The effect of interactions between a bacterial strain isolated from drinking water and a pathogen surrogate on biofilms formation diverged under static vs flow conditions.

AIMS: Interactions with water bacteria affect the incorporation of pathogens into biofilms and thus pathogen control in drinking water systems. This study was to examine the impact of static vs flow conditions on interactions between a pathogen and a water bacterium on pathogen biofilm formation under laboratory settings. METHODS AND RESULTS: A pathogen surrogate Escherichia coli and a drinking water isolate Stenotrophomonas maltophilia was selected for this study. Biofilm growth was examined under two distinct conditions, in flow cells with continuous medium supply vs in static microtitre plates with batch culture. E. coli biofilm was greatly stimulated (c. 2-1000 times faster) with the presence of S. maltophilia in flow cells, but surprisingly inhibited (c. 65-95% less biomass) in microtitre plates. These divergent effects were explained through various aspects including surface attachment, cellular growth, extracellular signals and autoaggregation. CONCLUSIONS: Interactions with the same water bacterium resulted in different effects on E. coli biofilm formation when culture conditions changed from static to flow. SIGNIFICANCE AND IMPACT OF STUDY: This study highlights the complexity of species interactions on biofilm formation and suggests that environmental conditions such as the flow regime can be taken into consideration for the management of microbial contamination in drinking water systems.

Quantification of genes and gene transcripts for microbial perchlorate reduction in fixed-bed bioreactors.

AIMS: Optimization of full-scale, biological perchlorate treatment processes for drinking water would benefit from knowledge of the location and quantity of perchlorate-reducing bacteria (PRB) and expression of perchlorate-related genes in bioreactors. The aim of this study was to quantify perchlorate removal and perchlorate-related genes (pcrA and cld) and their transcripts in bioreactors and to determine whether these genes or transcripts could serve as useful biomarkers for perchlorate treatment processes. METHODS AND RESULTS: Quantitative PCR (qPCR) assays targeting pcrA and cld were applied to two pilot-scale, fixed-bed bioreactors treating perchlorate-contaminated groundwater. pcrA and cld genes per microgram of DNA were two- to threefold higher and three- to fourfold higher, respectively, in the bioreactor showing superior perchlorate-removal performance. In a laboratory-scale bioreactor, quantities of pcrA and cld genes and transcripts were compared under two distinct performance conditions (c.60 and 20% perchlorate removal) for a 5-min empty bed contact time. cld genes per microgram of DNA were approximately threefold higher and cld transcripts per microgram of RNA were approximately sixfold higher under the higher perchlorate-removal condition. No differences in pcrA genes or transcripts per microgram of DNA or RNA, respectively, were detected between the c.60 and 20% perchlorate-removal conditions, possibly because these assays did not accurately quantify pcrA genes and transcripts in the mixed culture present. CONCLUSIONS: Quantities of cld genes and transcripts per microgram of DNA and RNA, respectively, were found to be higher when perchlorate removal was higher. However, quantities of pcrA and cld genes or transcripts were not found to directly correlate with perchlorate-removal rates. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this study represents the first application of qPCR assays to quantify perchlorate-related genes and transcripts in continuous-flow bioreactors. The results indicate that cld gene and transcript quantities can provide insights regarding the quantity, location and gene expression of PRB in bioreactors.

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters.

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.

Characterization of two Ashkenazi Jewish founder mutations in MSH6 gene causing Lynch syndrome.

Founder mutations are an important cause of Lynch syndrome and facilitate genetic testing in specific ethnic populations. Two putative founder mutations in MSH6 were analyzed in 2685 colorectal cancer (CRC) cases, 337 endometrial cancer (EnCa) cases and 3310 healthy controls of Ashkenazi Jewish (AJ) descent from population-based and hospital-based case­control studies in Israel, Canada and the United States. The carriers were haplotyped and the age of the mutations was estimated. MSH6*c.3984_3987dupGTCA was found in 8/2685 CRC cases, 2/337 EnCa cases, and 1/3310 controls, consistent with a high risk of CRC (odds ratio (OR) = 9.9, 95% confidence interval (CI) = 1.2­78.9, p = 0.0079) and a very high risk of EnCa (OR = 19.6, 95% CI = 1.8­217.2, p = 0.0006). MSH6*c.3959_3962delCAAG was identified in 3/2685 CRC cases, 2/337 EnCa cases and no controls. Each mutation was observed on separate conserved haplotypes. MSH6*c.3984_3987dupGTCA and MSH6*c.3959_3962delCAAG probably arose around 585 CE and 685 CE, respectively. No carriers were identified in Sephardi Jews (450 cases and 490 controls). Truncating mutations MSH6*c.3984_3987dupGTCA and MSH6*c.3959_3962delCAAG cause Lynch syndrome and are founder mutations in Ashkenazi Jews. Together with other AJ founder mutations, they contribute substantially to the incidence of CRC and EnCa and are important tools for the early diagnosis and appropriate management of AJ Lynch syndrome patients.

Characterization of microbial trophic structures of two anaerobic bioreactors processing sulfate-rich waste streams.

A multi-compartment anaerobic bioreactor, designated the anaerobic migrating blanket reactor (AMBR), did not perform well in terms of chemical oxygen demand (COD) removal after an increase in sulfate load, compared to a conventional upflow anaerobic sludge blanket (UASB) reactor. The trophic structures of the bioreactors were characterized by analyzing the electron flows, formation and consumption of fermentation intermediates and terminal product (methane and hydrogen sulfide) formation. Critical performance parameters were linked to operational perturbations such as increase in sulfate load and changes in flow reversal schemes in the AMBR. Both of these manipulations affected the microbial communities, which were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the bacterial and archaeal domains. The less stable AMBR did not produce granular biomass, and in response to increased sulfate concentrations, experienced a reversal in the distribution of hydrogenotrophic methanogens that correlated with a shift in electron flow from butyrate to propionate. As this shift occurred, bacterial populations such as butyrate-producing clostridia, became predominant, thus leading to reactor imbalance. The stable UASB reactor developed and retained granules and maintained a relatively stable archaeal community. Sulfate perturbation led to the selection of a novel bacterial group (Thermotogaceae), which was most likely well adapted to the increasingly sulfidogenic conditions in the bioreactor.

Modelling the effect of the antimicrobial tylosin on the performance of an anaerobic sequencing batch reactor.

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was fed a synthetic wastewater containing glucose to study the effects of the antimicrobial tylosin on treatment performance. Measurements of methane, volatile fatty acids, and COD concentrations suggested that the addition of 1.67 mg/L and 167 mg/l of tylosin to the synthetic wastewater inhibited propionate oxidizing syntrophic bacteria and aceticlastic methanogens. The latter is presumed to be an indirect effect. A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for microbial storage and hydrolysis of reserve carbohydrates, and tylosin liquid-solid mass transfer and inhibition adequately described the dynamic profiles observed in the ASBR.

Hydrogen production by anaerobic microbial communities exposed to repeated heat treatments.

Biological hydrogen production by anaerobic mixed communities was studied in laboratory-scale bioreactors using sucrose as the substrate. A bioreactor in which a fraction of the return sludge was exposed to repeated heat treatments performed better than a control bioreactor without repeated heat treatment of return sludge and produced a yield of 2.15 moles of hydrogen per mole of sucrose, with 50% hydrogen in the biogas. Terminal restriction fragment length polymorphism analysis showed that two different Clostridium groups (comprised of one or more species) were dominant during hydrogen production. The relative abundance of two other non-Clostridium groups increased during periods of decreased hydrogen production. The first group consisted of Bifidobacterium thermophilum, and the second group included one or more of Bacillus, Melissococcus, Spirochaeta, and Spiroplasma spp.

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes.

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.

Molecular ecology of anaerobic reactor systems.

Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and constitute a core facility in many waste treatment plants. Although much is known about the basic metabolism in different types of anaerobic reactors, little is known about the microbes responsible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms. This lack of knowledge is most clearly exemplified by the sometimes unpredictable and unexplainable failures and malfunctions of anaerobic digesters occasionally experienced, leading to sub-optimal methane production and wastewater treatment. Using a variety of molecular techniques, we are able to determine which microorganisms are active, where they are active, and when they are active, but we still need to determine why and what they are doing. As genetic manipulations of anaerobes have been shown in only a few species permitting in-situ gene expression studies, the only way to elucidate the function of different microbes is to correlate the metabolic capabilities of isolated microbes in pure culture to the abundance of each microbe in anaerobic reactor systems by rRNA probing. This chapter focuses on various molecular techniques employed and problems encountered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester systems.

Development of a sustainable treatment technology for domestic sewage.

We propose an integrated chemical-physical-biological treatment concept for the low-cost treatment of domestic wastewater. Domestic wastewater was subjected to a chemically enhanced primary sedimentation (CEPS), followed by treatment in an upflow anaerobic sludge blanket (UASB) reactor. In addition, a regenerable zeolite was used to remove NH4+, either after CEPS pretreatment or after biological treatment in the UASB reactor. The CEPS pretreatment consisted of the addition of a coagulant (FeCl3) and an anionic organic flocculant and removed on average 73% of the total chemical oxygen demand (CODt), 85% of the total suspended solids, and 80% of PO4(3-) present in the wastewater. The UASB system, which consequently received a low CODt input of approximately 140 mg/L, was operated using a volumetric loading rate of 0.4 g CODt/L.d (hydraulic retention time [HRT] = 10 h) and 0.7 g CODt/L.d (HRT = 5 h). For these conditions, the system removed about 55% of the CODt in its influent, thus producing an effluent with a low CODt of approximately 50 mg/L. The zeolite, when applied in batch mode before the UASB reactor, removed approximately 45% of the NH4+, whereas its application as a post-treatment cartridge resulted in almost 100% NH4+ removal. The simple design and relatively low operating costs, due to low costs of added chemicals and low energy input (estimated at Euro 0.07-0.1 per m3 wastewater treated), combined with excellent treatment performance, suggest that this system can be used as a novel domestic wastewater treatment system for developing countries. Therefore, the system is called a Low-cost, Integrated Sewage Treatment (LIST) system.

Who eats what? Classifying microbial populations based on diurnal profiles of rRNA levels.

Identifying the relationships between various bacterial populations and the substrates they consume is central to the understanding of population dynamics and to the development of process control in activated sludge. However, linking a heterotrophic population to its activity in situ is difficult because ribosomal RNA (rRNA) techniques, while allowing the rapid identification of populations, provide little information about their heterotrophic activity. Activated sludge models describe biodegradation kinetics by classifying substrates into two types: readily and slowly degradable substrates. Assuming that bacterial populations specialize in degrading one type of substrate, their growth rate should be affected differently if the COD loading rate varies diurnally as for a municipal activated sludge system. Modeling results suggested that the growth rates of populations consuming readily degradable substrates vary according to variations in COD loading rate. On the other hand, the growth rates of populations consuming slowly degradable substrates do not change despite the variation in COD loading rate. Since the cellular rRNA level is positively correlated with the growth rate, we hypothesized that the rRNA levels of some populations in municipal activated sludge should increase throughout the day, while they should stay constant for other populations. This hypothesis was verified by monitoring the rRNA level of Acinetobacter (a model population consuming readily degradable substrates) and Gordonia (a model population consuming slowly degradable substrates) in the mixed liquor of a full-scale municipal activated sludge reactor for three weeks.

Quantifying the impact of wastewater micronutrient composition on in situ growth activity of Acinetobacter spp.

Batch growth studies with pure cultures of Acinetobacter johnsoniiT and Acinetobacter johnsonii strain 210 on various media formulations were used to examine the effects of micronutrient composition on the growth rate of microbial populations in wastewater treatment systems. On nutrient rich Luria-Bertani medium, both strains of A. johnsonii grew with a doubling time of approximately 30 min. On a defined, minimal medium with acetate as the sole carbon source, the doubling time of A. johnsoniiT was 9.62 h and the doubling time of strain 210a was 2.6 h. Using a synthetic wastewater as a growth medium, the type strain had a doubling time of 56 h and strain 210a had a doubling time of 9.62 h. The concentration and redox state of iron appeared to be the principle growth limiting factors with higher doubling times occurring in media containing ferric iron as compared to ferrous iron. Additionally, grab samples from batch growth experiments were analyzed with oligonucleotide hybridization probes targeting the mature 16S ribosomal RNA (rRNA) and precursor 16S rRNA of Acinetobacter spp. Results showed that the precursor 16S rRNA levels responded more rapidly and to a greater extent than total 16S rRNA levels to changes in the micronutrient composition of the growth media. Precursor 16S rRNA levels increased in both strains when overnight cultures were diluted with fresh media and when micronutrient supplements were added to growing cultures. Our results show that the micronutrient composition of the influent wastewater can have a significant impact on the microbial community structure in wastewater treatment systems.

Oligonucleotide probe hybridization and modeling results suggest that populations consuming readily degradable substrate have high cellular RNA levels.

Analyses based on ribosomal RNA (rRNA)-targeted hybridization performed in our laboratory identified two types of bacterial populations: a population with a high RNA level per biomass and a population with a low level of RNA per biomass. To extend these descriptions, the diurnal dynamics of the RNA pool were monitored by rRNA-targeted oligonucleotide probe membrane hybridization. Under the typical diurnal variation in COD loading rate experienced by municipal wastewater treatment plants, the RNA level of the bacterial population with a high level of RNA per biomass varied with changes in the COD loading rate. Under the same conditions, the RNA level of the population with low RNA level per biomass remained constant. A structured biomass model was developed to describe these data. Substrate COD was divided into a readily biodegradable and a slowly biodegradable COD fraction. It was assumed that two specialized populations coexist in municipal activated sludge treatment systems. One population consumes readily degradable COD and the other consumes slowly degradable COD. According to model simulations, the population consuming readily degradable COD has a high level of RNA per biomass under variable substrate concentrations. Comparatively, the population consuming slowly degradable COD has a low level of RNA level per biomass. Furthermore, model simulations reproduced the two diurnal RNA profiles observed in a full-scale municipal activated sludge system. Therefore, we suggest that two populations can be distinguished in municipal activated sludge systems: a population consuming readily degradable substrate and a population consuming slowly degradable substrate.

Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--I. Digester performance.

The feasibility of codigestion of the organic fraction of municipal solid waste, primary sludge, and waste activated sludge was evaluated in mesophilic (37 degrees C), laboratory-scale digesters. In a first experiment, different startup strategies were compared using four digesters, operated under continuously mixed conditions. After two weeks, the experiment was continued under minimally mixed conditions. Results demonstrated that reducing the level of mixing improved digester performance. Therefore, in a second experiment, six digesters were operated to compare performance under continuous mixing and reduced mixing levels at various loading rates and solids levels. The continuously mixed digesters exhibited unstable performance at the higher loading rates, while the minimally mixed digesters performed well for all loading rates evaluated. In a third experiment, it was demonstrated that an unstable, continuously mixed digester was quickly stabilized by reducing the mixing level. These experiments confirmed that continuous mixing was not necessary for good performance and was inhibitory at higher loading rates. In addition, reduction of mixing levels may be used as an operational tool to stabilize unstable digesters.

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--II: Microbial population dynamics.

Microbial population dynamics were evaluated in anaerobic codigesters treating municipal solid waste and sewage sludge. Ribosomal RNA based oligonucleotide probes were used to characterize changes in population abundance of syntrophic volatile fatty acid degrading bacteria and methanogens. Changes in community structure were linked to traditional performance parameters during the recovery of previously unstable codigesters induced by a reduction in mixing levels. Methanosarcina spp. were the most abundant aceticlastic methanogens in unstable codigesters with high acetate concentrations, while Methanosaeta concilii was dominant in stable systems with low levels of acetate. Growth of Syntrophobacter wolinii was enhanced during stabilization of a codigester with a well-developed population of Methanobacteriaceae, possibly because the presence of adequate numbers of these hydrogenotrophic methanogens encouraged the syntrophic oxidation of propionate. Mesophilic saturated fatty acid beta-oxidizing syntrophs were most abundant in previously unstable codigesters. One minimally mixed reactor became unstable after switching to continuously mixed conditions. After the switch, total archaeal abundance decreased sharply, though Methanobacteriaceae and Methanosarcina spp. levels increased as the fermentation became unbalanced. Based on the results presented here, mixing appears to inhibit the syntrophic oxidation of volatile fatty acids, possibly by disrupting the spatial juxtaposition of syntrophic bacteria and their methanogenic partners.

Water quality factors affecting bromate reduction in biologically active carbon filters.

Biological removal of the ozonation by-product, bromate, was demonstrated in biologically active carbon (BAC) filters. For example, with a 20-min EBCT, pH 7.5, and influent dissolved oxygen (DO) and nitrate concentrations 2.1 and 5.1 mg/l, respectively, 40% bromate removal was obtained with a 20 microg/l influent bromate concentration. In this study, DO, nitrate and sulfate concentrations, pH, and type of source water were evaluated for their effect on bromate removal in a BAC filter. Bromate removal decreased as the influent concentrations of DO and nitrate increased, but bromate removal was observed in the presence of measurable effluent concentrations of DO and nitrate. In contrast, bromate removal was not sensitive to the influent sulfate concentration, with only a slight reduction in bromate removal as the influent sulfate concentration was increased from 11.1 to 102.7 mg/l. Bromate reduction was better at lower pH values (6.8 and 7.2) than at higher pH values (7.5 and 8.2), suggesting that it may be possible to reduce bromate formation during ozonation and increase biological bromate reduction through pH control. Biological bromate removal in Lake Michigan water was very poor as compared to that in tapwater from a groundwater source. Bromate removal improved when sufficient organic electron donor was added to remove the nitrate and DO present in the Lake Michigan water, indicating that the poor biodegradability of the natural organic matter may have been limiting bromate removal in that water. Biological bromate removal was demonstrated to be a sustainable process under a variety of water quality conditions, and bromate removal can be improved by controlling key water quality parameters.

The presence of humic substances and DNA in RNA extracts affects hybridization results.

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.

Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose.

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).

Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus.

About 60% of congenital hearing impairment cases in developed countries are due to genetic defects. Data on the molecular basis of hereditary hearing reflects vast genetic heterogeneity. There are >400 disorders in which hearing impairment is one of the characteristic traits of a syndrome. Linkage studies have identified more than 40 human chromosomal loci associated with non-syndromic hearing loss. So far, 16 of these 40 non-syndromic hearing impairment genes have been identified. We have studied the molecular basis of hearing impairment in four Druze families from the same village in Northern Galilee. The Druze are a small, isolated population in the Middle East practising endogamous marriage. Thus it was expected that a single mutation would account for hearing impairments in all these families. Our results show that at least four different genes are involved. Hearing impairment was caused in one family by a novel mutation in the recently identified OTOF (the DFNB9 gene), by a novel Pendred syndrome mutation (Thr193Ile) in another family, and by a GJB2 mutation (35delG also known as 30delG) in the third family. In the fourth family linkage was excluded from all known hearing impairments loci (recessive and dominant) as well as from markers covering chromosomes 11-22, pointing therefore to the existence of another non-syndromic recessive hearing loss (NSRD) locus on chromosomes 1-10.