°C (Centers for Disease Control and Prevention-CDC, 2005). PCR detection method Seven ml of the soil in the suspension were concentrated down to a final volume of 1.5 to 2 ml by centrifugation at 14800 rpm for 5 min in an IEC Micro CL 17 centrifuge (Thermo Fisher Scientific). The total genomic DNA was extracted from concentrates using ZR Soil Microbe DNA MiniPrep™ (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instruction manual. To determine whether L. pneumophila could also be present in the natural soil samples, a total of four Taqman detection systems were used for this study. The L. longbeachae system kit was acquired from QIAGEN® (microbial DNA qPCR assay kit BPID00555A, Qiagen, Toronto, ON, Canada). The other detection systems, namely, JFP/JRP: LegLC for Legionella spp (Jonas et al., 1995; Wellinghausen et al., 2001), PT69-PT70: LpneuFL for Legionella pneumophila (Macioszek et al., 1999), and P66/P65: Sg1 for L. pneumophila serotype 1 (Mérault et al., 2011), had previously been thoroughly validated and utilized in our lab, mainly on water samples from manufacturing processes and cooling towers (Marchand and Lacombe, 2015). The probes and primers came from Integrated DNA Technologies (Coralville, IO, USA) and their sequences can be found in Table 1. Target DNA from L. pneumophila ATCC 33152 and L. longbeachae 33462 were used as positive controls, and sterile PCR water as the negative control of each PCR plate. The TaqMan® Exogenous Internal Control from Applied Biosystems (Life Technologies, Austin, TX, USA) was added to the master mix to assess for possible inhibition. All qPCR were performed in a final volume of 25 µl with 2 µl of template DNA. Amplification was completed in an Eppendorf Mastercycle Realplex 2 thermocycler (Thermo Fisher Scientific). The DNA amplification programs for L. longbeachae started at 95°C for 15 min, followed by 40 cycles of 94°C for 15 s and 60°C 1 min, and then a final step of 72°C for 10 s. For the other Legionella, the program was 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 60.5°C for 30 s and 72°C for 15 s, with a final step of 72°C for 10 s. Table 1. Primes and probes sequences of the detection systems for Legionella sp, L. pneumophila, and L. pneumophila serogroup 1. Systems  Sequences  Legionella sp: JFP/JRP: LegLC   Primes  JFP  5-AGG GTT GAT AGG TTA AGA GC-3  JRP  5-CCA ACA GCT AGT TGA CAT CG-3   Probe  LegLC  5′-/56-FAM/TAC TGA CAC/ZEN/TGA GGC ACG AAA GCG T/3IABkFQ/-3  Legionella pneumophila: PT69-PT70: LpneuFL   Primes  PT69  5-GCA TTG GTG CCG ATT TGG-3  PT70  5-GCT TTG CCA TCA AAT CTT TCT GAA-3   Probe  LpneuFL  5′-/56-FAM/CCA CTC ATA/ZEN/GCG TCT TGC ATG CCT TTA/3IABkFQ/-3′  Legionella pneumophila sérogroupe 1: P66/P65: LegSg1   Primes  P66  5-CAA ACA CCC CAA CCG TAA TCA-3  P65  5-CAA AGG GCG TTA CAG TCA AAC C-3   Probe  LegSg1  5′-/56-FAM/TCC TGG GAT/ZEN/TGG GTT GGG TTA TTT TAA CTC CT/3IABkFQ/-3′  Systems  Sequences  Legionella sp: JFP/JRP: LegLC   Primes  JFP  5-AGG GTT GAT AGG TTA AGA GC-3  JRP  5-CCA ACA GCT AGT TGA CAT CG-3   Probe  LegLC  5′-/56-FAM/TAC TGA CAC/ZEN/TGA GGC ACG AAA GCG T/3IABkFQ/-3  Legionella pneumophila: PT69-PT70: LpneuFL   Primes  PT69  5-GCA TTG GTG CCG ATT TGG-3  PT70  5-GCT TTG CCA TCA AAT CTT TCT GAA-3   Probe  LpneuFL  5′-/56-FAM/CCA CTC ATA/ZEN/GCG TCT TGC ATG CCT TTA/3IABkFQ/-3′  Legionella pneumophila sérogroupe 1: P66/P65: LegSg1   Primes  P66  5-CAA ACA CCC CAA CCG TAA TCA-3  P65  5-CAA AGG GCG TTA CAG TCA AAC C-3   Probe  LegSg1  5′-/56-FAM/TCC TGG GAT/ZEN/TGG GTT GGG TTA TTT TAA CTC CT/3IABkFQ/-3′  View Large Culture method Pre-treatment and inoculation To enhance the odds of isolating L. longbeachae in natural soil, three treatments (acid, heat, and acid/heat) were applied to all samples in suspension before plating them on buffer charcoal yeast extracts (BCYE) and BMPA (Oxoid Inc, Nepean, ON, Canada). Heat treatment was performed on 15 ml of extracted soil, which was deposited in a water bath at 50 ± 2°C for 30 ± 2 minutes. Directly afterward, suspensions were diluted (1:10 and 1:100) and plated on both culture media, except for 1 ml that was reprocessed with the acid treatment to obtain a combined acid/heat effect. The acid treatment was performed by adding 1 ml of the sample extract to 1 ml of HCL/KCl pH 2.2 ± 0.2 solution, and after 10 min, the samples were diluted in PBS and 200 µl of the suspensions and 1:10 and 1:100 dilutions were immediately inoculated. The 1:100 sample extract was plated in triplicate, 1:10 in duplicate and the direct suspension was inoculated once on each media. Finally, for each sample, a total of 48 petri dishes were plated. A fresh suspension of L. pneumophila ATCC 33152 was processed in each treatment as a positive control, with sterile water as the negative control. Confirmation of L. longbeachae in culture After 4 days of incubation at 35°C with the addition of 5% CO2, all petri dishes were observed under a Nikon SMZ18 stereomicroscope at magnifications of 10X to 135X (Nikon Instruments Inc., Melville, NY, USA). All colonies that appeared to be Legionella were isolated on BCYE and TSA blood agar (Oxoid Inc.). In the first trial, a total of 127 colonies were isolated for prospective confirmation. The colonies came from all the dilutions, both media and the three treatments. Following the positive PCR results, a second plating trial was performed only on the four positive samples. 1:1000 and 1:10000 dilutions of the suspension, maintained for 12 days at 4°C were re-plated on BMPA and BCYE. After incubation, this second plating resulted in 38 new prospective isolated colonies. All isolate colonies that did not grow on TSA with blood agar were analyzed using the Sherlock fatty acid methyl ester method (GC-FAME) (version 6.1) (MIDI Inc., Newark, NY, USA) following the manufacturer’s instructions and using the IBA1 database for the profiles matching the Legionella species. Dendrogram The dendrogram was used to compare the fatty acid methyl ester (GC-FAME) profiles of the strains and determine the degree of relatedness between the isolates. The seven strains analyzed by the MIDI Sherlock MIS (MIDI Inc.) were the strains of the two patients, isolated from their lung sputum, the soil strain came from sample 183, one reference strain of L. longbeachae (ATCC-33462), and three other ATCC non-L. longbeachae strains of Legionella. Isolated strains linked at a Euclidean distance of 10 units generally belong to the same microbial species; isolates linked at a Euclidean distance of 6 units generally belong to the same subspecies; and isolates linked at a Euclidean distance between 2 and 4 generally are considered to be the same strain (Leonard et al., 1995; Smith and Siegel, 1996; Sasser, 2001; Hinton et al., 2004). Results To our surprise, when analyzed with the qPCR method, all 16 samples were positive for Legionella spp (Table 2). Also surprising was that qPCR could detect L. longbeachae in four of them, but only one had a concentration above the analytical quantification limit (6000 GU/g). After retesting those four qPCR positive samples with the culture method, Legionella spp was finally recovered in one sample. Table 2. Concentration of Legionella spp, L. pneumophila, and L. longbeachae (GU/g) measured by qPCR in natural soil samples collected from the metal recycling plant.   Legionella spp  L. pneumophila  L. pneumophila serogroup 1  L. longbeachae  Samples  GU/g  GU/g  GU/g  GU/g  176  8200  120  <6000  <6000  178  2600  <6000  <6000  <6000  179  9500  <6000  <6000  <6000  180  73000  <6000  <6000  <6000  181  31000  600  <6000  <6000  182  47000  <6000  <6000  <6000  183  690000  500  65  10000  190  370000  <6000  <6000  <6000  192  57000  <6000  <6000  <6000  193  83000  <6000  <6000  <6000  195  360000  <6000  <6000  <6000  196  58000  700  <6000  <6000  197  58000  <6000  <6000  <6000  198  27000  250  <6000  <6000  199  75000  500  65  <6000  200  36000  700  140  <6000    Legionella spp  L. pneumophila  L. pneumophila serogroup 1  L. longbeachae  Samples  GU/g  GU/g  GU/g  GU/g  176  8200  120  <6000  <6000  178  2600  <6000  <6000  <6000  179  9500  <6000  <6000  <6000  180  73000  <6000  <6000  <6000  181  31000  600  <6000  <6000  182  47000  <6000  <6000  <6000  183  690000  500  65  10000  190  370000  <6000  <6000  <6000  192  57000  <6000  <6000  <6000  193  83000  <6000  <6000  <6000  195  360000  <6000  <6000  <6000  196  58000  700  <6000  <6000  197  58000  <6000  <6000  <6000  198  27000  250  <6000  <6000  199  75000  500  65  <6000  200  36000  700  140  <6000  View Large The cultivable Legionella spp was confirmed in one sample at a concentration of ~2.0 × 105 CFU/g. The extremely high concentrations of non-Legionella colonies on the culture media, even with the combined acid/heat treatment, render isolation of the Legionella spp difficult. Of the total 165 colonies isolated in pure culture on the BCYE media, only one isolate strain could be confirmed to be L. longbeachae with the GC-FAME and the qPCR methods. The relatedness between the patients (strains 26 and 46) and the environmental strain (183-17) is shown in Fig. 1. The Euclidean distance calculated for all strains of L. longbeachae tested (reference, patient and soil) is below 5 units. Figure 1. View largeDownload slide Dendrogram displaying the Euclidean distance obtained from the fatty acid profile produced by GC-FAME analysis of the strains isolated from the occupational environment and the lungs of the two patients. Figure 1. View largeDownload slide Dendrogram displaying the Euclidean distance obtained from the fatty acid profile produced by GC-FAME analysis of the strains isolated from the occupational environment and the lungs of the two patients. Discussion For the great majority of sporadic legionellosis cases, the source of infection remains unknown (Den Boer et al., 2008; van Heijnsbergen et al., 2014; Currie and Beattie, 2015). Usually, it is because no environmental investigation has been attempted. When cases are related, sampling may be considered but not attempted due to known deficiencies in sampling or analytical protocol but another reason could be that the pathogenic strain goes undetected because of in the sampling and or analytical protocols used. Obtaining a culture of L. longbeachae from the soil in the work environment was a major coup in this occupational investigation. O’Connor et al. (2007) point out that the presence of Legionella spp in the media does not necessarily indicate that people handling it will become infected. Obtaining a Legionella strain from a culture made it possible to compare the clinical strains obtained from the two workers’ lungs with the environmental soil strain found in the recycling plant. Although most Legionella species are reported in aquatic environments, their presence has also been described in compost, humus, and soil by many authors (Hughes and Steele, 1994; Koide et al., 2001; Casati et al., 2009, 2010; Travis et al., 2012; van Heijnsbergen et al., 2014). In Australia, Steele et al. (1990) reported that Legionella spp could be isolated from as much as 73% of the potting soil produced. In the Netherlands, van Heijnsbergen et al. (2014) found that 30% (6/30) of the natural soil analyzed was positive for Legionella spp. Travis et al. (2012) reported isolation of Legionella spp from 56% of damp soil samples from Thailand, but they did not recover any that tested positive for L. longbeachae. In fact, with the exception of Koide et al. (2001), who reported that 37% of the samples tested positive for L. longbeachae in compost and potting soils without distinction, other authors reported in soil samples none to very sparse occurrences (4.3%) of L. longbeachae. Successful isolation of a cultivable L. longbeachae strain from the soil was considered to be a challenge by most people involved in the investigation and most did not encourage the environmental investigation and microbial analysis, in part due to the unreported presence of L. longbeachae in Canadian soil, and to the poor nature of the soil at the investigation site. This reluctance to gather environmental samples and to perform analyses may possibly be part of the explanation for the failures reported in identifying the source of sporadic cases of legionellosis. The combined approach applied in this study undoubtedly improves the likelihood of finding the environmental source of the strain. The qPCR analysis was used effectively to target samples containing L. longbeachae DNA. Once the samples possibly containing L. longbeachae were identified, our chances of recovering the strain from the soil of the work environment was somewhat improved. From more than 150 colonies isolated, only one was identified as Legionella spp by the GC-FAME method. The identification of this positive strain, obtained by culture, was confirmed with the qPCR using markers specific to L. longbeachae. This study also demonstrates that a dendrogram performed on the GC-FAME profiles obtained from the clinical and the environmental strains can provide preliminary appreciation of the relatedness between isolates. The Euclidean distances calculated between the four Legionella species included in this study are 10, 14, and 16 units, those distances correspond well to other inter-species distances reported in the literature (Sasser, 2001; Hinton et al., 2004). However, the distance found between the 4 L. longbeachae strains is smaller than that reported by Diogo et al. (1999). In fact, the Euclidean distance they reported among the six strains of L. longbeachae tested was 13 units, compared to only 5 units in this study for all tested strains (reference, patients and soil). Furthermore, the isolated strains of L. longbeachae from this study, and a Euclidean distance of only 2.4 units showed a very high relatedness among those three strains. As reported by Picard-Masson (2016), the strains were subsequently analyzed by pulsed-field gel electrophoresis, which revealed that they had the same genotype. Detecting the presence of L. longbeachae using the qPCR method made it possible to target the right samples to enable the cultivable strain of L. longbeachea to be isolated from the soil of the metal recycling plant. The complementarity of the two methods was established. This paper demonstrates the significance of making the proper analytical choices in order to successfully link microorganisms in the workplace to infectious illness; it also highlights the potential occupational risks associated with L. longbeachae in soil. Declaration Genevieve Marchand, Carole Pépin, and Nancy Lacombe are employed by the Institut de recherche Robert Sauvé en santé et en sécurité du travail (IRSST) who provided funding for this study. The authors designed and executed the study and have sole responsibility for the writing and content of the manuscript. Judith Lord is employed by the Direction de la santé public, CISSS de la Montérégie-Centre who provided funding for this study. The authors designed and executed the study and have sole responsibility for the writing and content of the manuscript. Conflict of Interest The authors declare no conflict of interest relating to the material presented in this article. References Association of Water Technologies. ( 2003) Legionella 2003- an update and statement by the association of water technologies (AWT) . Rockville, MD: Association of Water Technologies. Bartram J (ed.). ( 2007) Legionella and the prevention of legionellosis. World Health Organization . Casati S, Conza L, Bruin Jet al.  . ( 2010) Compost facilities as a reservoir of Legionella pneumophila and other Legionella species. Clin Microbiol Infect ; 16: 945– 7. 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Journal

Annals of Work Exposures and Health (formerly Annals Of Occupational Hygiene)Oxford University Press

Published: Apr 1, 2018

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