Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

Control of biofouling on seawater reverse osmosis (SWRO) membranes is a major challenge as treatments can be expensive, damage the membrane material and often biocides do not remove the polymers in which bacteria are embedded. Biological control has been largely ignored for biofouling control. The objective of this study was to demonstrate the effectiveness of xanthine oxidase enzyme against complex fouling communities and then identify naturally occurring bacterial strains that produce the free radical generating enzyme. Initially, 64 bacterial strains were isolated from different locations of the Perth Seawater Desalination Plant. In our preceding study, 25/64 isolates were selected from the culture collection as models for biofouling studies, based on their prevalence in comparison to the genomic bacterial community. In this study, screening of these model strains was performed using a nitroblue tetrazolium assay in the presence of hypoxanthine as substrate. Enzyme activity was measured by absorbance. Nine of 25 strains tested positive for xanthine oxidase production, of which Exiguobacterium from sand filters and Microbacterium from RO membranes exhibited significant levels of enzyme production. Other genera that produced xanthine oxidase were Marinomonas, Pseudomonas, Bacillus, Pseudoalteromonas and Staphylococcus. Strain variations were observed between members of the genera Microbacterium and Bacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes. Bacterial xanthine oxidase production in the presence of hypoxanthine may prove to be a cost effective, in situ method for alleviation of fouling.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater.

Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 degrees C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen.

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.

Isolation and identification of ruminal methanogens from grazing cattle.

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H(2)/CO(2) and formate, and optimally at 38 degrees C and pH 6.5-7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H(2)/CO(2), acetate, and methyl-containing compounds, with optimal growth at 40 degrees C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management.

The role of exopolysaccharides in dual species biofilm development.

A plasmid encoding the green fluorescent protein (GFP) of Aequorea victoria was transformed into a biofilm-forming strain of Enterobacter agglomerans originally isolated from an industrial environment. The transformed strain, EntGFP, could then be identified in dual species biofilms by direct visualization, plate counts and quantitiative fluorescence measurements. A variety of cell constituents and products may be involved in the adhesion and accumulation process and exopolysaccharides (EPS) represent one of these factors. The involvement of EPS in the initial adhesion events and the role in dual species biofilm development was investigated. Cells of EntGFP and Klebsiella pneumoniae Gl interact forming biofilms more successfully in a mixture than in isolation. The co-resistance results in enhanced biofilm formation and increased resistance to disinfection. Microscopic examination showed that the two species were often closely juxtaposed in microcolonies, suggesting the interactions involve surface-associated macromolecules. Fluorescence was used to measure the adhesion of EntGFP cells to Kleb, pneumoniae Gl (Gl) EPS. The results showed EntGFP adhered better to Gl EPS that Ent EPS. Polysaccharde depolymerases isolated from a bacteriophage for Ent. agglomerans were used to degrade Ent EPS specifically. Following polysaccharase treatment, the adhaesion of EntGFP to Gl cells was reduced. This suggests both types of EPS mediate adhesion. The two types of EPS were dissolved in dimethylsulphoxide and when mixed, their viscosity increased, reaching a maximum after ∼+40 min. This may partially explain the increased protection of dual species biofilms from disinfectants. The depolymerases were used to treat dual species biofilms and this resulted in the effective removal of both species from the surface. This may suggest Ent contributes more EPS to the biofilm matrix. The EPS play an important role in EntGFP and Gl dual species biofilm formation both as adhesins and as the EPS interact, changing their physical properties.

Isolation of nontuberculous, non-avium mycobacteria from patients infected with human immunodeficiency virus.

Mycobacterium avium serovars account for 97% of typeable M. avium complex (MAC) organisms causing infection in patients with AIDS. We reviewed 216 consecutive cultures that yielded nontuberculous mycobacteria (NTM) from 212 patients. Only the first isolate of each species of NTM recovered from each patient was analyzed in the study. Among the 92 patients infected with the human immunodeficiency virus, 96 NTM organisms were identified; M. avium was recovered from 50 (77%) of the 65 NTM-positive cultures of blood or bone marrow, while Mycobacterium intracellular and other non-avium NTM accounted for 18% and 5% of the isolates, respectively. Little difference in the susceptibility of isolates to antibiotics was noted between HIV-positive and HIV-negative patients or between M. avium and M. intracellulare. These data demonstrate that HIV-positive patients develop disseminated disease with NTM other than M. avium more frequently than has been previously reported and that these patients do not appear to be infected with NTM that are more resistant to antimicrobial agents than are NTM isolated from HIV-negative patients.

Infection due to Mycobacterium haemophilum identified by whole cell lipid analysis and nucleic acid sequencing.

A patient with indolent, non-Hodgkin's lymphoma developed a pretibial soft tissue abscess caused by a fastidious mycobacterium. Because the organism could not be definitively identified by standard microbiologic testing, whole cell fatty acid analysis and 16S rDNA sequencing were performed. These procedures identified the organism as Mycobacterium haemophilum. We review the diagnostic considerations with regard to this pathogen.

In vitro susceptibility of clinical isolates of Mycobacterium avium and M. intracellulare to folate antagonists.

An agar dilution method against trimethoprim, sulfamethoxazole, sulfisoxazole, and trimethoprim-sulfamethoxazole was used to test clinical isolates of Mycobacterium intracellulare (MI) and M. avium (MA) from both HIV-infected and non-infected patients. MI and MA isolates demonstrated similar susceptibility data and were inhibited by concentrations of sulfamethoxazole achievable in serum.

Rapid diagnosis of coccidioidomycosis with a DNA probe to ribosomal RNA.

BACKGROUND: Although nucleic acid probe hybridization assays were previously exclusively used as a tool in the research setting, such assays have recently become commercially available for the detection of a variety of infectious microorganisms. OBSERVATIONS: We used a commercially available DNA hybridization probe test that targets organism-specific ribosomal RNA sequences to rapidly diagnose a patient with disseminated coccidioidomycosis. The natural amplification inherent to such DNA:RNA probe systems obviates the need for electrophoretic separation and amplification steps, which are often required in more traditional DNA:DNA probe assays. With this probe, culture confirmation was obtained within 48 hours after the clinical specimens were received. CONCLUSION: Rapid DNA hybridization probe techniques have wide application in infectious diseases, especially those characterized by slow culture growth of pathogens such as deep fungi and atypical mycobacteria.

"You'll feel me touching you, sweetie": vaginal examinations during the second stage of labor.

Videotapes of women during the second stage of labor and their caregivers were examined in this descriptive study to determine how caregivers performed sterile vaginal examinations. Conversational analysis techniques were used to analyze the data. Results showed that the examinations were performed in a ritualistic manner by all caregivers, and the way the ritual was enacted repeatedly demonstrated the power of the caregivers over the women. The most common reason for performing the procedure, to help the woman push better, seems to be specific to the second stage of labor and is not described in the literature. The authors offer suggestions for better use of the examination, and recommend that it be performed far less frequently than is common.