Cryptosporidium parvum decay during air drying and stockpiling of mesophilic anaerobically digested sewage sludge in a simulation experiment and oocyst counts in sludge collected from operational treatment lagoons in Victoria, Australia.

The inactivation of Cryptosporidium species oocysts during sewage sludge treatment is important to protect human health when the residual biosolids are applied to agricultural land. Quantifying the decay of Cryptosporidium species during sludge treatment for microbiological assurance purposes is difficult if low numbers are present in wastewater. The rate of decay of Cryptosporidium parvum oocysts during solar/air drying treatment and in sludge stockpiles in temperate environment conditions was simulated in laboratory inoculation experiments using sludge sampled from a mesophilic anaerobic digester. Oocyst numbers were also determined in settled lagoon sludge samples collected from three operational rural wastewater treatment plants (WWTPs). C. parvum oocysts were enumerated by immunomagnetic separation followed by staining with vital dyes and examination by confocal laser scanning microscopy. An air-drying/storage period equivalent to 11 weeks was required for a 1 log10 reduction of viable oocysts inoculated into digested sludge. Oocyst viability in air-dried and stored digested sludge decreased with time, but was independent of sludge desiccation and dry solids (DS) content. No oocysts were detected in sludge samples collected from the anaerobic digester, and the average concentration of oocysts found in settled lagoon sludge from the rural WWTP was 4.6 × 102 oocysts/g DS.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Factors affecting decay of Salmonella Birkenhead and coliphage MS2 during mesophilic anaerobic digestion and air drying of sewage sludge.

Factors affecting the decay of Salmonella Birkenhead and coliphage, as representatives of bacterial and viral pathogens, respectively, during mesophilic anaerobic digestion (MAD) and air drying treatment of anaerobically digested sewage sludge were investigated. Controlled concentrations of S. Birkenhead were inoculated into non-sterile, autoclaved, γ-irradiated and nutrient-supplemented sludge and cultures were incubated at 37 °C (MAD sludge treatment temperature) or 20 °C (summer air drying sludge treatment temperature). Nutrient limitation caused by microbial competition was the principal mechanism responsible for the decay of S. Birkenhead by MAD and during air drying of digested sludge. The effects of protease activity in sludge on MS2 coliphage decay in digested and air dried sludge were also investigated. MS2 coliphage showed a 3.0-3.5 log10 reduction during incubation with sludge-protease extracts at 37 °C for 25 h. Proteases produced by indigenous microbes in sludge potentially increase coliphage inactivation and may therefore have a significant role in the decay of enteric viruses in sewage sludge. The results help to explain the loss of viability of enteric bacteria and viral pathogens with treatment process time and contribute to fundamental understanding of the various biotic inactivation mechanisms operating in sludge treatment processes at mesophilic and ambient temperatures.

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity.

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.

Effects of erythromycin on the phenotypic and genotypic biofilm expression in two clinical Staphylococcus capitis subspecies and a functional analysis of Ica proteins in S. capitis.

The ica operon encoding polysaccharide intercellular adhesion, which facilitates biofilm formation in staphylococci, has been extensively studied in Staphylococcus epidermidis and Staphylococcus aureus. Based on in silico analysis, we suggest the following functional model for Ica proteins in S. capitis. IcaA is responsible for polysaccharide synthesis. IcaA and IcaD complete transferring the growing sugar chain to the cell surface; IcaB is a deacetylase, with the same function as IcaB of S. epidermidis. IcaC mainly modifies the synthesized glucan by acetylation. We also examined the effects of subinhibitory concentrations of erythromycin on phenotypic biofilm expression and transcription of biofilm-related genes, using isolates representing the two subspecies of Staphylococcus capitis and different biofilm and resistance phenotypes. On induction with erythromycin, biofilm density was strongly elevated in two erythromycin-resistant S. capitis, but not in three susceptible isolates. In the representative erythromycin-resistant S. capitis subsp. urealyticus, there were significant upregulations of the icaA gene and its positive regulator sarA on transition to the stationary phase without erythromycin induction. There were also significant increases in the transcription levels of icaA, rsbU and sigB corresponding to a very strong biofilm phenotype in the stationary phase on erythromycin stress. In contrast, the representative erythromycin-susceptible S. capitis subsp. capitis displayed upregulation only of altE on entry into the stationary phase with erythromycin induction, but this change was not associated with enhancement of biofilm production. These findings suggest that the two subspecies of S. capitis adopt different pathogenesis and survival strategies to adapt to a hostile environment.

Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3µg of plasmid DNA was mixed with 70-80µL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen.

Evolution of the first genetic cells and the universal genetic code: a hypothesis based on macromolecular coevolution of RNA and proteins.

A qualitative hypothesis based on coevolution of protein and nucleic acid macromolecules was developed to explain the evolution of the first genetic cells, from the likely organic chemical-rich environment of early earth, through to the Last Universal Common Ancestor (LUCA). The evolution of the first genetic cell was divided into three phases, proto-genetic cells I, II and III, and the transition to each milestone is described, based on development of chemical cross-catalysis, bio-cross-catalysis, and the universal genetic code, respectively. Selection of macromolecular properties of both peptides and nucleic acids, in response to environmental factors, was likely to be a key aspect of early evolution. The development of hereditable nucleic acids with various key functions; translation, transcription and replication, is described. These functions are envisaged to have coevolved with protein enzymes, from simple organic precursors. Genetically heritable nucleotides may have developed after the local earth environment had cooled below 63 °C. Around this temperature G-C bases would have been preferentially utilized for nucleotide synthesis. Under these conditions RNA type nucleotides were then likely selected from a range of different types of nucleotide backbones through template-based synthesis. Initial development of the genetic coding system was simplified by the availability of proto-messenger RNA sequences that contained only G and C bases, and the need to encode only four amino acids. The step-wise addition of further amino acids to the code was predicted to parallel the growing metabolic complexity of the proto-genetic cell. On completion of this evolutionary process the proto-genetic cell is envisaged to have become the LUCA, the last common ancestor of bacteria, eukaryote and archaea domains. Key issues addressed by the model include: (a) the transition from non-hereditable random sequences of peptides and nucleic acids to specific proteins coded by hereditable nucleotide sequences, (b) the origin of homochiral amino acids and sugars, and (c) the mutation limits on the sizes of early nucleic acid genomes. The first genome was limited to a size of about 200 base pairs.

Differences between two clinical Staphylococcus capitis subspecies as revealed by biofilm, antibiotic resistance, and pulsed-field gel electrophoresis profiling.

Coagulase-negative staphylococci have been identified as major causes of late-onset neonatal bacteremia in neonatal intensive care units. Sixty isolates of Staphylococcus capitis obtained from blood cultures of neonates between 2000 and 2005 were examined in this study. Biochemical analysis confirmed that 52 of these isolates belonged to the subsp. urealyticus, and the remaining 8 belonged to the subsp. capitis. Isolates of the predominant subsp. urealyticus clones were characterized by their resistance to penicillin, erythromycin, and oxacillin and their biofilm formation ability, whereas subsp. capitis isolates were generally antibiotic susceptible and biofilm negative. Pulsed-field gel electrophoresis (PFGE) after SacII digestion separated the 60 isolates into five major clusters. Sequence analysis showed that, in S. capitis, the ica operon plus the negative regulator icaR was 4,160 bp in length. PCRs demonstrated the presence of the ica operon in all isolates. Further analysis of five isolates (two biofilm-positive subsp. urealyticus, one biofilm-negative subsp. urealyticus, and two biofilm-negative subsp. capitis) revealed that the ica operons were identical in all of the biofilm-positive subsp. urealyticus strains; however, the biofilm-negative isolates showed variations. The distinctive phenotypic and genotypic characteristics revealed by this study may affect the epidemiology of the two subspecies of S. capitis in the clinical setting. These results may provide a better understanding of the contribution of these two species to bloodstream infections in neonates.

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs.

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Arg(87)respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368)and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Arg(368)and Asn(459)/Arg(87)pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.

Effects of pesticide toxicity, salinity and other environmental variables on selected ecosystem functions in streams and the relevance for ecosystem services.

Effects of anthropogenic and environmental stressors on freshwater communities can propagate to ecosystem functions and may in turn impede ecosystem services. We investigated potential shifts in ecosystem functions that provide energy for freshwater ecosystems due to pesticides and salinity in 24 sites in streams of southeast Australia. First, effects on allochthonous organic matter (AOM) breakdown using three different substrates (leaves, cotton strips, wood sticks) in coarse and fine bags were investigated. Second, we examined effects on stream metabolism that delivers information on the ecosystem functions of gross primary production and ecosystem respiration. We found up to a fourfold reduction in AOM breakdown due to exposure to pesticides and salinity, where both stressors contributed approximately equally to the reduction. The effect was additive as, no interaction or correlation between the two stressors was found. Leaf breakdown responded strongly and exclusively to exposure to pesticides and salinity, whereas cotton strip breakdown was less sensitive and responded also to other stressors such as nutrients. No functional redundancy for the effects of pesticides and salinity on leaf breakdown was observed. For wood stick breakdown, no relationship to environmental gradients was found, however, the sample size was lower. We did not detect effects of pesticides or salinity on gross primary production or ecosystem respiration. A reduction in AOM breakdown by pesticides and salinity may impair the ecosystem services of food provision and possibly water purification. Hence, future studies should examine the spatial extent of these effects.

Microbial safety of air-dried and rewetted biosolids.

To assess microbial safety of treated sewage sludge (biosolids), we examined the inactivation of microbial indicators for potential bacterial, viral and protozoan pathogens. The levels of indicators were determined throughout the air-drying and storage phases of anaerobically digested sewage sludge. Samples were collected from two wastewater treatment plants (WWTPS) in Victoria, Australia. Established methods were applied for analysis of bacteria and coliphages, based on membrane filtration and layered plates, respectively. In the pan drying phase, the prevalence of Escherichia coli was reduced by >5 log10 compared with sludge entering the pan. Thus, after pan drying of 8-11 months at WWTP A and 15 months at WWTP B, the numbers of E. coli were reduced to below 10(2) cfu/g dry solids (DS). This level is acceptable for unrestricted use in agriculture in Australia (P1 treatment grade), the UK (enhanced treatment status) and the USA (Class A pathogen reduction). Coliphage numbers also decreased substantially during the air-drying phase, indicating that enteric viruses are also likely to be destroyed during this phase. Clostridium perfringens appeared to be an overly conservative indicator. Survival, but not regrowth, of E. coli or Salmonella was observed in rewetted biosolids (15-20% moisture content), after being seeded with these species, indicating a degree of safety of stored biosolids upon rewetting by rain.

Densely adherent growth mode, rather than extracellular polymer substance matrix build-up ability, contributes to high resistance of Staphylococcus epidermidis biofilms to antibiotics.

OBJECTIVES: (i) To evaluate the role of the adherent growth mode and extracellular polymer substance build-up in biofilm resistance to antibiotics. (ii) To re-assess various mechanisms leading to biofilm resistance to antibiotics. METHODS: We compared the biofilm MICs, biofilm MBCs using the viable count method, biofilm MBCs based on broth recovery methods and minimum biofilm eradication concentrations (MBECs) of antistaphylococcal antibiotics for multilayer biofilms formed by 'biofilm-positive' S. epidermidis strains and monolayer biofilms formed by their 'biofilm-negative' mutants/variants. Bacterial densities and the quantity of persister cells in both multilayer and monolayer biofilms were assessed to evaluate their roles in biofilm resistance. RESULTS: Monolayer and multilayer biofilms presented similar susceptibilities to multiple antibiotics, based on biofilm MIC, broth recovery-based biofilm MBC and MBEC results. Multilayer biofilms demonstrated higher viable count-based MBCs than monolayer biofilms. Both monolayer and multilayer biofilms had very high bacterial densities of approximately 10(11-12) cfu/mL. Persister cells were found in both monolayer and multilayer biofilms, but not in planktonic cultures at log phase. The presence of persister cells in monolayer and multilayer biofilms appeared to be strain and antibiotic dependent. CONCLUSIONS: The adherent growth mode, rather than the ability to build up a typical multilayer biofilm structure, contributes to the high resistance of biofilms to antibiotics, and therefore might be the main virulence factor of coagulase-negative staphylococci (CoNS) with respect to antibiotic resistance. The presence of persister cells in CoNS biofilms plays an important role in antibiotic resistance. Growth at high bacterial densities is another significant factor in biofilm resistance.

Comparison of various antimicrobial agents as catheter lock solutions: preference for ethanol in eradication of coagulase-negative staphylococcal biofilms.

Coagulase-negative staphylococci (CoNS) are the main causative agents of bacteraemia in infants managed in neonatal intensive care units (NICUs). Intraluminal colonization of long-term central venous catheters by these bacteria and subsequent biofilm formation are the prerequisites of the bloodstream infections acquired in NICUs. The catheter lock technique has been used to treat catheter colonization; however, the optimum choice of antimicrobial agents and their corresponding concentrations and exposure times have not been determined. The effectiveness of catheter lock solutions (CLSs) was assessed by determining the minimal biofilm eradication concentration of antimicrobial agents against CoNS biofilms. Five conventional antibiotics (oxacillin, gentamicin, vancomycin, ciprofloxacin and rifampicin) alone or in combination, as well as ethanol, were evaluated. Ethanol was found to be superior to all of these conventional antibiotics when used as a CLS. A time-kill study and confocal laser scanning microscopy revealed that exposure to 40 % ethanol for 1 h was sufficient to kill CoNS biofilm cells. To our knowledge, this is the first in vitro study to provide solid evidence to support the rationale of using ethanol at low concentrations for a short time as a CLS, instead of using conventional antibiotics at high concentrations for a long period to treat catheter-related bloodstream infections.

Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter.

All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K(m) value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter.

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.

Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter.

This study represents the first attempt to investigate the molecular mechanisms by which nitrate, an anion of significant ecological, agricultural, and medical importance, is transported into cells by high-affinity nitrate transporters. Two charged residues, R87 and R368, located within hydrophobic transmembrane domains 2 and 8, respectively, are conserved in all 52 high-affinity nitrate transporters sequenced thus far. Site-directed replacements of either of R87 or R368 residues by lysine were found to be tolerated, but such residue changes increased the K(m) for nitrate influx from micromolar to millimolar values. Seven other amino acid substitutions of R87 or R368 all led to loss of function and lack of growth on nitrate. No evidence was obtained of R87 or R368 forming a salt-bridge with conserved acidic residues. Remarkably, the phenotype of loss-of-function mutant R87T was found to be alleviated by an alteration to lysine of N459, present in the second copy of the nitrate signature (transmembrane domain 11), suggesting a structural or functional interplay between residues R87 and N459 in the three-dimensional NrtA protein structure. Failure of the potential reciprocal second site suppressor N168K (in the first nitrate signature copy of transmembrane domain 5) to revert R368T was observed. Taken with recent structural studies of other major facilitator superfamily proteins, the results suggest that R87 and R368 are involved in substrate binding and probably located in a region of the protein close to N459.