Melissa H. BrownBrownBrown

Effective wastewater treatment is critical for public health and environmental protection. In regional communities, where resources are limited, there is a need for sustainable and low-cost wastewater treatment solutions. Commonly used waste stabilisation ponds, have large land requirements, inconsistent treatment performance and high rates of evaporative water loss. High rate algal ponds (HRAPs) offer a smaller area footprint and consequentially reduced capital expenditure, enhanced treatment performance and a low maintenance alternative. HRAPs are commonly operated as continuously stirred tank reactors, at shallow depth (0.2–0.5 m) mixed by a paddlewheel. Effective wastewater treatment is then achieved by a consortium of naturally occurring, harmless microalgae and bacteria. However, there is a need to further improve their operation and the quality of the treated effluent to enhance water reuse opportunities and alleviate water insecurity concerns in rural communities. Here we uniquely propose two different operational strategies for HRAPs as the next step forward for this treatment technology. The two strategies require operation as sequencing batch reactors, which enables independent management, of hydraulic retention time and solids retention time, providing additional operational management strategies. Significantly, this offers the potential to develop influent feeding and mixing strategies to develop biofilm like assemblages of photogranules or to selectively enrich and maintain filamentous algal populations. The increased density of either photogranules or filamentous algae will enable efficient biosolids separation yielding an effluent low in suspended solids. The biomass separation may also be achieved within the HRAP avoiding the need to construct and manage additional infrastructure. The enhanced treated effluent quality increases opportunities for added value beneficial water reuse in climate change related water stressed communities. Future research is needed to validate this approach and the optimum operating conditions to achieve treatment and efficient in situ biomass separation.

Amid the antibiotic resistance crisis, biocides, including antiseptics and disinfectants, are indispensable tools to limit the spread of nosocomial infections and extensively drug-resistant bacteria. Thus, it is crucial that they remain effective. Extruding biocides from the cell using efflux systems is one mechanism by which bacteria can survive in their presence. The CepA protein from Klebsiella pneumoniae belongs to a family of heavy metal exporters but has been reported to be associated with chlorhexidine resistance, a common biocide. This study aims to elucidate the function of CepA as a biocide transporter and clarify its substrate profile in Escherichia coli . The results support the role of CepA in the transport of Fe ²⁺ , Mn ²⁺ , and possibly Cu ²⁺ and Zn ²⁺ to a lesser extent. The transporter also contributes to cell tolerance to iron and manganese stress. However, in contrast to previous reports, no significant impact of the expression of this protein on bacterial resistance to quaternary ammonium biocides was detected. Therefore, we propose that the main role of K. pneumoniae CepA is as a heavy metal transporter. IMPORTANCE: Understanding the structure and function of efflux pumps is crucial for addressing efflux-mediated resistance. Transporters that pump out biocides or heavy metals are not uncommon. However, to date, no transport protein capable of extruding both types of substrates has been reported. Initially, it was proposed that Klebsiella pneumoniae CepA was associated with chlorhexidine resistance and subsequently considered a biocide efflux pump. That notion also hinted at a novel group of drug efflux pumps that can extrude both biocides and heavy metals, a direct mechanism of cross-resistance between the two. The findings of this study indicate that it is more plausible that K. pneumoniae CepA is involved in metal homeostasis rather than in chlorhexidine biocide resistance in the recombinant Escherichia coli .

The use of synchrotron macro ATR-FTIR micro-spectroscopy to reveal the antibacterial mechanism of silver ions against S. aureus and P. aeruginosa.

Objectives: To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. Methods: A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. Results: Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. Conclusions: TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.

Plant-derived pharmacological agents have been used extensively to dissect the structure–function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABA_A receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC₅₀ for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Acinetobacter baumannii is an opportunistic human pathogen responsible for numerous severe nosocomial infections. Genome analysis on the A. baumannii clinical isolate 04117201 revealed the presence of 13 two-component signal transduction systems (TCS). Of these, we examined the putative TCS named here as StkSR. The stkR response regulator was deleted via homologous recombination and its progeny, ΔstkR, was phenotypically characterized. Antibiogram analyses of ΔstkR cells revealed a two-fold increase in resistance to the clinically relevant polymyxins, colistin and polymyxin B, compared to wildtype. PAGE-separation of silver stained purified lipooligosaccharide isolated from ΔstkR and wildtype cells ruled out the complete loss of lipooligosaccharide as the mechanism of colistin resistance identified for ΔstkR. Hydrophobicity analysis identified a phenotypical change of the bacterial cells when exposed to colistin. Transcriptional profiling revealed a significant up-regulation of the pmrCAB operon in ΔstkR compared to the parent, associating these two TCS and colistin resistance. These results reveal that there are multiple levels of regulation affecting colistin resistance; the suggested ‘cross-talk’ between the StkSR and PmrAB two-component systems highlights the complexity of these systems.

Opportunistic premise plumbing pathogens (OPPP) are microorganisms that are native to the plumbing environment and that present an emerging infectious disease problem. They share characteristics, such as disinfectant resistance, thermal tolerance, and biofilm formation. The colonisation of domestic water systems presents an elevated health risk for immune-compromised individuals who receive healthcare at home. The literature that has identified the previously described OPPPs (Aeromonas spp., Acinetobacter spp., Helicobacter spp., Legionella spp., Methylobacterium spp., Mycobacteria spp., Pseudomonas spp., and Stenotrophomonas spp.) in residential drinking water systems were systematically reviewed. By applying the Preferred reporting items for systematic reviews and meta-analyses guidelines, 214 studies were identified from the Scopus and Web of Science databases, which included 30 clinical case investigations. Tap components and showerheads were the most frequently identified sources of OPPPs. Sixty-four of these studies detected additional clinically relevant pathogens that are not classified as OPPPs in these reservoirs. There was considerable variation in the detection methods, which included traditional culturing and molecular approaches. These identified studies demonstrate that the current drinking water treatment methods are ineffective against many waterborne pathogens. It is critical that, as at-home healthcare services continue to be promoted, we understand the emergent risks that are posed by OPPPs in residential drinking water. Future research is needed in order to provide consistent data on the prevalence of OPPPs in residential water, and on the incidence of waterborne homecare-associated infections. This will enable the identification of the contributing risk factors, and the development of effective controls.

Marine sponges are an ideal source for isolating as yet undiscovered microorganisms with some sponges having about 50% of their biomass composed of microbial symbionts. This study used a variety of approaches to investigate the culturable diversity of the sponge-associated bacterial community from samples collected from the South Australian marine environment. Twelve sponge samples were selected from two sites and their bacterial population cultivated using seven different agar media at two temperatures and three oxygen levels over 3 months. These isolates were identified using microscopic, macroscopic, and 16S rRNA gene analysis. A total of 1234 bacterial colonies were isolated which consisted of four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, containing 21 genera. The diversity of the bacterial population was demonstrated to be influenced by the type of isolation medium, length of the incubation period and temperature, sponge type, and oxygen level. The findings of this study showed that marine sponges of South Australia can yield considerable bacterial culturable diversity if a comprehensive isolation strategy is implemented. Two sponges, with the highest and the lowest diversity of culturable isolates, were examined using next-generation sequencing to better profile the bacterial population. A marked difference in terms of phyla and genera was observed using culture-based and culture-independent approaches. This observed variation displays the importance of utilizing both methods to reflect a more complete picture of the microbial population of marine sponges. Key points: Improved bacterial diversity due to long incubations, 2 temperatures, and 3 oxygen levels. Isolates identified by morphology, restriction digests, and 16S rRNA gene sequencing. At least 70% of culturable genera were not revealed by NGS methods.