Research in Our Laboratory

Investigation of the bacterial microflora of the human intestinal tract

The composition of the human large intestinal microflora has a profound influence on health and disease through its involvement in nutrition, pathogenesis, and immunology of the host. A proper comprehension of the diversity of species present and their numerical preponderance is therefore of utmost importance. Although this microflora has been the subject of intensive investigation, using anaerobic culturing techniques combined with phenotypic methods of characterisation, there is now a universal recognition that these approaches have provided a vastly incomplete picture of the predominant gut flora diversity and as much as 80% may have eluded scientific description. Molecular phylogenetic studies are now being employed to provide as complete as possible inventory of the microbial biodiversity present.

Gastrointestinal microflora studies in children with late-onset autism

Autism is characterized by delays in understanding and use of language, unusual response to sensory stimuli, resistance to change and emphasis on routines and with difficulties in normal social interactions. The disease usually manifests itself in early infancy but in at least one third of patients, the onset is delayed until 18-24 months of age. In approximately 10% of cases a genetic disposition is apparent but in the majority of patients no underlying aetiology as been determined. Therapy centred on speech, language, psychological and behavioural themes have been shown to give some benefits, but these are both labour-intensive and expensive.

We hypothesize that antimicrobial compounds taken for early childhood illnesses, may disrupt the normally protective indigenous intestinal flora and allow colonization by a rare organism(s) that produce a metabolite or neurotoxin leading to the brain impairment presented by children diagnosed with this particular form of autism. The hypothesis is based on the following observations. First, onset of the disease often follows antimicrobial therapy. Second, gastrointestinal symptoms are common at onset and often persist. Third, other antimicrobials (e.g., oral vancomycin) may lead to a clear-cut response and relapse may occur when the vancomycin is discontinued. Fourth, some patients have responded to several courses of vancomycin and relapsed each time it will be discontinued.

In collaboration with Sydney Finegold, of the Department of Medicine, UCLA School of Medicine our previous studies have used traditional culture-based methodologies in concert with molecular tools used for the identification of a number of novel clostridium-like organisms. However, culture-independent, molecular genetic-based techniques (e.g. direct 16S rRNA gene/ PCR cloning/sequencing) have shown that the human gut harbors an immense diversity of organisms, most of which cannot be cultured by current techniques. Detailed phylogenetic inventories are necessary that will facilitate the identification of bacterial population shifts or individual neurotoxin-producing organisms associated with the autistic condition. While continuing this work at OU we recently isolated a novel organism that presented us with an opportunity to reclassify a number of misclassified ruminococci in an important clostridial rRNA cluster (XIVa). In addition to this successful strategy of the cultivation and characterization of organisms I now wish to use additional molecular methods to more rigorously investigate the microflora of autistic children. Preliminary studies in our laboratory using 16S rRNA gene clone libraries have shown that autistic children have a higher proportion of Firmicutes (a division of bacteria that contain the clostridia) present in their GI tracts. Currently we are using the recent advance of “454 pyrosequencing” that gives a many fold increase in the coverage of sequences obtained in a single sequencing run. This provides a much more comprehensive inventory of sequences representing both dominant but equally important minor populations of organisms that have previously eluded identification using traditional sequencing methods. We are very excited about our current studies and believe we are making important contributions to a better understanding of this devastating disease and may lead to treatment strategies. There is evidence that some cases of autism, notably late onset or regressive autism, may involve abnormal intestinal flora since the oral administration of the antibiotic vancomycin, which is very poorly absorbed, leads to significant improvement in some children with this form of autism. Molecular phylogenetic studies in collaboration with Prof. Sydney Finegold, of the Department of Medicine, UCLA School of Medicine USA, are now being employed to investigate the intestinal flora of children with late onset autism undergoing this treatment.

Biofuels

Search for Novel Microorganisms Capable Of Producing Ethanol and Other End Products

TWorld energy consumption is projected to increase by 71% from 2003-2030 contributing to a potential crippling energy crisis. Combined with the CO2 emissions and climate change issues now becoming ever more prevalent, there is now a great need to use and develop alternative fuels. One such example is biofuels whereby the chemical energy in biomass can be converted to chemical energy in the form of ethanol, hydrogen or other microbial end products. In order to capitalize on these developments and manage the projects in an organized manner, the Oklahoma Center for Bioenergy (OCB) was established. I have become involved in two OCB sponsored projects.

1. Isolation and characterization of novel microbial catalysts for direct fermentation of lignocellulose to ethanol.

   One of the key problems hindering efficient utilization of lignocellulosic biomass and conversion to ethanol is the low susceptibility of lignocellulose to hydrolysis, a critical step for generating sugars for microbial fermentation. Pretreatment of cellulosic biomass to break down the cellulose structure using various mechanical, physical and chemical approaches is required prior to the subsequent steps of hydrolysis and fermentation. Furthermore, due to the inhibitory effect of end products and/or inhibitive reagents used in pretreatment processes, the final ethanol yield, generally is low, and does not meet the industrial requirements. An ideal solution is to develop biological processes that do not require pretreatment of plant materials. In addition, the current technology for conversion of cellulosic biomass to ethanol is not efficient or economically viable - a key bottleneck in using cellulosic biomass as an energy source. Thus, novel technologies are urgently needed for efficient ethanol production from cellulosic biomass. Hydrolysis of lignocellulosic materials by fungi generates a mix of sugars including both 5- and 6-carbon sugars. An objective of this project will be the isolation of bacteria capable of ethanol production from fungi-degraded plant biomass.

   Some unexpected outcomes of biofuels research are coming into the public arena. For example, the competition of energy companies and food companies for the same starting materials, such as of corn, is leading to vastly increased food prices. Secondly, the destruction of the South American rainforests to plant sugar-beets as a starting material for biofuels is also causing much concern. Moreover, in response to the above problems, in July 2008, the UK government and other European countries announced a slowing down in the adoption of biofuels in preference for other alternative fuels. However there are alternative starting materials for biofuels for which there is no competition with foodstuffs.

2. Indirect fermentation of biomass to ethanol and other products.

   Biomass can be first burned to produce a gas called “syngas” composed of carbon monoxide, hydrogen and carbon dioxide that is then passed into a bioreactor where bacteria ferment the syngas and produce ethanol. The advantage of this process is that any biomass that can be burned can be used as starting material. For example, switch grass, a plant that can be grown on marginal land where normal agricultural practices are not possible, city garbage recycling plants, and paper and pulp mills are all potential sources for the generation of “syngas” for renewable energy purposes. Dr. Ralph Tanner has long been at the forefront of this technology and we are currently involved into the search for new microbial catalysts that may be more efficient in the above process and may in turn be enhanced by molecular genetic manipulate techniques.

Biocorrosion

In 2006 I was very pleased to be approached by Dr. Joseph M. Suflita to become involved in some studies arising from the oil pipe breakage that occurred on the North Slope, Alaska. We initiated some preliminary studies into the role of microorganisms in microbially influenced corrosion (MIC). To explore the fundamental scientific issues that lead to new knowledge, understanding and technology for the diagnosis and mitigation of fuel biodeterioration and biocorrosion problems. This collaboration with a number of OU-base laboratories as led to formation of a Biodeterioration and Biocorrosion Center here at OU, sponsored by ConocoPhillips and the University. The University recognized this research with the prestigious designation of University Strategic Organization, and therefore ear-marking this as an important area of future research. This exciting development should lead to long-term funding with other energy-based companies being encouraged to take part and address the problems being encountered with MIC.

Molecular identification and phylogenetic analysis of unique microorganisms from swine feces and manure storage pits

Intensive farming practices have resulted in huge amounts of manure being produced. Odious compounds such as phenolics, indols, volatile amines and ammonia produced by microorganisms can be detrimental to the health of agricultural works, animals and the general population. ln collaboration with Drs. Cotta/ Whitehead, Agricultural Research Service, USDA, we have begun to unravel the complex ecosystems and organisms present in swine manure and the storage facilities of this waste material. This work has resulted in a number of publications describing novel bacterial taxa from manure sources. We propose to extend this work to specifically look at samples from several difference facilities here in Oklahoma. Initial studies will employ a culture-independent molecular approach to create a clone library to produce a comprehensive inventory of the representative organisms present. Organisms will be targeted for directed cultivation work involving the isolation of organisms that will be subjected to further analysis for more complete characterizations. Strategies can then be developed to decrease or eliminate organisms producing noxious compounds. In addition to detecting harmful compounds, samples will be analyzed for the presence of substances such as alcohols and solvents that may be beneficial to industry and renewable energy programs such as bioethanol production and effective waste recycling projects.

Identification of novel emerging human and animal pathogens

An increasing number of novel organisms, that have previously eluded identification by traditional methods, including many potentially pathogenic bacteria, are now being revealed by modern molecular methodologies. Many of these organisms are anaerobes co-existing in complex community structures; some may act as opportunistic pathogens causing veterinary or clinical problems only when particular circumstances arise. Some organisms are present as a “reservoir” in the environment from where they may infect both man and animals when contact is made and/or the appropriate conditions arise. In my laboratory in collaboration with a number of international groups we are adding to the knowledge of base line microbial communities in man and animals and organisms that arise and cause disease processes.

Consultancy / Miscellaneous Projects

In addition to the studies outlined above, because of my long-standing collaborations with a network of national and international research groups, I am often approached for my opinion on unusual or difficult to identify microorganisms.

Microbial Identification Service

Unidentified bacteria that require a rapid identification are taken as pure cultures or isolated DNA. Samples are subjected to 16S rDNA sequence analysis. A report is then generated providing details of the analysis prior to further characterization if requested.