From laboratory tests to field trials: a review of cathodic protection and microbially influenced corrosion.

Cathodic protection (CP), an electrochemical method for managing corrosion, is widely used in many industries in both marine and buried environments. However, literature surrounding cathodic protection and its ability to prevent microbially influenced corrosion (MIC) is mixed. This review describes the mechanics of CP, how CP may influence MIC, and collates and summarises tests on CP and MIC reported in literature. The aim of the review is to identify any trends and knowledge gaps requiring further study. While the outcomes of CP testing are generally mixed, some trends can be seen and, overall, MIC is detrimental to the protective effects of CP, with CP being less effective when used according to current international standards. Tests conducted in the field or with mix communities of microbes showed that CP could be effective at preventing MIC, while tests with sulfate-reducing bacteria generally proved CP to be highly ineffective. It was commonly seen that the effectiveness of CP can be improved by increasing polarization, to potentials as low as -1000 mV (Ag/AgCl). However, a balance does need to be met via careful monitoring to ensure negative side effects of over protection do not become a major problem.

Seasonal metabolic analysis of marine sediments collected from Moreton Bay in South East Queensland, Australia, using a multi-omics-based approach.

Anthropogenic effects of urban density have altered natural ecosystems. Such changes include eutrophication of freshwater and adjoining coastal habitats, and increased levels of inorganic nutrients and pollutants into waterways. In Australia, these changes are intensified by large-scale ocean-atmospheric events, leading to considerable abiotic stress on the natural flora and fauna. Bacterial communities in marine sediments from Moreton Bay (South East Queensland, Australia) were examined in order to assess the impact of rainfall changes, chemical pollution, and subsequent abiotic stress on living organisms within a marine ecosystem. Sediments were collected during the wet and dry seasons and analyzed using bacterial metagenomics and community metabolomics techniques. Physicochemical data were also analyzed to account for biological variance that may be due to non-rainfall-based abiotic stresses. Wet-dry seasonality was the dominant control on bacterial community structure and metabolic function. Changes in the availability of nutrients, organic matter and light appeared to be the major seasonal stressors. In contrast, urban and industrial pollutants appeared to be minor stressors at the sites sampled. During the wet season, the bacterial community composition reflected organisms that utilize biogeochemical pathways with fast kinetics, such as aerobic metabolism, direct assimilation of inorganic compounds, and primary production. The transition to the dry season saw the bacterial community composition shift towards organisms that utilize more complex organic energy sources, such as carbohydrates and fatty acids, and anaerobic redox processes.

A multi-omics based ecological analysis of coastal marine sediments from Gladstone, in Australia's Central Queensland, and Heron Island, a nearby fringing platform reef.

The impact of anthropogenic factors arising from point and non-point pollution sources at a multi commodity marine port and its surrounding ecosystems were studied using sediment samples collected from a number of onshore (Gladstone Harbour and Facing Island) and offshore (Heron Island and Fitzroy Reefs) sites in Australia's Central Queensland. Sediment samples were analyzed for trace metals, organic carbon, polycyclic aromatic hydrocarbons (PAH), emerging chemicals of concern (ECC) and sterols. Similarly, the biological and biochemical interaction between the reef and its environment was analyzed by the multi-omic tools of next-generation sequencing characterization of the bacterial community and microbial community metabolic profiling. Overall, the trace elements were observed at the lower end of the Australian environmental guideline values at the offshore sites, while higher values were observed for the onshore locations Nickel and copper were observed above the high trigger value threshold at the onshore sites. The levels of PAH were below limits of detection across all sites. However, some of the ECC and sterols were observed at higher concentrations at both onshore and offshore locations, notably, the cholesterol family sterols and 17α-ethynylestradiol. Multi-omic analyses also indicated possible thermal and photo irradiation stressors on the bacterial communities at all the tested sites. The observed populations of γ-proteobacteria were found in combination with an increased pool of fatty acids that indicate fatty acid synthesis and utilisation of the intermediates of the shikimate pathways. This study demonstrates the value of applying a multi-omics approach for ecological assessments, in which a more detailed assessment of physical and chemical contaminants and their impact on the community bacterial biome is obtained.

Determination of Ancylostoma caninum ova viability using metabolic profiling.

Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.

An 'omics' approach towards the characterisation of laboratory scale anaerobic digesters treating municipal sewage sludge.

In this study, laboratory scale digesters were operated to simulate potential shocks to the Anaerobic Digestion (AD) process at a 350 ML/day wastewater treatment plant. The shocks included high (42 °C) and low (32 °C) temperature (either side of mesophilic 37 °C) and a 20% loading of fats, oil and grease (FOG; 20% w:v). These variables were explored at two sludge retention times (12 and 20 days) and two organic loading rates (2.0 and 2.5 kgTS/m(3)day OLR). Metagenomic and metabolomic approaches were then used to characterise the impact of operational shocks in regard to temperature and FOG addition, as determined through monitoring of biogas production, the microbial profile and their metabolism. Results showed that AD performance was not greatly affected by temperature shocks, with the biggest impact being a reduction in biogas production at 42 °C that persisted for 32 ± 1 days. The average biogas production across all digesters at the completion of the experiment was 264.1 ± 76.5 mL/day, with FOG addition observed to significantly promote biogas production (+87.8 mL/day). Metagenomic and metabolomic analyses of the digesters indicated that methanogens and methane oxidising bacteria (MOB) were low in relative abundance, and that the ratio of oxidising bacteria (methane, sulphide and sulphate) with respect to sulphate reducing bacteria (SRB) had a noticeable influence on biogas production. Furthermore, increased biogas production correlated with an increase in short chain fatty acids, a product of the addition of 20% FOG. This work demonstrates the application of metagenomics and metabolomics to characterise the microbiota and their metabolism in AD digesters, providing insight to the resilience of crucial microbial populations when exposed to operational shocks.

Enhancing a search for traditional medicinal plants with anthelmintic action by using wild type and stress reporter Caenorhabditis elegans strains as screening tools.

Traditional healers in Sarawak, Malaysia, use plants such as Picria fel-terrae, Linariantha bicolor and Lansium domesticum to treat gastrointestinal infections. This study aimed to test whether their nematocidal activities could be confirmed in vitro using highly standardised Caenorhabditis elegans models. We applied eight different ethanol solubilised plant extracts and two commercial anthelmintic drugs to larval and adult stages of C. elegans in vitro. Seven C. elegans strains were evaluated, one wild type and six strains with GFP-tagged stress response pathways to help characterise and compare the pathways affected by plant extracts. Our in vitro screen confirmed that both of the commercial anthelmintic drugs and five of the eight traditionally used plant extracts had significant nematocidal activity against both larval and adult C. elegans. The most effective extracts were from P. fel-terrae. The plant extracts triggered different stress response pathways from the commercial anthelmintic drugs. This study showed that using traditional knowledge of plant medicinal properties in combination with a C. elegans in vitro screen provided a rapid and economical test with a high hit rate compared with the random screening of plants for nematocidal activities. The use of transgenic C. elegans strains may allow this approach to be refined further to investigate the mode of action of active extracts.

Metabolic profiling of biofilm bacteria known to cause microbial influenced corrosion.

This study builds upon previous research that demonstrated the simplicity of obtaining metabolite profiles of bacteria in urban water networks, by using the metabolic profile of bacteria extracted from a reticulation pipe biofilm, which is known to cause microbial influenced corrosion (MIC). The extracellular metabolites of the isolated bacteria, and those bacteria in consortium, were analysed in isolation, and after exposure to low levels of copper. Applying chemometric analytical methodologies to the metabolomic data, we were able to better understand the profile of the isolated biofilm bacteria, which were differentiated according to their activity and copper exposure. It was found that the metabolic activity of the isolated bacteria and the bacteria in consortium varied according to the bacterium's ability to metabolise copper. This demonstrates the power of metabolomic techniques for the discrimination of water reticulation biofilms comprising similar bacteria in consortium, but undergoing different physico-chemical activities, such as corrosion and corrosion inhibition.

Application of metabolomics to understanding biofilms in water distribution systems: a pilot study.

Biofilms formed in pipes are known to contribute to waterborne diseases, accelerate corrosion and cause aesthetic taste and odour issues within the potable water supply network. This paper describes a pilot study, undertaken to assess the potential of using metabolomics to monitor bacterial activity in biofilms of an urban water network. Using samples from a water mains flushing programme, it was found that a profile of intracellular and extracellular metabolites associated with microbial activity could be obtained by analysing samples using gas chromatography mass spectrometry. Chemometric analysis of the chromatograms in conjunction with data from the mass spectrometer showed that it is possible to differentiate between biofilms from different pipe materials and planktonic bacteria. This research demonstrates that metabolomics has the potential for investigating biofilms and other microbial activity within water networks, and could provide a means for enhancing monitoring programmes, understanding the source of water quality complaints, and optimising water network management strategies.

Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005.

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5+11] (15.4%). G10P[11] and G6+G10P[5] were each detected at a rate of 7.7%, and G6+G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.

Antibacterial activity of traditional Australian medicinal plants.

Fifty-six ethanolic extracts of various parts of 39 plants used in traditional Australian Aboriginal medicine were investigated for their antibacterial activities against four Gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pyogenes) and four Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium) bacterial species. In a plate-hole diffusion assay, 12 extracts inhibited the growth of one or more of the bacteria, with five extracts showing broad spectrum antibacterial activity against Gram-positive bacteria. B. cereus was the most susceptible bacterium, with all 12 extracts displaying activity against this organism. Extracts from the leaves of Eremophila species (Myoporaceae) were the most active, with Eremophila duttonii exhibiting the greatest activity (against Gram-positive bacteria). The antibacterial effects of E. duttonii were further investigated by time-course growth assays which showed that significant growth inhibition was observed in cultures incubated in the presence of the extract within 1 h for B. cereus, E. faecalis and S. aureus and 2 h for S. pyogenes.

Genetic relatedness of VP1 genes of Australian and Taiwanese rotavirus isolates.

Gene 1 (which encodes the viral RNA-dependent RNA polymerase, VP1) of an atypical human reassortant rotavirus strain, E210 (serotype G2P1B), is unrelated to genes 1 of standard human rotaviruses. To ascertain the origin of this gene, we determined a partial sequence and found that it exhibited greatest identity to gene 1 of a Taiwanese isolate, TE83, which is representative of G2 strains that caused an epidemic of gastroenteritis in 1993. Limited sequence identity to genes 1 of standard human and animal viruses was observed. This was confirmed by phylogenetic analysis. However, hybridization analysis using an E210 gene 1-specific probe indicated that a related gene was found among other Australian G2 isolates and in a Japanese strain isolated in the 1970s.

Rotavirus encephalopathy: pathogenesis reviewed.

Two cases of rotavirus gastroenteritis associated with neurological involvement, one with encephalitis (defined by abnormal neurological signs, cerebrospinal fluid (CSF) pleocytosis and detection of rotavirus genomic nucleic acid in the CSF) and one with a non-inflammatory encephalopathy (defined by abnormal neurological signs, an entirely normal CSF and detection of rotavirus genomic nucleic acid in the CSF), are presented and used as a basis to review and explore potential pathogenetic mechanisms, including direct viral replication within neurons and indirect effects of the newly described rotavirus 'enterotoxin'.

Characterisation and phylogenetic analysis of the VP7 proteins of serotype G6 and G8 human rotaviruses.

Serotype G6 and G8 rotaviruses are rarely found in man and may have originated in animals. Human serotype G6 and G8 rotaviruses, isolated from hospitalised children at various locations in Australia, were characterised. Deduced amino acid sequences of the major neutralising antigen, V7, showed significant identity to the cognate proteins of prototype human and bovine G6 and G8 viruses, respectively, and the strains reacted with G6 and G8 serotype-specific neutralising monoclonal antibodies, respectively, in an enzyme immunoassay. The VP4 type was determined as P[14] for all strains tested. Phylogenetic analysis of these and other human and bovine VP7 sequences suggested that a single inter-species transmission event, possibly from cattle, may have led to the emergence of G6 viruses in man. In contrast, the exchange of genes between human and bovine G8 viruses may have occurred onmore than one occasion, or these genes may have originated in a different host.

Molecular characterization of porcine rotaviruses from the southern region of Brazil: characterization of an atypical genotype G[9] strain.

The G (VP7) and P (VP4) serotype distribution of Brazilian porcine rotaviruses was determined using reverse transcription-PCR genotyping methods. Common porcine G types G3, G4, and G5 were detected in combination with P types [6] and [7]. The detection of nonporcine G types and unusual G-P combinations and the characterization of an atypical virus indicated that interspecies transmission may contribute to the genetic diversity of porcine rotaviruses.

Epidemiology of astrovirus infection in young children hospitalized with acute gastroenteritis in Melbourne, Australia, over a period of four consecutive years, 1995 to 1998.

The incidence of astrovirus infection in children less than 5 years of age hospitalized with acute gastroenteritis in Melbourne, Australia, from 1995 to 1998 was determined. Astrovirus was detected in 40 of 449 specimens tested by Northern hybridization, and astrovirus infection was confirmed by reverse transcription-PCR with or without culture in CaCO-2 cells. This represented 3.0% (40 of 1, 327) of all children tested for enteric pathogens, including viral, bacterial, and parasitic pathogens, over the survey period. The incidences of astrovirus infection in each year were 4.4% (1995), 2. 2% (1996), 3.9% (1997), and 1.4% (1998). In 1995 and 1997, the incidences of astrovirus infection were greater than the incidence of infection with all individual bacterial pathogens and were either greater than or equal to the incidence of adenovirus infection. Astrovirus exhibited an unusual biennial winter peak of incidence that correlated with a greater incidence of serotype 1 virus and an increased rate of hospitalization of children aged 6 to 12 months. Uncommon (serotype 2 and 4) and rare (serotype 8) serotypes were detected during the survey period. Genetic analysis of ORF2 (which encodes the astrovirus capsid precursor) of Melbourne isolates showed nucleotide sequence variation from year to year. This was not accompanied by significant amino acid substitutions. However, geographical variation was apparent by comparison of Melbourne astrovirus isolates with prototype strains identified in the United Kingdom.

Detection of astrovirus gastroenteritis in children.

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.

Characterisation of a "European-like" serotype G8 human rotavirus isolated in Australia.

An atypical human rotavirus strain, DG8, was isolated from a 13-month-old child hospitalised with acute gastro-enteritis in Australia. The virus could not be serotyped by enzyme immunoassay (EIA) using standard reagents specific for common Group A human rotavirus G serotypes. The deduced amino acid sequence of the outer capsid glycoprotein, VP7, indicated that this strain belonged to the uncommon human serotype G8. This was confirmed by EIA incorporating a G8-specific neutralising monoclonal antibody (NMAb). The VP4 genotype of DG8 was determined as P[14], equivalent to P serotype P3B, by sequence analysis and confirmed by EIA incorporating a P3B-specific NMAb. Electrophoretic analysis of DG8 genomic dsRNA indicated that the virus exhibited a "long" electropherotype. Northern hybridisation analysis (using a whole-genome probe derived from DG8) indicated that DG8 shared overall homology with the European serotype G8 strain, HAL1166 (11 of 11 genes). In contrast, only 9 of 11 genes of DG8 hybridised with the Asian serotype G8 strain, B37, and with the bovine G8 strain, A5. Hence, DG8 displayed features reminiscent of the human serotype G8 rotaviruses isolated in Europe in the mid-1980s rather than the geographically local G8 Asian strains isolated a decade earlier. It is possible that DG8 arose through reassortment between human and bovine rotaviruses.

Genetic and antigenic diversity of human rotaviruses: potential impact on the success of candidate vaccines.

Licensing of the first human rotavirus vaccine raises the hope of a reduction in the burden of paediatric diarrheal disease. However, the less than optimal performance of this vaccine in trials carried out in developing countries indicates that improvements in vaccine design are necessary. Analysis of the genetic and antigenic diversity of rotavirus isolates collected from various geographical locations suggests that future vaccine formulations may need to include a broader spectrum of strains. This may increase vaccine efficacy by providing comprehensive coverage against circulating viruses. Continued surveillance and genetic analysis of the rotavirus population prior to and after the introduction of routine vaccination will reveal if the diversity of this pathogen will impact on the success of vaccine programs.