Abstract
Legionella longbeachae is the predominant causative agent of Legionnaires’ disease (LD) in New Zealand with a peak infection period during the spring and summer months. This is a plant-associated microbe that can be found throughout the environment and is associated with potting mix and compost. LD is difficult to diagnose because of difficulty with obtaining respiratory samples.
Acid pre-treatment followed by culture is commonly used for the isolation of Legionella species in both clinical and environmental samples but has limited sensitivity because of high contamination loads. Detecting volatile organic compounds (VOCs) in breath is a minimally invasive technique that could be used to investigate patients with possible LD.
The aims of the current study were to:
a) develop isolation methods to improve identification of L. longbeachae in clinical and environmental settings,
b) develop an immunomagnetic separation (IMS) method to improve culture and PCR,
c) identify possible VOCs using gas chromatography-mass spectrometry (GC-MS) that can be used as diagnostic biomarkers for LD.
d) investigate potting mix productions for the presence of L. longbeachae,
e) study the potential influence of physico-chemical characteristics of potting mix products that permit L. longbeachae to survive,
To develop the IMS method, a polyclonal antibody was raised in rabbits by immunising them with heat-killed L. longbeachae antigens with several booster injections. The antibody produced was separated from serum and purified by ion-exchange chromatography. This polyclonal antibody had high specificity and sensitivity in capturing L. longbeachae, with minimum cross-reactivity with other species. This antibody was coupled to immunomagnetic beads and used to separate organisms in stored respiratory samples for both culture and PCR. Feedstock samples used in the manufacture of the potting mix were taken from three sites around New Zealand for PCR and culture.
Culture results of sputum samples were significantly improved by using IMS. qPCR was used for this preliminary screening of environmental samples. The results showed that bark and bark-containing products had the highest number of PCR positive samples for L. longbeachae.
Culture results for these PCR positive samples were negative. Several isolation methods were developed to improve the culturability of L. longbeachae from environmental samples. The results showed that the combination of the Legionella selective GVPC antibiotic suspension with IMS reduces the contamination of cultures with other organisms and also improves the recovery of L. longbeachae from the samples.
Physico-chemical methods were used to determine the elemental make up of a subgroup of feedstock samples. The results showed that there were higher concentrations of boron and sulfur in PCR negative bark samples than PCR positive samples.
Finally, the headspace of the culture of L. longbeachae was pre-concentrated and analysed by GC-MS to generate a volatile profile of these bacteria. A peak of interest was found in this profile. Further work needs to be undertaken to identify the precise chemical structure of this compound.
In conclusion, this work has demonstrated that the rate of positive cultures of stored sputum samples is higher with IMS separation. This result needs validating in prospective studies in a diagnostic laboratory to determine if it can become a useful diagnostic tool. The sensitivity of culture of heavily contaminated environmental samples can be improved using IMS and GVPC decontamination and these techniques can be validated in future studies. Addition of elements such as sulphur and boron to potting mix should be evaluated as a method of manufacturing a safer potting mix product. Further studies are needed to precisely identify the chemical structure of a VOC identified and clinical trials conducted to determine whether this is of diagnostic value.