Sorption of Cellulases in Biofilm Enhances Cellulose Degradation by \u3ci\u3eBacillus subtilis\u3c/i\u3e

Biofilm commonly forms on the surfaces of cellulosic biomass but its roles in cellulose degradation remain largely unexplored. We used Bacillus subtilis to study possible mechanisms and the contributions of two major biofilm components, extracellular polysaccharides (EPS) and TasA protein, to submerged biofilm formation on cellulose and its degradation. We found that biofilm produced by B. subtilis is able to absorb exogenous cellulase added to the culture medium and also retain self-produced cellulase within the biofilm matrix. The bacteria that produced more biofilm degraded more cellulose compared to strains that produced less biofilm. Knockout strains that lacked both EPS and TasA formed a smaller amount of submerged biofilm on cellulose than the wild-type strain and also degraded less cellulose. Imaging of biofilm on cellulose suggests that bacteria, cellulose, and cellulases form cellulolytic biofilm complexes that facilitate synergistic cellulose degradation. This study brings additional insight into the important functions of biofilm in cellulose degradation and could potentiate the development of biofilm-based technology to enhance biomass degradation for biofuel production

Acoustically evoked potentials in two cephalopods inferred using the auditory brainstem response (ABR) approach

It is still a matter of debate whether cephalopods can detect sound frequencies above 400 Hz. So far there is no proof for the detection of underwater sound above 400 Hz via a physiological approach. The controversy of whether cephalopods have a sound detection ability above 400 Hz was tested using the auditory brainstem response (ABR) approach, which has been successfully applied in fish, crustaceans, amphibians, reptiles and birds. Using ABR we found that auditory evoked potentials can be obtained in the frequency range 400 to 1500 Hz (Sepiotheutis lessoniana) and 400 to 1000 Hz (Octopus vulgaris), respectively. The thresholds of S. lessoniana were generally lower than those of O. vulgaris

Development of a Probe Hybridization Method to Facilitate Detection of Noroviruses in Oysters

Centers for Disease Control (CDC) reports that at least 50% of all foodborne outbreaks of gastroenteritis are due to noroviruses (NoV). Since NoV is mainly transmitted through the fecal-oral route and the infectious dose may be as low as 10 viral particles, the risk of infection after consumption of raw or improperly cooked seafood or after exposure to contaminated water is considered high. Although highly sensitive methods to detect NoV using RT-PCR are already available, isolation of either NoV RNA or virions from shellfish remains a cumbersome process. We developed a new hybridization method to extract NoV RNA from contaminated shellfish that is much faster compared to existing methods. Using the new method, NoV detection includes three basic steps: an initial extraction of total RNA using TRIZol, followed by isolation of NoV RNA using biotinylated DNA probe hybridization and then NoV detection by TaqMan RT-PCR. With oyster (Crassostrea virginica) homogenate spiked with 100 PCR detection units (PDU) of NoV, the virus can be detected with CT values at about 30. Compared to published methods that require an initial virus purification step, the new method is much faster, requiring approximately 3 hr compared to at least 8 hr using conventional methods. Coupled with TaqMan RT-PCR, the new method can be used to detect NoV in contaminated oysters and clams (Corbicula fluminea) within 8 hr. The detection limit was 100 PDU of NoV in spiked oyster tissue samples. The method has been successfully used to detect NoV in oysters artificially contaminated in the laboratory and in rare cases, oysters collected from the field

A Comparison of Methods to Concentrate Viruses from Environmental Waters Using MS2 as a Model

Viruses such as Rotavirus, Adenovirus and Norovirus are important etiological agents of gastroenteritis worldwide. With the high sensitivity and specificity of PCR, it is now possible to develop PCR-based methods to detect and quantify pathogenic viruses in environmental water samples. To develop reliable methods however, an effective procedure to concentrate viruses from large volumes of water is required. Because of the scale of concentration required, the procedure often requires two steps. The first to reduce tens of liters of water to less than half a liter and then a second to concentrate the sample to a final volume of less than 10 mL for RNA/DNA extraction. The objectives of the study were to compare the efficacy of hollow fiber ultrafiltration (HFUF) using F200B to that of an adsorption/elution method (AEM) using positively charged filters for concentrating viruses for the first step and to compare polyethylene glycol (PEG) precipitation to centrifugal ultrafiltration for the second step. A third objective was to determine the viral detection limit using real-time RT-PCR. Using beach water spiked with a singlestranded RNA bacteriophage (MS2) as a model, our results show a virus recovery rate of 84±6% and 18±8% for the HFUF method and AEM, respectively. For the second concentration step, we obtained a recovery rate of 49±5 % and 87±7% using PEG precipitation and centrifugal ultrafiltration, respectively. A potential limiting factor to more widespread using of HFUF is the higher cost and we found that cost can be reduced by using reusable filters. We were able to sanitize and reuse the same filter at least six times without affecting the virus recovery rate or the processing time

Evaluation of Methanobrevibacter smithii as a Human-Specific Marker of Fecal Pollution

Microbial source tracking has historically focused on the origin of traditional enteric indicators including coliforms, enterococci, or Escherichia coli. Recently, questions of genetic variability and environmental persistence have encouraged researchers to search for additional animal specific indicators of fecal pollution. To date only eubacteria have been utilized as markers of human and animal-specific pollution. In this study we developed a molecular primer pair specific for Methanobrevibacter smithii, a methanogen found only in the human intestine. PCR primers for the nifH gene of M. smithii were designed, tested, and used to detect the presence or absence of the organism in fecal and environmental samples. Product amplification was observed in 28.6% of all human fecal samples and 93% of sewer samples, and water contaminated with human sewage. No amplification was observed when primers were tested against 43 bacterial stock cultures and fecal samples from 204 animals. Sequencing of PCR products from sewers and M. smithii cells demonstrated that the 222bp product amplified was the nifH gene of M. smithii. Sensitivity assays demonstrated a detection limit of 10ng in human feces, 10ng in fecally contaminated water, and 5 ng in sewer samples. In addition, samples seeded with M. smithii established a lower detection limit of 13 cells/ml. The Mnif method for M. smithii detection appears to be a rapid, inexpensive, and reliable test for determining the presence or absence of human fecal pollution in recreational waters

Methanogens as Ruminant-Specific Indicators of Fecal Pollution

Tracking the source of fecal pollution in surface waters has traditionally focused on the origin of enteric indicators including coliforms, enterococci, or E. coli. Recently, questions of genetic variability and environmental persistence have encouraged researchers to investigate additional animal specific indicators of fecal pollution. To date only eubacteria have been utilized as markers of human and animal-specific pollution. Here we report domestic ruminant-specific markers of fecal pollution utilizing methanogens found in the rumen. PCR primers for the mcrA gene of Methanomicrobium mobile (MMmcrA) and the nifH gene of Methanobrevibacter ruminantium (Mrnif-1) were designed, tested, and used to detect ruminant-specific pollution in fecal and environmental samples. The MMmcrA amplicons were detected in expected fecal and environmental samples (71% cow, 92% sheep, 50% goat, 100% cow lagoon samples, and 100% creek contaminated with cow lagoon waste), and were observed in only 5% of human and deer fecal samples. Mrnif-1 amplification was seen in 80% cow, 100% sheep, and 54% goat fecal samples; only 2% individual human samples were positive. No PCR amplification was observed when the MMmcrA and Mrnif-1 primers were tested against 47 bacterial stock cultures and fecal samples from 134 non-ruminant animals. More importantly, no amplification was observed in sewer samples using either primer pair. Sensitivity assays using MMmcrA primers demonstrated a detection limit of 0.01ng total DNA in bovine feces, 10ng in fecally contaminated surface water, and 5ng in cow lagoon samples

Detection of Salmonella spp. in Recreational Waters Using a Real-Time PCR Assay

The U.S. Environmental Protection Agency water quality guidelines for assessing bacteriological recreational water quality is currently based on Escherichia coli and enterococci counts as indicators of the presence of fecal pollution. Although these indicator organisms have been widely used, detection of specific pathogens rather than their surrogates may be preferable in the future for assessing health risks. This necessitates development of rapid, sensitive and reliable methods for pathogen detection in environmental waters. A quantitative PCR-based assay for detecting Salmonella spp., an important enteric pathogen, in recreational water and beach sediment samples was developed in the present study. The assay is based on the amplification of a 172 bp fragment in the invA gene which is quantified using a molecular beacon specific for Salmonella. The assay tests for the presence of Salmonella in 50 ml of environmental water samples and relies on the use of a 6 hr culture enrichment procedure. It has a lower detection limit of one CFU per mL and works within the 1 to 10 6 CFU per mL concentration range tested thus far. Due to the possible presence of PCR inhibitors in environmental samples, an internal amplification control, using the same primers but a different molecular beacon target sequence, was created to be included in each amplification reaction to distinguish true from false negative results. Preliminary studies reveal the presence of Salmonella in several coastal water samples along the northern Gulf of Mexico

Overcoming the Inhibitor Problem in PCR Detection of Salmonella in Recreational Waters: A Volume Based Approach

Fecal contamination of environmental or recreational waters is a world wide health concern. Methods currently endorsed by the U.S. EPA for monitoring fecal contamination are based on plate counts of fecal surrogates such as E. coli or Enterococci spp. Due to the sensitivity and specificity of PCR, there is growing interest in using PCR-based methods to detect and quantitate pathogens directly. However, a major impediment to more widespread use of PCR-based methods is the presence of inhibitors that frequently contaminate DNA isolated from environmental sources, especially water rich in organic matter. The major objectives of the present project are to ascertain the extent of this inhibitor problem and to develop alternative approaches. Using Salmonella as a model, real-time PCR and an Amplification Control (AC), we demonstrate that PCR inhibition occurs to varying degrees with all environmental water samples. Inhibition can be ameliorated by using less template DNA but the level of dilution required to permit amplification of the AC is often too severe to permit detection of the pathogen. Our results show that because the severity of the inhibitor problem is stochastic and there is not a reliable method to correct for the effect of the inhibitor, it is not feasible to convert real-time PCR data to bacterial counts. Instead, we propose a volume based approach to quantitate pathogens in recreational waters. The method is based on realtime PCR detection of bacteria, Salmonella in this case, in a specific volume of water. It includes a short enrichment step in a non-selective medium prior to DNA isolation and PCR. The enrichment step increases the amount of template DNA isolated without a concomitant increase in inhibitor level and solves the inhibitor problem because the ratio of inhibitors to template DNA used in PCR is greatly reduced. The detection limit of the method was 10 CFU/dL using a 6 hr enrichment period. Because the risk of exposure to a particular waterborne pathogen is directly related to its concentration, we believe this volume based approach provides a useful method to assess exposure risk in the future

Detection of Salmonella spp. in Coastal Waters

The U.S. Environmental Protection Agency published guidelines in 1986 on recommended water quality criteria for bacteria to protect bathers from gastrointestinal illness in recreational waters. The criteria, based on Escherichia coli and enterococci counts as indicators of the presence of fecal pollution, are still in use today. With the availability of PCR-based methods for the detection and quantification of specific pathogens, it might be possible in the future to base recreational water quality standards on the level of specific pathogens instead of indicator counts. Because most pathogen detection methods were developed for clinical samples, research is needed to adopt such methods to environmental samples where the presence of PCR-inhibitors is a common problem. The purpose of the current project was to determine the best method to process beach water samples for the detection and quantification of Salmonella spp. by qPCR. Although Salmonella is considered a low-grade pathogen that does not persist in the environment, it is one of the most common causes of enteric diseases and several PCR-based methods for its detection have already been developed. Our findings are that filtration through a coarse filter to remove debris followed by centrifugation (5 min at 5,000 x g) was an efficient method to concentrate samples. Detection limit can be lowered to 10 cfu/dL using sample enrichment in either a nonselective medium such as Brain Heart Infusion or a selective medium such as Rappaport Vassiliadis Soya Peptone. DNA purification prior to PCR increases the frequency of false negatives probably as a result of co-precipitation of PCR inhibitors with bacterial DNA. A simple boiling lysis procedure was found to be the most efficient method to prepare samples for PCR. Our conclusion is that it is now feasible to use a PCR-based method to detect and semi-quantitate Salmonella in environmental water samples

Enhancing PCR Amplification of DNA From Recalcitrant Plant Specimens Using a Trelahose-Based Additive

Premise of the study: PCR amplification of DNA extracted from plants is sometimes difficult due to the presence of inhibitory compounds. An effective method to overcome the inhibitory effect of compounds that contaminate DNA from difficult plant specimens is needed. Methods and Results: The effectiveness of a PCR additive reagent containing trehalose, bovine serum albumin (BSA), and polysorbate‐20 (Tween‐20) (TBT‐PAR) was tested. PCR of DNA extracted from fresh, silica‐dried, and herbarium leaf material of species of Achariaceae, Asteraceae, Lacistemataceae, and Samydaceae that failed using standard techniques were successful with the addition of TBT‐PAR. Conclusions: The addition of TBT‐PAR during routine PCR is an effective method to improve amplification of DNA extracted from herbarium specimens or plants that are known to contain PCR inhibitors