Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Determination and localisation of in situ substrate uptake by anaerobic wastewater treatment granular biofilms.

Radiotracer incubation experiments and beta microimaging, along with fluorescent in situ hybridisation (FISH), are proposed as a complementary approach to specific methanogenic activity testing and measurement of in vitro substrate utilisation rates to understand better the ecophysiology of anaerobic granular biofilms from wastewater treatment reactors.

High nitrification rate at low pH in a fluidized bed reactor with chalk as the biofilm carrier.

A typical steady state bulk pH of about 5 was established in a nitrifying fluidized bed with chalk as the only buffer agent. In spite of the low pH, high rate nitrification was observed with the nitrification kinetic parameters in the chalk reactor similar to those of biological reactors operating at pH>7. Various methods were used to determine the reasons for high rate nitrification at such low pH including (i) determination of bacterial species, (ii) microsensor measurements in the biofilm, and (iii) comparison of nitrification performance at low pH with a non-chalk fluidized bed reactor. Fluorescence in situ hybridization (FISH) using existing 16S rRNA-targeted oligonucleotide probes showed common nitrifying bacteria in the low pH chalk reactor. The prevalent nitrifying bacteria were identified in the Nitrosomonas oligotropha, Nitrosomonas europeae/eutropha, Nitrosospira and Nitrospira related groups, all well known nitrifiers. Microelectrode measurements showed that the pH in the biofilm was low and similar to that of the bulk pH. Finally, reactor performance using a non-chalk biofilm carrier (sintered glass) with the same bacterial inoculum also showed high rate nitrification below pH 5. The results suggest that inhibition of nitrification at low pH is highly overestimated.

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm.

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.

A marine microbial consortium apparently mediating anaerobic oxidation of methane.

A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm.

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea-like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1-2 orders of magnitude lower than those of N. europaea. Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic-anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.