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Presence of Legionella Longbeachae in Pine but not Composted Green Waste Used in Manufacture of Potting Soils in New Zealand

Ali Mohammadi, Stephen T. Chambers*, Sandy Slow, Trevor Anderson, Jonathan Williman, Pippa Scott, David Murdoch, Amy Scott-Thomas

Department of Pathology and Medical Science, University of Otago, Christchurch, Christchurch New Zealand

Abstract: In this research we compared the proportion of samples taken at different stages in the manufacturing process of potting soils that were qPCR positive for Legionella longbeachae. This included samples of composted feed stock, mixing sites and bagged products. Feedstock, manufacturing sites, and final retail products at three locations across the North and South Islands of New Zealand were tested for L. longbeachae DNA by qPCR. Positive tests were found on samples of composted pine bark 14/360 (3.9%), pine sawdust 5/185 (2.7%) and pre-bagged or bagged potting soils 9/200 (4.5%). All samples from peat (n = 20), green waste (n = 100), and mixing areas (n = 73) were negative. Samples that contained pine bark or pine sawdust were more likely to be qPCR positive (28/818, 3.4%) than other samples (0/355, 0%; P<0.001). L. longbeachae probably reaches the manufacturing sites from fresh pine bark and persists through the composting and manufacturing process, leading to a wide distribution following the purchase.

Keywords: Legionella longbeachae, compost, pine, Legionnaires’ disease, LD, potting soil

Received April 17, 2021; Revised May 22, 2021; Accepted May 31, 2021

Citation: Ali Mohammadi, Stephen T. Chambers, Sandy Slow, Trevor Anderson, Jonathan Williman, Pippa Scott, David Murdoch, Amy Scott-Thomas, Presence of Legionella Longbeachae in Pine but not Composted Green Waste Used in Manufacture of Potting Soils in New Zealand. Journal of Applied & Environmental Microbiology, 9 (1). 1-4. January 2021. doi:10.12691/jaem-9-1-1

1. Introduction

Legionella longbeachae was first isolated from three patients with pneumonia in California and one from Georgia in 1980 [1], and subsequently it has been found in patients in Australia, New Zealand, Japan, Europe, United States and Asia. [2,3,4,5,6,7,8] A recent nationwide study of Legionnaires’ disease (LD) demonstrated that L. longbeachae caused 150 (63%) of 238 cases of LD in New Zealand and was widely distributed across the country. [9] There was a marked seasonal variation in LD incidence with most cases occurring in spring and early summer (October to January) confirming previous observations. [10] Case control studies in New Zealand and Australia have linked the recent use, tipping, or trowelling of commercial potting soil, and poor hygiene practices with L. longbeachae infection [11,12,13], as well as exposure to aerosolised commercial potting soil with Pontiac fever. [14] Infection with L. longbeachae, has also been associated with contact with compost and potting soil in Asia, Europe and North America. [7,15,16,17,18,19,20,21]

Steele et al. isolated living L. longbeachae in Australian made potting soils that contained composted pine, eucalyptus and pine sawdust. [22,23] Subsequent environmental studies in Australia, Europe, Japan and North America, have demonstrated that L. longbeachae has been identified in compost, soil and contaminated water bodies. [24,25,26,27] In Europe L. longbeachae has never been identified in peat, and studies on green waste reported variable results where L. longbeachae was identified in composted but not fresh green waste. [27,28,29] It is possible that green waste becomes contaminated from external sources such as rain and wind-blown material during the composting process. [28]

We have recently demonstrated that L. longbeachae DNA is present on the bark of most living pine species including Pinus radiata, but was seldom found on bark from non-pine species. [30] This suggests that living pine species provide a reservoir for these organisms. Bark and sawdust from P. radiata are a major component of feed stocks used in the manufacture of potting soils in New Zealand. Because of this we thought that L. longbeachae would be present in pine bark, at the potting soil manufacturing site and persist in pine containing manufactured products. Our aim was to determine if L. longbeachae could be found in the feedstock at these manufacturing plants. If so, P. radiata products may be the major source of L. longbeachae in commercial potting soils and could provide a control point for reducing L. longbeachae content in such products, thereby reducing the incidence of human disease.

2. Materials and Methods

A point prevalence study conducted at three commercial potting soil and compost production sites widely dispersed across the North and South Islands of New Zealand. Sampling of feed stock was unrestricted but was conducted within the manufacturing site’s health and safety protocols.

2.1. Potting Mix Manufacturing Sites

Production site 1 was located in the lower South Island. Samples were taken from large windrows of graded pine bark or piles of pine sawdust outside the plant, the indoor mixing area, and locally produced peat that had been stored inside the plant. Samples were taken at approximately 3 m intervals across the surface and edges of the feedstock at depths between 5 and 50 cm, from a grid at 1 m intervals over the floor of the mixing area, and from bagged finished product stored at the site. There was no composted green waste on site. Samples (N=348) were taken on 12 April 2017.

Production site 2 was in the upper South Island providing access to large piles of pine bark, partially composted sawdust, some imported peat, the mixing area and finished product but no green waste. All feedstock, mixed composted bagged material and not yet bagged product was located outside. The mixing area was indoors. Samples (N=255) were taken on 17 April 2017.

Production site 3 was in the upper North Island of New Zealand. Most of the feedstock was located offsite and could not be sampled at that location. Feedstock that could be sampled was brought inside to a temporary storage area and included locally produced bark products and composted green-waste. Peat was imported from Europe. Mixing and bagging of the products was done inside, and the bagged product was stored outside with bags on large plastic covered pallets awaiting shipping. Samples (N=570) were taken on 31 May 2017.

2.2. Quantitative PCR (qPCR)

The samples were pulverised and 5 g were mixed with 50 mL of ultrapure distilled water (Invitrogen, Thermo Fisher Scientific, MA, USA) and shaken for 5 minutes. Two hundred microliters of the supernatant were used for DNA extraction using the GenElute Bacterial DNA extraction kit (Sigma, MO, USA) as per the manufacturer’s instructions. The presence of L. longbeachae DNA was detected using the qPCR parameters and the primer and probes designed and validated as specific for L. longbeachae as previously described [10,30]. Positive and negative controls were included in each run and negative qPCR results were validated by using internal controls.

The crossing threshold (CT) cut off of ≤44 cycles for the lower limit of detection and quantification was determined by spiking samples of sterilised potting mix with dilutions of a pure culture of L. longbeachae sg1 ATCC33462 from x10¹-x10⁷ cfu/mL (R² = 0.9964 y = -1.7ln(x) + 48.811). The lower limit of quantification was 10 CFU/mL.

2.3. Statistical Analysis

The prevalence rates of samples meeting qPCR CT values for L. longbeachae were summarised by site and type as counts and percentages with 95% binomial (Wilson) confidence intervals. Differences in rates were compared across subgroups using Fisher’s exact tests. All statistical testing was conducted using R software (version 4.0.3) [31] and P values < 0.05 were considered significant.

3. Results

Fourteen (4%) pine bark samples were qPCR positive, with all but one coming from production site 1 (Table 1). There were significant differences in the percentages of qPCR positive samples between the manufacturing sites (18/348 from site 1, 3/255 from site 2 and 7/570 from site 3; Fisher’s exact p<0.001).

None of the samples from the mixing area, which is the beginning of the blending and bagging cycle, were qPCR positive but 9 of 200 (4.5%) samples taken from pre-bagged or bagged compost or potting mix products were qPCR positive. These were predominantly from samples taken at production site 3.

Table 1. qPCR results of samples collected from three widely separated potting mix production sites across both the North and South Islands of New Zealand in Autumn 2017

Overall of the 1153 samples 28 (2.4 %) were qPCR positive (Table 1). There was a significant difference in the proportion of qPCR positive samples from bark and bark containing products (pine bark, pine sawdust, mixing area and bagged product, 28/818, 3.4%) compared with non-bark containing samples (peat, green waste and pumice 0/355, 0%; Fisher exact p<0.001).

4. Discussion

In this study we have found that L. longbeachae DNA could be detected in pine bark and pine sawdust feedstock, or pine bark containing potting soil products from three widely separated production sites across New Zealand. In contrast, L. longbeachae DNA was not detected from peat and green waste feedstock. As pine bark is a major constituent of potting soils, it is likely to be the major source of L. longbeachae in these products. In addition, pine products may facilitate the persistence of L. longbeachae in the manufactured potting soil products.

The presence L. longbeachae DNA in pine bark and pine sawdust is consistent with the finding that this organism was present on bark from living P. radiata and with studies from Steele et al. who found living Legionella spp. in fresh pine sawdust and composted bark. [22,30] The lack of evidence for L. longbeachae in composted green waste is compatible with other studies that have reported variable results. In Switzerland, Casati et al found multiple pathogenic Legionella species, (e.g. Legionella pneumophila sg 1-15, Legionella bozemanii, Legionella cincinnatiensis, Legionella jamestowniensis, Legionella micdadei and Legionella oakridgensis) but not L. longbeachae in samples from stored composted green waste. In contrast they found only a single legionella isolate (L. cincinnatiensis) in samples taken from fresh green waste at three separate sites. [28] They suggested that green waste becomes contaminated from other environmental sources such as wind and rain. In the Netherlands, Huss et al., found that many legionella species were present in 97 (68%) of both fresh and composted green waste but there was only one isolate of L. longbeachae identified (0.7%). [29] In Scotland Currie et al., did not find any Legionella species in the single green waste product they had sampled, but found multiple isolates of many species including L. longbeachae in peat containing composts. [27] As Legionella species have never been isolated from peat the organisms are likely to have come from another source.

L. longbeachae was found on a relatively low number of samples of pine bark and pine containing products and there were differences in the proportion of positive samples between the sites. This may have been related to multiple factors. Firstly, the study was done in winter when there is both a low rate of clinical LD caused by L. longbeachae and where we have previously shown that only a low proportion of bark samples from living trees had detectable levels of DNA present. [9,10,30] L. longbeachae favours growth over a temperature range of 4°C – 25°C rather than higher temperatures, but will persist at temperatures down to -20 °C in potting mix for months. [22,32] Secondly, the time of sampling may be important as the load of L. longbeachae may vary with local variations in temperate and humidity. This has previously been reported for L. pneumophila and it is likely, these factors will also influence L. longbeachae in similar manner. [33,34] Thirdly, variation in accessibility of samples may also have contributed to differences found between production sites. The feedstock at production sites 1 and 2 were outside, so that samples could only be taken close to the surface of the windrows. In contrast, at production site 3 samples could be taken from the indoor low capacity depots that were refilled by a high capacity digger that mixed variable portions from across the windrow types. Similarly, L. longbeachae may have been inhibited in inner portions where composting produces high temperatures, while outer portions are cooler with temperatures that are supportive of L. longbeachae replication, either in the environment or within protozoa or nematodes. [33,34,35]

Other limitations to this study include the use of qPCR as the primary endpoint. Our findings do not exclude the presence of L. longbeachae in numbers below the level of detection. The samples have not been cultured and positive cultures would have added more certainty to our findings but it is less sensitive than qPCR. [36] Secondly, the green waste tested was produce by a well-controlled commercial process that may have eliminated L. longbeachae so that these results may not apply to green waste compost produced by other processes.

5. Conclusion

Pine bark provides an important niche for L. longbeachae and the use of pine bark in the manufacture of potting soils is likely to be a major source, but not necessarily the only source of this organism. Failure to find L. longbeachae in peat and composted green waste suggests that control measures on P. radiata feedstock at the site of manufacture may offer a way to reduce the rates of human disease. In addition, P. radiata is planted in extremely large commercial plantations in several countries and there are also large industries based on other pine species around the world that could pose a hazard to human health. The use of pine based product as a substitute for peat, as this resource becomes scarce, needs careful consideration.

Acknowledgements

We sincerely thank all of the manufacturing site staff for access to sites and samples. We have no actual or potential competing interests regarding this study. This work was funded by an unrestricted grant from the Canterbury Medical Research Foundation, a University of Otago Scholarship grant and an unrestricted grant from Tui Products Ltd, Tauranga, New Zealand.

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