Michael Taylor

Legionella, Protozoa, and Biofilms: Interactions Within Complex Microbial Systems

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.

Legionella detection by culture and qPCR: Comparing apples and oranges

Abstract Legionella spp. are the causative agent of Legionnaire’s disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples

Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the techniques inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes.

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores. Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.

Human strongyloidiasis: identifying knowledge gaps, with emphasis on environmental control

Abstract Strongyloides is a human parasitic nematode that is poorly understood outside a clinical context. This article identifies gaps within the literature, with particular emphasis on gaps that are hindering environmental control of Strongyloides. The prevalence and distribution of Strongyloides is unclear. An estimate of 100–370 million people infected worldwide has been proposed; however, inaccuracy of diagnosis, unreliability of prevalence mapping, and the fact that strongyloidiasis remains a neglected disease suggest that the higher figure of more than 300 million cases is likely to be a more accurate estimate. The complexity of Strongyloides life cycle means that laboratory cultures cannot be maintained outside of a host. This currently limits the range of laboratory-based research, which is vital to controlling Strongyloides through environmental alteration or treatment. Successful clinical treatment with antihelminthic drugs has meant that controlling Strongyloides through environmental control, rather than clinical intervention, has been largely overlooked. These control measures may encompass alteration of the soil environment through physical means, such as desiccation or removal of nutrients, or through chemical or biological agents. Repeated antihelminthic treatment of individuals with recurrent strongyloidiasis has not been observed to result in the selection of resistant strains; however, this has not been explicitly demonstrated, and relying on such assumptions in the long-term may prove to be shortsighted. It is ultimately naive to assume that continued administration of antihelminthics will be without any negative long-term effects. In Australia, strongyloidiasis primarily affects Indigenous communities, including communities from arid central Australia. This suggests that the range of Strongyloides extends beyond the reported tropical/subtropical boundary. Localized conditions that might result in this extended boundary include accumulation of moisture within housing because of malfunctioning health hardware inside and outside the house and the presence of dog fecal matter inside or outside housing areas.

An Evaluation of Antifungal Agents for the Treatment of Fungal Contamination in Indoor Air Environments

Fungal contamination in indoor environments has been associated with adverse health effects for the inhabitants. Remediation of fungal contamination requires removal of the fungi present and modifying the indoor environment to become less favourable to growth. This may include treatment of indoor environments with an antifungal agent to prevent future growth. However there are limited published data or advice on chemical agents suitable for indoor fungal remediation. The aim of this study was to assess the relative efficacies of five commercially available cleaning agents with published or anecdotal use for indoor fungal remediation. The five agents included two common multi-purpose industrial disinfectants (Cavicide® and Virkon®), 70% ethanol, vinegar (4.0%−4.2% acetic acid), and a plant-derived compound (tea tree (Melaleuca alternifolia) oil) tested in both a liquid and vapour form. Tea tree oil has recently generated interest for its antimicrobial efficacy in clinical settings, but has not been widely employed for fungal remediation. Each antifungal agent was assessed for fungal growth inhibition using a disc diffusion method against a representative species from two common fungal genera, (Aspergillus fumigatus and Penicillium chrysogenum), which were isolated from air samples and are commonly found in indoor air. Tea tree oil demonstrated the greatest inhibitory effect on the growth of both fungi, applied in either a liquid or vapour form. Cavicide® and Virkon® demonstrated similar, although less, growth inhibition of both genera. Vinegar (4.0%–4.2% acetic acid) was found to only inhibit the growth of P. chrysogenum, while 70% ethanol was found to have no inhibitory effect on the growth of either fungi. There was a notable inhibition in sporulation, distinct from growth inhibition after exposure to tea tree oil, Virkon®, Cavicide® and vinegar. Results demonstrate that common cleaning and antifungal agents differ in their capacity to inhibit the growth of fungal genera found in the indoor air environment. The results indicate that tea tree oil was the most effective antifungal agent tested, and may have industrial application for the remediation of fungal contamination in residential and occupational buildings.

Rickettsia Detected in the Reptile Tick Bothriocroton hydrosauri from the Lizard Tiliqua rugosa in South Australia

Rickettsiosis is a potentially fatal tick borne disease. It is caused by the obligate intracellular bacteria Rickettsia, which is transferred to humans through salivary excretions of ticks during the biting process. Globally, the incidence of tick-borne diseases is increasing; as such, there is a need for a greater understanding of tick–host interactions to create more informed risk management strategies. Flinders Island spotted fever rickettsioses has been identified throughout Australia (Tasmania, South Australia, Queensland and Torres Strait Islands) with possible identifications in Thailand, Sri Lanka and Italy. Flinders Island spotted fever is thought to be spread through tick bites and the reptile tick Bothriocroton hydrosauri has been implicated as a vector in this transmission. This study used qPCR to assay Bothriocroton hydrosauri ticks collected from Tiliqua rugosa (sleepy lizard) hosts on mainland South Australia near where spotted fever cases have been identified. We report that, although we discovered Rickettsia in all tick samples, it was not Rickettsia honei. This study is the first to use PCR to positively identify Rickettsia from South Australian Bothriocroton hydrosauri ticks collected from Tiliqua rugosa (sleepy lizard) hosts. These findings suggest that B. hydrosauri may be a vector of multiple Rickettsia spp. Also as all 41 tested B. hydrosauri ticks were positive for Rickettsia this indicates an extremely high prevalence within the studied area in South Australia.

Airborne fungal profiles in office buildings in metropolitan Adelaide, South Australia: Background levels, diversity and seasonal variation

The presence of bioaerosols in indoor non-industrial workplace environments has become an increasing concern to indoor air quality assessors and Occupational Health and Safety professionals. The paucity of workplace survey information and national standards limits the comparisons that can be made when investigating suspected indoor fungal contamination. Data are needed on typical non-problem conditions, thereby providing background survey information. This study examined viable fungi in 128 air samples (89 indoor: 39 outdoor) from office buildings in Adelaide, South Australia, which has an arid Mediterranean climate. Results across four consecutive seasons show that the viable airborne fungal concentrations in indoor air were on average 75% lower than those in outdoor air. A seasonal influence was noted with higher fungal levels in autumn and summer compared with winter and spring. The most common culturable airborne fungi, across all seasons and conditions, were Penicillium, Aspergillus, Cladosporium and Alternaria. A weak correlation between fungal spore concentration in indoor air and carbon dioxide was observed (r = 0.26). No other correlations with indoor air quality parameters were noted. This study provides a profile of airborne fungal diversity and abundance in non-problem indoor environments and practical guidance to indoor air quality assessors on the interpretation of indoor fungal monitoring data.

Spatial arrangement of legionella colonies in intact biofilms from a model cooling water system.

There is disagreement among microbiologists about whether Legionella requires a protozoan host in order to replicate. This research sought to determine where in biofilm Legionellae are found and whether all biofilm associated Legionella would be located within protozoan hosts. While it is accepted that Legionella colonizes biofilm, its life cycle and nutritional fastidiousness suggest that Legionella employs multiple survival strategies to persist within microbial systems. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) demonstrated an undulating biofilm surface architecture and a roughly homogenous distribution of heterotrophic bacteria with clusters of protozoa. Legionella displayed 3 distinct spatial arrangements either contained within or directly associated with protozoa, or dispersed in loosely associated clusters or in tightly packed aggregations of cells forming dense colonial clusters. The formation of discreet clusters of tightly packed Legionella suggests that colony formation is influenced by specific environmental conditions allowing for limited extracellular replication. This work represents the first time that an environmentally representative, multispecies biofilm containing Legionella has been fluorescently tagged and Legionella colony morphology noted within a complex microbial system.

Independent validation and regulatory agency approval for high rate algal ponds to treat wastewater from rural communities

Despite the many recognised benefits, the application of high rate algal ponds (HRAP) to manage wastewater treatment in small communities has been limited. To be incorporated into the South Australian Community Wastewater Management Scheme (CWMS), new wastewater treatment systems are required to undergo validation and obtain regulatory approval from the South Australian Department of Health, Wastewater Management Group. A HRAP system at Kingston on Murray, South Australia, underwent validation to be incorporated into the CWMS. The process was consistent with the Australian National Guidelines which requires the demonstration of the log10 reduction values (LRV) for indicator organisms achieved by the wastewater treatment system. These were required to be measured twice weekly, over a 10 week period in below average solar radiation and temperature conditions, by an independent National Association of Testing Authorities accredited laboratory. The Australian Water Quality Centre was commissioned to assess the removal of Escherichia coli, F-RNA bacteriophage and aerobic spore-forming bacteria. Flinders University of South Australia concurrently monitored the removal of the same organisms and other standard wastewater parameters. While ASFB were shown to be unsuitable indicators of protozoa in natural pond systems, the system effectively removed E. coli and F-RNA bacteriophage with the treated effluent meeting the limits set by the guidelines for effluent reuse for non-food crop irrigation: a 5th percentile LRV of >1.0 for F-RNA bacteriophage and a median E. coli concentration of <4.0 log10E. coli MPN 100 mL−1. Based on these results two configurations of HRAP systems were approved to be incorporated into the CWMS.