Microbial risk from rainwater tanks in South East Queensland

Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to pathogens from potable and non-potable uses of roof-harvested rainwater in South East Queensland (SEQ). A total of 84 rainwater samples were analysed for the presence of faecal indicators (using culture based methods) and zoonotic bacterial and protozoan pathogens using binary and quantitative PCR (qPCR). The concentrations of Salmonella invA, and Giardia lamblia β-giradin genes ranged from 65-380 genomic units/1000 mL and 9-57 genomic units/1000 mL of water, respectively. After converting gene copies to cell/cyst number, the risk of infection from G. lamblia and Salmonella spp. associated with the use of rainwater for bi-weekly garden hosing was calculated to be below the threshold value of 1 extra infection per 10,000 persons per year. However, the estimated risk of infection from drinking the rainwater daily was 44-250 (for G. lamblia) and 85-520 (for Salmonella spp.) infections per 10,000 persons per year. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically discussed. Nevertheless, it would seem prudent to disinfect rainwater for potable use.

Signs, symptoms and comorbidities in contact lens-related microbial keratitis

Purpose: The aim of this study was to characterize the clinical signs, symptoms, and ocular and systemic comorbidities in a large case series of contact lens-related microbial keratitis. Methods: Two hundred ninety-seven cases of contact lens-related microbial keratitis, aged between 15 and 64 years were detected through surveillance of hospital and community based ophthalmic practitioners in Australia and New Zealand. Full clinical data were available for 190 cases and 90 were interviewed by telephone. Clinical data included the size, location, and degree of anterior chamber response. Symptom data were available from the practitioner and from participant self-report. Associations between symptoms and disease severity were evaluated. Data on ocular and systemic disease were collected from participants and practitioners. The frequency of comorbidities was compared between the different severities of disease and to population norms. Results: More severe disease was associated with greater symptom severity and pain was the most prevalent symptom reported. Ninety-one percent of cases showed progression of ocular symptoms after lens removal, and symptom progression was associated with all severities of disease. Twenty-five percent of cases reported prior episodes requiring emergency attention. Thyroid disease (p 0.05) and self-reported poor health (p 0.001) were more common in cases compared with age-matched population norms. Discussion: Information on the signs, symptoms, and comorbidities associated with contact lens-related microbial keratitis may be useful in patient education and for practitioners involved in the fitting of lenses and management of complications. Although pain was the most common symptom experienced, progression of symptoms despite lens removal was close to universal. Poor general health, particularly respiratory disease and thyroid disease was more common in cases than in the general population, which may prompt practitioners to recommend flexibility in wear schedules when in poor health or the selection of a lower risk wear schedule in at risk patients

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Bioavailability of soil organic carbon and Fe as influenced by forestry practices in a subtropical coastal catchment

Potential impacts of plantation forestry practices on soil organic carbon and Fe available to microorganisms were investigated in a subtropical coastal catchment. The impacts of harvesting or replanting were largely limited to the soil top layer (0–10 cm depth). The thirty-year-old Pinus plantation showed low soil moisture content (Wc) and relatively high levels of soil total organic carbon (TOC). Harvesting and replanting increased soil Wc but reduced TOC levels. Mean dissolved organic carbon (DOC) and microbial biomass carbon (MBC) increased in harvested or replanted soils, but such changes were not statistically significant (P > 0.05). Total dithionite-citrate and aqua regia-extractable Fe did not respond to forestry practices, but acid ammonium oxalate and pyrophosphate-extractable, bioavailable Fe decreased markedly after harvesting or replanting. Numbers of heterotrophic bacteria were significantly correlated with DOC levels (P < 0.05), whereas Fe-reducing bacteria and S-bacteria detected using laboratory cultivation techniques did not show strong correlation with either soil DOC or Fe content.

Health risk from the use of roof-harvested rainwater in Southeast Queensland, Australia, as potable or nonpotable water, determined using quantitative microbial risk assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analysed for the zoonotic bacterial and protozoan pathogen using real-time binary PCR and quantitative PCR (qPCR). Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from potable and non-potable uses of roof-harvested rainwater. Of the 214 samples tested, 10.7%, 9.8%, and 5.6%, and 0.4% samples were positive for Salmonella invA, Giardia lamblia β-giardin , Legionella pneumophila mip, and Campylobacter jejuni mapA genes. Cryptosporidium parvum could not be detected. The estimated numbers of viable Salmonella spp., G. lamblia β-giradin, and L. pneumophila genes ranged from 1.6 × 101 to 9.5 × 101 cells, 1.4 × 10-1 to 9.0 × 10-1 cysts, and 1.5 × 101 to 4.3 × 101 per 1000 ml of water, respectively. Six risk scenarios were considered from exposure to Salmonella spp., G. lamblia and L. pneumophila. For Salmonella spp., and G. lamblia, these scenarios were: (1) liquid ingestion due to drinking of rainwater on a daily basis (2) accidental liquid ingestion due to garden hosing twice a week (3) aerosol ingestion due to showering on a daily basis, and (4) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were: (5) aerosol inhalation due to showering on a daily basis, and (6) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeds this threshold value, and indicates that if undisinfected rainwater were ingested by drinking, then the gastrointestinal diseases of Salmonellosis and Giardiasis is expected to range from 5.0 × 100 to 2.8 × 101 (Salmonellosis) and 1.0 × 101 to 6.4 × 101 (Giardiasis) cases per 10,000 persons per year, respectively. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for potable use.

Networked aquatic microbial observing systems: An overview

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Ability of silver-impregnated contact lenses to control microbial growth and colonisation

Purpose: To examine the ability of silver nano-particles to prevent the growth of Pseudomonas aeruginosa and Staphylococcus aureus in solution or when adsorbed into contact lenses. To examine the ability of silver nano-particles to prevent the growth of Acanthamoeba castellanii. ----- ----- Methods: Etafilcon A lenses were soaked in various concentrations of silver nano-particles. Bacterial cells were then exposed to these lenses, and numbers of viable cells on lens surface or in solution compared to etafilcon A lenses not soaked in silver. Acanthamoeba trophozoites were exposed to silver nano-particles and their ability to form tracks was examined. ----- ----- Results: Silver nano-particle containing lenses reduced bacterial viability and adhesion. There was a dose-dependent response curve, with 10 ppm or 20 ppm silver showing > 5 log reduction in bacterial viability in solution or on the lens surface. For Acanthamoeba, 20 ppm silver reduced the ability to form tracks by approximately 1 log unit. ----- ----- Conclusions: Silver nanoparticles are effective antimicrobial agents, and can reduce the ability of viable bacterial cells to colonise contact lenses once incorporated into the lens.----- ----- Resumen: Objetivos: Examinar la capacidad de las nanopartículas de plata para prevenir el crecimiento de Pseudomonas aeruginosa y Staphylococcus aureus en soluciones para lentes de contacto o cuando éstas las adsorben. Examinar la capacidad de las nanopartículas de plata para prevenir el crecimiento de Acanthamoeba castellanii.----- ----- Métodos: Se sumergieron lentes etafilcon A en diversas concentraciones de nanopartículas de plata. Las células bacterianas fueron posteriormente expuestas a dichas lentes, y se compararon cantidades de células viables en la superficie de la lente o en la solución con las presentes en lentes etafilcon A que no habían sido sumergidas en plata. Trofozoítos de Acanthamoeba fueron expuestos a nanopartículas de plata y se examinó su capacidad para formar quistes.----- ----- Resultados: Las lentes que contienen nanopartículas de plata redujeron la viabilidad bacteriana y la adhesión. Hubo una curva de respuesta dependiente de la dosis, en la que 10 ppm o 20 ppm de plata mostró una reducción logarítmica > 5 en la viabilidad bacteriana tanto en la solución como en la superficie de la lente. Para Acanthamoeba, 20 ppm de plata redujeron la capacidad de formar quistes en aproximadamente 1 unidad logarítmica.----- ----- Conclusiones: Las nanopartículas de plata son agentes antimicrobianos eficaces y pueden reducir la capacidad de células bacterianas viables para colonizar las lentes de contacto una vez que se han incorporado en la lente.

Incorporating parameter uncertainty into quantitative microbial risk assessment (QMRA)

Modern statistical models and computational methods can now incorporate uncertainty of the parameters used in Quantitative Microbial Risk Assessments (QMRA). Many QMRAs use Monte Carlo methods, but work from fixed estimates for means, variances and other parameters. We illustrate the ease of estimating all parameters contemporaneously with the risk assessment, incorporating all the parameter uncertainty arising from the experiments from which these parameters are estimated. A Bayesian approach is adopted, using Markov Chain Monte Carlo Gibbs sampling (MCMC) via the freely available software, WinBUGS. The method and its ease of implementation are illustrated by a case study that involves incorporating three disparate datasets into an MCMC framework. The probabilities of infection when the uncertainty associated with parameter estimation is incorporated into a QMRA are shown to be considerably more variable over various dose ranges than the analogous probabilities obtained when constants from the literature are simply ‘plugged’ in as is done in most QMRAs. Neglecting these sources of uncertainty may lead to erroneous decisions for public health and risk management.

Tubal damage, infertility and tubal ectopic pregnancy : chlamydia trachomatis and other microbial aetiologies

Infertility is a worldwide health problem with one in six couples suffering from this condition and with a major economic burden on the global healthcare industry. Estimates of the current global infertility rate suggest that 15% of couples are infertile (Zegers-Hochschild et al 2009) defined as: (1) failure to conceive after 12 months of unprotected sexual intercourse (i.e. infertility); (2) repeated implantation failure following ART cycles; or (3) recurrent miscarriage without difficulty conceiving (natural conceptions). Tubal factor infertility is among the leading causes of female factor infertility accounting for 7-9.8% of all female factor infertilities. Tubal disease directly causes from 36% to 85% of all cases of female factor infertility in developed and developing nations respectively and is associated with polymicrobial aetiologies. One of the leading global causes of tubal factor infertility is thought to be symptomatic (and asymptomatic in up to 70% cases) infection of the female reproductive tract with the sexually transmitted pathogen, Chlamydia trachomatis. Infection-related damage to the Fallopian tubes caused by Chlamydia accounts for more than 70% of cases of infertility in women from developing nations such as sub-Saharan Africa (Sharma et al 2009). Bacterial vaginosis, a condition associated with increased transmission of sexually transmitted infections including those caused by Neisseria gonorrhoeae and Mycoplasma genitalium is present in two thirds of women with pelvic inflammatory disease (PID). This review will focus on (1) the polymicrobial aetiologies of tubal factor infertility and (2) studies involved in screening for, and treatment and control of, Chlamydial infection to prevent PID and the associated sequelae of Fallopian tube inflammation that may lead to infertility and ectopic pregnancy.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

Iron biogeochemistry and associated greenhouse gas evolution in a forested subtropical Australian coastal catchment : Poona Creek, Southeast Queensland

Iron (Fe) is the fourth most abundant element in the Earth’s crust. Excess Fe mobilization from terrestrial into aquatic systems is of concern for deterioration of water quality via biofouling and nuisance algal blooms in coastal and marine systems. Substantial Fe dissolution and transport involve alternate Fe(II) oxidation followed by Fe(III) reduction, with a diversity of Bacteria and Archaea acting as the key catalyst. Microbially-mediated Fe cycling is of global significance with regard to cycles of carbon (C), sulfur (S) and manganese (Mn). However, knowledge regarding microbial Fe cycling in circumneutral-pH habitats that prevail on Earth has been lacking until recently. In particular, little is known regarding microbial function in Fe cycling and associated Fe mobilization and greenhouse (CO2 and CH4, GHG) evolution in subtropical Australian coastal systems where microbial response to ambient variations such as seasonal flooding and land use changes is of concern. Using the plantation-forested Poona Creek catchment on the Fraser Coast of Southeast Queensland (SEQ), this research aimed to 1) study Fe cycling-associated bacterial populations in diverse terrestrial and aquatic habitats of a representative subtropical coastal circumneutral-pH (4–7) ecosystem; and 2) assess potential impacts of Pinus plantation forestry practices on microbially-mediated Fe mobilization, organic C mineralization and associated GHG evolution in coastal SEQ. A combination of wet-chemical extraction, undisturbed core microcosm, laboratory bacterial cultivation, microscopy and 16S rRNA-based molecular phylogenetic techniques were employed. The study area consisted primarily of loamy sands, with low organic C and dissolved nutrients. Total reactive Fe was abundant and evenly distributed within soil 0–30 cm profiles. Organic complexation primarily controlled Fe bioavailability and forms in well-drained plantation soils and water-logged, native riparian soils, whereas tidal flushing exerted a strong “seawater effect” in estuarine locations and formed a large proportion of inorganic Fe(III) complexes. There was a lack of Fe(II) sources across the catchment terrestrial system. Mature, first-rotation plantation clear-felling and second-rotation replanting significantly decreased organic matter and poorly crystalline Fe in well-drained soils, although variations in labile soil organic C fractions (dissolved organic C, DOC; and microbial biomass C, MBC) were minor. Both well-drained plantation soils and water-logged, native-vegetation soils were inhabited by a variety of cultivable, chemotrophic bacterial populations capable of C, Fe, S and Mn metabolism via lithotrophic or heterotrophic, (micro)aerobic or anaerobic pathways. Neutrophilic Fe(III)-reducing bacteria (FeRB) were most abundant, followed by aerobic, heterotrophic bacteria (heterotrophic plate count, HPC). Despite an abundance of FeRB, cultivable Fe(II)-oxidizing bacteria (FeOB) were absent in associated soils. A lack of links between cultivable Fe, S or Mn bacterial densities and relevant chemical measurements (except for HPC correlated with DOC) was likely due to complex biogeochemical interactions. Neither did variations in cultivable bacterial densities correlate with plantation forestry practices, despite total cultivable bacterial densities being significantly lower in estuarine soils when compared with well-drained plantation soils and water-logged, riparian native-vegetation soils. Given that bacterial Fe(III) reduction is the primary mechanism of Fe oxide dissolution in soils upon saturation, associated Fe mobilization involved several abiotic and biological processes. Abiotic oxidation of dissolved Fe(II) by Mn appeared to control Fe transport and inhibit Fe dissolution from mature, first-rotation plantation soils post-saturation. Such an effect was not observed in clear-felled and replanted soils associated with low SOM and potentially low Mn reactivity. Associated GHG evolution post-saturation mainly involved variable CO2 emissions, with low, but consistently increasing CH4 effluxes in mature, first-rotation plantation soil only. In comparison, water-logged soils in the riparian native-vegetation buffer zone functioned as an important GHG source, with high potentials for Fe mobilization and GHG, particularly CH4 emissions in riparian loam soils associated with high clay and crystalline Fe fractions. Active Fe–C cycling was unlikely to occur in lower-catchment estuarine soils associated with low cultivable bacterial densities and GHG effluxes. As a key component of bacterial Fe cycling, neutrophilic FeOB widely occurred in diverse aquatic, but not terrestrial, habitats of the catchment study area. Stalked and sheathed FeOB resembling Gallionella and Leptothrix were limited to microbial mat material deposited in surface fresh waters associated with a circumneutral-pH seep, and clay-rich soil within riparian buffer zones. Unicellular, Sideroxydans-related FeOB (96% sequence identity) were ubiquitous in surface and subsurface freshwater environments, with highest abundance in estuary-adjacent shallow coastal groundwater water associated with redox transition. The abundance of dissolved C and Fe in the groundwater-dependent system was associated with high numbers of cultivable anaerobic, heterotrophic FeRB, microaerophilic, putatively lithotrophic FeOB and aerobic, heterotrophic bacteria. This research represents the first study of microbial Fe cycling in diverse circumneutral-pH environments (terrestrial–aquatic, freshwater–estuarine, surface–subsurface) of a subtropical coastal ecosystem. It also represents the first study of its kind in the southern hemisphere. This work highlights the significance of bacterial Fe(III) reduction in terrestrial, and bacterial Fe(II) oxidation in aquatic catchment Fe cycling. Results indicate the risk of promotion of Fe mobilization due to plantation clear-felling and replanting, and GHG emissions associated with seasonal water-logging. Additional significant outcomes were also achieved. The first direct evidence for multiple biomineralization patterns of neutrophilic, microaerophilic, unicellular FeOB was presented. A putatively pure culture, which represents the first cultivable neutrophilic FeOB from the southern hemisphere, was obtained as representative FeOB ubiquitous in diverse catchment aquatic habitats.