IRAK3 Knockout and Wildtype THP-1 Monocytes as Models for Endotoxin Detection Assays and Fusobacterium nucleatum Bacteriophage FNU1 Cytokine Induction.

Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor - alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations.

Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees.

Chalkbrood infection caused by the fungus Ascosphaera apis currently has a significant impact on Australia's apicultural industry. We investigated the genetic variation of A. apis and colony and apiary level conditions to determine if an emerging, more virulent strain or specific conditions were responsible for the prevalence of the disease. We identified six genetically distinct strains of A. apis, four have been reported elsewhere and two are unique to Australia. Colonies and individual larvae were found to be infected with multiple strains of A. apis, neither individual strains, combinations of strains, or obvious colony or apiary characteristics were found to be predictive of hive infection levels. These results suggest that host genotype plays an important role in colony level resistance to chalkbrood infection in Australia.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia.

BACKGROUND: Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. METHODS: Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia-telangiectasia mutated (ATM). RESULTS: Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. CONCLUSIONS: The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

Isolation and characterization of bacteriophage NTR1 infectious for Nocardia transvalensis and other Nocardia species.

We describe here the isolation and characterization of the bacteriophage, NTR1 from activated sludge. This phage is lytic for Nocardia transvalensis, Nocardia brasiliensis and Nocardia farcinica. NTR1 phage has a genome sequence of 65,275 bp in length, and its closest match is to the Skermania piniformis phage SPI1 sharing over 36% of its genome. The phage belongs to the Siphoviridae family, possessing a long non-contractile tail and icosahedral head. Annotation of the genome reveals 97 putative open reading frames arranged in the characteristic modular organization of Siphoviridae phages and contains a single tRNA-Met gene.

The mediating role of sleep in the relationship between Indigenous status and body mass index in Australian school-aged children.

AIM: Associations between sleep duration and obesity and between obesity and chronic illness are established. Current rates of obesity for all Australian people are rising. Recent reports indicate that high body mass index (BMI) is a leading contributor to overall burden of disease for Indigenous Australians. Understanding the factors that contribute to higher rates of obesity in Indigenous people is critical to developing effective interventions for reducing morbidity and premature mortality in this population. To explore the effect of sleep duration on the relationship between Indigenous status and BMI in Australian children. METHODS: 716 non-Indigenous and 186 Indigenous children aged 5-12 years in the Australian Health Survey 2011-2013. Primary carers were interviewed regarding children's sleep times; BMI was derived from measurement. RESULTS: Analysis of covariance revealed that regardless of a number of demographic and socio-economic status markers, sleep duration and Indigenous status were independent predictors of BMI. However when both predictors were considered together, only sleep duration remained predictive of BMI. CONCLUSIONS: Sleep duration plays an important mediating role in the relationship between Indigenous status and BMI in this Australian sample. Modification of sleep duration for Indigenous children may lead to longer-term positive health outcomes.

Pharmacogenomics in Papua New Guineans: unique profiles and implications for enhancing drug efficacy while improving drug safety.

Papua New Guinea (PNG) can be roughly divided into highland, coastal and island peoples with significant mitochondrial DNA differentiation reflecting early and recent distinct migrations from Africa and East Asia, respectively. Infectious diseases such as tuberculosis, malaria and HIV severely impact on the health of its peoples for which drug therapy is the major treatment and pharmacogenetics has clinical relevance for many of these drugs. Although there is generally little information about known single nucleotide polymorphisms in the population, in some instances, their frequencies have been shown to be higher than anywhere worldwide. For example, CYP2B6*6 is over 50%, and CYP2C19*2 and *3 are over 40 and 25%, respectively. Conversely, CYP2A6*9, 2B6*2, *3, *4 and *18, and 2C8*3 appear to be much lower than in Whites. CYP2D6 known variants are unclear, and for phase II enzymes, only UGT2B7 and UGT1A9 data are available, with variant frequencies either slightly lower than or similar to Whites. Although almost all PNG people tested are rapid acetylators, but which variant(s) define this phenotype is not known. For HLA-B*13:01, HLA-B*35:05 and HLA-C*04:01, the frequencies show some regioselectivity, but the clinical implications with respect to adverse drug reactions are not known. There are minimal phenotype data for the CYPs and nothing is known about drug transporter or receptor genetics. Determination of genetic variants that are rare in Whites or Asians but common in PNG people is a topic of both scientific and clinical importance, and further research needs to be carried out. Optimizing the safety and efficacy of infectious disease drug therapy through pharmacogenetic studies that have translation potential is a priority.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Association between short sleep duration and body mass index in Australian Indigenous children.

AIM: Associations between short sleep duration and obesity and the relationship between obesity and chronic illness are well documented. Obese children are likely to become obese adults. To date, there is a paucity of information regarding sleep duration and quality for Indigenous Australian people. It may be that poor-quality, short sleep is contributing to the gap in health outcomes for Indigenous people compared with non-Indigenous adults and children. This study sought to investigate the possibility that poor sleep quality may be contributing to health outcomes for Indigenous children by exploring associations between sleep duration and body mass index (BMI). METHODS: Participants included 1253 children aged 7-12 years in Wave 7 of the national Longitudinal Study of Indigenous Children survey. Interviewers asked primary carers about children's sleep times. BMI was derived from measurements of children made by researchers. RESULTS: Regardless of age, relative socio-economic disadvantage and level of remoteness, unhealthy weight was associated with less sleep duration than healthy weight for Indigenous children. CONCLUSION: The relationship between short sleep duration and BMI in Indigenous children has important implications for their future health outcomes. Both overweight conditions and short sleep are established modifiable risk factors for metabolic dysfunction and other chronic illnesses prominent in the Indigenous population. It is important to consider strategies to optimise both for Indigenous children in an attempt to help 'close the gap' in health outcomes and life expectancy between Indigenous and non-Indigenous people.

Omega 3 polyunsaturated fatty acids and the treatment of depression.

Depression is a common, recurrent, and debilitating illness that has become more prevalent over the past 100 years. This report reviews the etiology and pathophysiology of depression, and explores the role of omega 3 polyunsaturated fatty acids (n-3 PUFA) as a possible treatment. In seeking to understand depression, genetic factors and environmental influences have been extensively investigated. Research has led to several hypotheses for the pathophysiological basis of depression but a definitive pathogenic mechanism, or group thereof, has hitherto remained equivocal. To date, treatment has been based on the monoamine hypothesis and hence, selective serotonin reuptake inhibitors have been the most widely used class of medication. In the last decade, there has been considerable interest in n-3 PUFAs and their role in depression. These fatty acids are critical for development and function of the central nervous system. Increasing evidence from epidemiological, laboratory, and randomized placebo-controlled trials suggests deficiency of dietary n-3 PUFAs may contribute to development of mood disorders, and supplementation with n-3 PUFAs may provide a new treatment option. Conclusions based on systematic reviews and meta-analyses of published trials to date vary. Research into the effects of n-3 PUFAs on depressed mood is limited. Furthermore, results from such have led to conflicting conclusions regarding the efficacy of n-3 PUFAs in affecting reduction in symptoms of depression. PUFAs are generally well tolerated by adults and children although mild gastrointestinal effects are reported. There is mounting evidence to suggest that n-3 PUFAs play a role in depression and deserve greater research efforts.

A brief review of the application and pharmacology of ethnomedicines of Indigenous Australians.

OBJECTIVE: Indigenous Australians suffer higher mortality and have statistically worse outcomes for many chronic disease states than the non-Indigenous population. Although many of these people are prescribed pharmaceutical drugs for their illnesses, some still use medicines that were traditional to their culture. This report reviews some of the traditional medicines used for ailments seen in Indigenous Australian communities. DESIGN: A literature search was conducted, with the period between the publication of an 'Aboriginal Pharmacopoeia' in 1988 and 'current' our target interval for searching. The ethics of publishing knowledge belonging to Aboriginal people is briefly discussed in this context. RESULTS: Ailments were grouped into communicable diseases, pain and inflammation, skin disorders and gastrointestinal disorders. Although cancer is regarded as a disease of the 'white man', it is briefly discussed in the context that several traditional medicines and foods may have provided some protective effects. Where known, the ethnopharmacology of these medicines is discussed, as well as a brief description of their preparation and application. CONCLUSION: Some Indigenous Australians continue to use traditional medicines. We have tabulated these according to ailment, and have listed pharmacological actions where known. What is not known, however, is their potential to interact with pharmaceutical drugs. Further study in this area is needed to optimise health outcomes for Indigenous Australians, especially those in remote communities.

Three of a Kind: Genetically Similar Tsukamurella Phages TIN2, TIN3, and TIN4.

Three Tsukamurella phages, TIN2, TIN3, and TIN4, were isolated from activated sludge treatment plants located in Victoria, Australia, using conventional enrichment techniques. Illumina and 454 whole-genome sequencing of these Siphoviridae viruses revealed that they had similar genome sequences, ranging in size between 76,268 bp and 76,964 bp. All three phages shared 74% nucleotide sequence identity to the previously described Gordonia phage GTE7. Genome sequencing suggested that phage TIN3 had suffered a mutation in one of its lysis genes compared to the sequence of phage TIN4, to which it is genetically very similar. Mass spectroscopy data showed the unusual presence of a virion structural gene in the DNA replication module of phage TIN4, disrupting the characteristic modular genome architecture of Siphoviridae phages. All three phages appeared highly virulent on strains of Tsukamurella inchonensis and Tsukamurella paurometabola.

ZOLEDRONIC ACID THERAPY OF PATIENTS WITH PAGET DISEASE OF BONE RESISTANT TO OR WITH UNSUSTAINED REMISSION FOLLOWING PRIOR BISPHOSPHONATE THERAPY.

OBJECTIVE: To evaluate the effect of zoledronic acid (ZA) in patients with Paget disease (PD) who had not had a biochemical remission with prior bisphosphonate therapy or had a remission ≤12 months. METHODS: The effects of ZA therapy were studied in 14 patients aged 54 to 90. Serum alkaline phosphatase (ALP) levels were elevated to at least 40% above the normal reference range, and glomerular filtration rates (GFRs) were ≥40 mL/minute. ZA (5 mg) was infused over 15 minutes. ALP and urine N-telopeptide/creatinine (NTx/Cr) were obtained before therapy and at 3, 6, 9, and 12 months, and thereafter at 4-month intervals. RESULTS: At baseline, ALP ranged from 141 to 1,009 U/L. In 13 patients, ALP fell to normal following ZA administration. Remissions occurred in 9 patients who had not previously had a remission. Remissions varied from 12 to 60 months and were more prolonged in 4 patients with prior remissions ≤12 months. ZA failed to induce a remission in 1 patient. Ten to 12 days after therapy in 3 asymptomatic patients, serum calcium levels fell to 7.9, 8, and 8.3 mg/dL. Other than flu-like symptoms in 3 patients after ZA infusion, there were no other adverse effects. CONCLUSION: Therapy with ZA induced remissions in 13/14 patients and induced more prolonged remissions in patients who previously had remissions ≤12 months. The lack of remission in 1 patient despite 2 courses of therapy is evidence of a continuing therapeutic challenge for some patients with a more resistant form of PD.

Evaluation of bioactive compounds from Ficus carica L. leaf extracts via high-performance thin-layer chromatography combined with effect-directed analysis.

This study compares different solvent systems with the use of spontaneous fermentation on the phytochemical composition of leaf extracts from a locally grown white variety of common fig (Ficus carica Linn.). The aim was to detect and identify bioactive compounds that are responsible for acetylcholinesterase (AChE), α-amylase and cyclooxygenase-1 (COX-1) enzyme inhibition, and compounds that exhibit antimicrobial activity. Bioactive zones in chromatograms were detected by combining High-performance thin-layer chromatography (HPTLC) with enzymatic and biological assays. A new experimental protocol for measuring the relative half-maximum inhibitory concentration (IC50) was designed to evaluate the potency of the extracts compared to the potency of known inhibitors. Although the IC50 of the fig leaf extract for α-amylase and AChE inhibition were significantly higher when compared to IC50 for acarbose and donepezil, the COX-1 inhibition by the extract (IC50 = 627 µg) was comparable to that of salicylic acid (IC50 = 557 µg), and antimicrobial activity of the extract (IC50 = 375-511 µg) was similar to ampicillin (IC50 = 495 µg). Four chromatographic zones exhibited bioactivity. Compounds from detected bioactive bands were provisionally identified by comparing the band positions to coeluted standards, and by Fourier transform infrared (FTIR) spectra from eluted zones. Flash chromatography was used to separate selected extract into fractions and isolate fractions that are rich in bioactive compounds for further characterisation with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis. The main constituents identified were umbelliferon (zone 1), furocoumarins psoralen and bergapten (zone 2), different fatty acids (zone 3 and 4), and pentacyclic triterpenoids (calotropenyl acetate or lupeol) and stigmasterol (zone 4).

Still unseen and ignored: Tracking community knowledge and attitudes about child abuse and child protection in Australia.

In September 2003, we released the first results of a national community attitude tracking study about child abuse and child protection. At that time, we concluded that as a community, violence against children was tolerated. The community did not understand or appreciate the seriousness, size and cost of child abuse in Australia. There was evidence that child abuse was not viewed as an important challenge facing children in Australia. A second study conducted in 2006 found that nothing much had changed, indeed community engagement with the issue of child abuse may have even deteriorated. A third study in 2010 found that the community actively avoids the problem of child abuse rating it less concerning than high petrol prices. In 2021, 18 years after the first report was published, we have concluded again that child abuse remains out of sight and out of mind as a community concern. This article describes the findings of this fourth iteration of our survey and analyses the implications for ensuring that individuals are more engaged and committed to taking action to preventing child abuse and/or protecting children from violation.

The Microbiome in Pancreatic Cancer-Implications for Diagnosis and Precision Bacteriophage Therapy for This Low Survival Disease.

While the mortality rates for many cancers have decreased due to improved detection and treatments, that of pancreatic cancer remains stubbornly high. The microbiome is an important factor in the progression of many cancers. Greater understanding of the microbiome in pancreatic cancer patients, as well as its manipulation, may assist in diagnosis and treatment of this disease. In this report we reviewed studies that compared microbiome changes in pancreatic cancer patients and non-cancer patients. We then identified which bacterial genera were most increased in relative abundance across the oral, pancreatic, duodenal, and faecal tissue microbiomes. In light of these findings, we discuss the potential for utilising these bacteria as diagnostic biomarkers, as well as their potential control using precision targeting with bacteriophages, in instances where a causal oncogenic link is made.

Hyperparathyroidism in a Fracture Population.

Primary hyperparathyroidism (PHPT) is a common endocrine disorder that results in excess parathyroid hormone (PTH) secretion and hypercalcemia. PHPT is usually caused by an adenoma and its presentation is often asymptomatic, though it can negatively impact the skeleton via osteoporosis mostly affecting cortical bone and fracture. The diagnosis of PHPT is made by clinical presentation and biochemical and hormonal assessment. Surgical treatment guided by ultrasound sonography and/or 99mTc-sestamibi scintigraphy is generally curative. Normocalcemic hyperparathyroidism (NPHPT) is a variant of hyperparathyroidism defined by normal serum calcium and persistently elevated serum PTH levels. Limited data exist on NPHPT's effects on the skeleton, though current evidence suggests a positive correlation between the disorder and the presence of osteoporotic fractures. Taken together, patients affected by the various manifestations of hyperparathyroidism and their associated homeostatic disturbances represent a not insignificant portion of fracture patients seen in a fracture liaison service.

The bioprofiling of antibacterials in olive leaf extracts via thin layer chromatography-effect directed analysis (TLC-EDA).

In this study, effect-directed analysis (EDA) (i.e. TLC hyphenated with an in situ MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) antimicrobial assay), was used for screening and identification of antimicrobials in olive leaf extract. EDA detected that the same compounds exhibited significant antimicrobial activity against bacterial species of the genera Enterococcus (E. faecalis), Escherichia (E. coli), Streptococcus (S. mutans) and Staphylococcus (S. aureus). Flash chromatography-fractionation was used to isolate antimicrobial compounds in olive leaf extract. The active compounds were identified as maslinic acid and oleanolic acid by comparing RF values of the detected active bands with the standard reference materials, with identity confirmed with NMR and ATR-FTIR spectroscopy. Maslinic and oleanolic acids were tested on the E. faecalis strain (which displayed the highest sensitivity in the MTT assay) to determine their inhibiting concentration 50% (IC50) and minimum bactericidal concentrations.

Association between indigenous status and Body Mass Index (BMI) in Australian adults: Does sleep duration affect the relationship?

BACKGROUND: Overweight/obesity is a well-defined risk factor for a variety of chronic cardiovascular and metabolic diseases. Sleep duration has been associated with overweight/obesity and other cardio metabolic and neurocognitive problems. Notably, overweight/obesity and many of the associated comorbidities are prevalent in Indigenous Australians. Generally, sleep duration has been associated with BMI for Australian adults but information about Australian Indigenous adults' sleep is scant. A recent report established that sleep is a weak predictor of obesity for Indigenous Australian adults. AIM: To determine whether sleep remains a predictor of obesity when physical activity, diet and smoking status are accounted for; and to determine whether sleep duration plays a mediating role in the relationship between Indigenous status and BMI. METHODS: Statistical analyses of 5,886 Australian adults: 5236 non-Indigenous and 650 Indigenous people aged over 18 years who participated in the Australian Health Survey 2011-2013. Demographic and lifestyle characteristics were described by χ2 and t-tests. ANOVA was used to determine the variables that significantly predicted BMI and sleep duration. Stepwise regression analyses were performed to determine the strongest significant predictors of BMI. Sleep duration was self-reported; BMI was calculated from measurement. RESULTS: The study revealed two main findings: (i) short sleep duration was an independent predictor of obesity (adjusted-R2 = 0.056, p <0.0001); and (ii) controlling for sleep duration and other possible confounders, Indigenous status was a significant predictor of BMI overweight/obesity. Sleep duration played a weak, partial mediator role in this relationship. Increased BMI was associated with lower socioeconomic status and level of disadvantage of household locality for non-remote Indigenous and non-Indigenous people. CONCLUSION: Indigenous status strongly predicted increased BMI. The effect was not mediated by the socioeconomic indicators but was partially mediated by sleep duration.

Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

In situ profiling of microbial communities in full-scale aerobic sequencing batch reactors treating winery waste in australia.

On-site aerobic sequencing batch reactor (SBR) treatment plants are implemented in many Australian wineries to treat the large volumes of associated wastewater they generate. Yet very little is known about their microbiology. This paper represents the first attempt to analyze the communities of three such systems sampled during both vintage and nonvintage operational periods using molecular methods. Alphaproteobacterial tetrad forming organisms (TFO) related to members of the genus Defluviicoccus and Amaricoccus dominated all three systems in both operational periods. Candidatus 'Alysiosphaera europaea' and Zoogloea were codominant in two communities. Production of high levels of exocellular capsular material by Zoogloea and Amaricoccus is thought to explain the poor settleability of solids in one of these plants. The dominance of these organisms is thought to result from the high COD to N/P ratios that characterize winery wastes, and it is suggested that manipulating this ratio with nutrient dosing may help control the problems they cause.