Rapid Detection and Enumeration of Pseudomonas in Metalworking Fluids using FISH

Corey Hecksel, Ratul Saha, and Susan Bagley, Biological Sciences, Michigan Technological University

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial contamination, which leads to degradation and biofouling of the MWFs. MWFs harbor a wide variety of microorganisms of which different species of rRNA group I Pseudomonas are dominant. It is important to study the distribution of these Pseudomonas in MWFs because they act as both potential pathogens and deteriorgens. The actual distribution of Pseudomonas in MWF is difficult to study using standard culturing techniques as most of them fail to grow on the commonly used Pseudomonas Isolation Agar (PIA), thus underestimating their abundance. To overcome the limitations associated with traditional culturing techniques, molecular-based methods such as Fluorescent in situ Hybridization (FISH) were implemented to study the distribution and the overall diversity of Pseudomonas belonging to the rRNA group I. FISH aids in the direct visualization of the bacterial cells and also reduces the time of detection. After analysis of all the available 16S rRNA sequences, a fluorescent molecular probe was designed targeting a conserved signature sequence found common to all rRNA group I Pseudomonas using the ARB software package. The designed molecular probe (Pseudo120) was fluorescently labeled with Cy3 and the universal bacterial probe (EUB338) was labeled with FAM. The CELLC software was used for analysis of images. The specificity of the probe was evaluated by performing hybridization experiments with whole cells of P. oleovorans, P. pseudoalcaligenes, P. alcaliphila, P. mendocina, P. fluorescens and P. aeruginosa using a stringency of 40% formamide at 46 °C. The sensitivity of the probe was determined to be 103 CFU/ml. The probe was successful in detecting and enumerating the abundance and distribution of Pseudomonas indicating levels between 3.20 (±1.11)x10^6 and 4.98 (±2.31)x10^6 CFU/ml in different contaminated MWF samples collected from industry (using stringency of 30% formamide at 46 °C). On the basis of these observations, the molecular probe can be successfully used for rapid detection, enumeration and study of spatial distribution of rRNA group I Pseudomonas in contaminated MWFs.

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Associations between Livestock Farms and Rice Creek’s Health: A Microbiological Examination

Molly Estill and Ola Olapade, Albion College

The Rice Creek Watershed, covering eastern Calhoun County and western Jackson County, serves as a major agricultural drain for those Michigan counties. Preliminary studies on the Rice Creek Watershed have shown that some aspects of the pollution in Rice Creek, particularly excess nutrients, influxes of E. coli, and increased sedimentation, stem from the presence of livestock farms along the channel of the creek.  In this study, the impact of livestock farms adjacent to Rice Creek on the health of the creek was quantified using standard microbiological techniques.  The amount of bacterial populations, specifically of E. coli and other coliforms, indicators of fecal contamination, in the creek were measured using membrane filter [MF] and viable plate counts approaches.  Water parameters, such as temperature and oxygen content, were measured using the YSI multi-parameter meter.  Concentrations of inorganic nutrients (i.e. nitrogen and phosphorous) within the creek were also determined colorimetrically.  In summary, bacterial abundance was observed to increase between sampling locations both upstream and downstream of livestock farms and also during periods of precipitation (with concomitant increase in nutrient concentrations).  Each of the three river sites examined appeared to have  unique patterns of bacterial and nutrient dynamics.  At the study sites “Ferris”, “Hicks” and “24 mile”, the study’s averages of total coliform for the sites were, respectively, 1,970, 1,294, and 1,527 total coliforms per 100 mL water.  Generally, it appeared from the results that there were constant influxes of nutrients and bacterial cells, particularly fecal bacteria populations, into Rice Creek, often exceeding the limit of 1000 E. coli cells/ 100 mL water for partial body contact threshold, as issued by the Michigan Quality Water Standards.
Supported by: FURSCA (Foundation for Undergraduate Research, Scholarship, and Creative Activity)

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Establishment of a Plant Pathogen Effector Model System and its use to Identify Cellular Targets

Vanessa Revindran and J.R. Geiser, Western Michigan University, Kalamazoo, MI

We have created a model system to study the action of plant pathogen effectors in furtherance of our desire to find ways to stop bacterial pathogen infections. We have established an inducible effector expression system in Saccharomyces cerevisiae that contains an inducible GAL1 promoter and a V5 epitope for visualization. We are screening effector proteins of Pseudomonas syringae to determine which ones are lethal in Saccharomyces cerevisiae. We created our expression constructs by PCR amplification followed by moving the genes into the appropriate Gateway SystemTM vector. We have successfully transferred 3 of the effectors, HopM1, HopAO1 and HopAF1, to the yeast expression plasmid and have transformed them into S. cerevisiae for examination of phenotypes. HopM1 when expressed in yeast is lethal on solid media whereas HopAO1 and HopAF1 are not. Examination of the lethal effect of HopM1 using a titer assay has not duplicated the effect seen on solid media, suggesting the effect seen is a delay or arrest of growth but not an outright cytotoxic effect. Surprisingly, HopM1 requires two plasmids to be lethal at 21°C, but is not lethal at 30°C and 37°C with any combination of plasmids. We have also examined expression levels of the HopM1 protein at all three temperatures and have discovered that at 21°C, HopM1 shows maximal expression at 24 hours, and at 30°C maximal expression is at hour 3. These results suggest that HopM1 protein may be unstable in our system. In an attempt to identify the cellular targets of HopM1 and to understand the function of HopM1, we have begun to isolate spontaneous suppressor strains that survive the HopM1 imposed lethality at 21°C. At present, we have identified 30 suppressor strains that survive at 21°C.Characterization of these strains are continuing and results will be presented to show growth rates, HopM1 protein expression in both wild type and suppressor strains and the effect of the suppressors on viability of HopM1 strains at 21°C.
Poster will also be presented at the 109th ASM General Meeting in Philadelphia.

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Use of nanotechnology to target acutely and persistently Chlamydia-infected cells for therapy and imaging in vitro and in vivo

Kawanjit Sekhon*, Susan Wykes*, Udaya Toti#, Mirabela Hali*, Herve Gérard*, Alan Hudson*, Jayanth Panyam#, Judith Whittum-Hudson*, *Dept. of Immunology & Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, #Dept. of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455

We have tested whether folate-conjugated nanoparticles preferentially target and enter chlamydia-infected cells and their potential to deliver agents to inclusions to better reduce infectious load. Nanoparticles fabricated from biodegradable poly(lactide-co-glycolide) polymer were surface functionalized with folic acid as targeting ligand. For some experiments, 6 coumarin was incorporated in nanoparticles (6C-NP) to allow cell or animal imaging under fluorescence. 6C-NP rapidly entered acutely and persistently (PenG induced) C trachomatis-infected HEp2 cells and targeted inflamed tissues in our mouse model of genital infection and reactive arthritis. Clear association of 6C-NP with chlamydial inclusion and reticulate body membranes was observed with acutely and persistently infected HEp2 cells proving rapid entry of NP into inclusions; folate did not seem to alter in vitro localization. We compared 6C-NP-FA vs 6C-NP targeting to infected tissues 7-14dpi by whole animal live-imaging (Kodak). Ex vivo imaging and HPLC analysis showed that FA-NP significantly increased localization in inflamed tissues (eg, genital tract, knees, p<0.05). Rifampin- and azithromycin-loaded nanoparticles each reduced in vitro infectious loads significantly better than respective free drugs and at lower drug concentrations (eg, p<0.02 at 100 ng/ml azithromycin and p<0.001 rifampin). Our in vivo targeting data suggest that FA will improve delivery of therapeutics to infected tissues. NP provide a new basic science tool to image living chlamydia-infected cells and in vivo/ex vivo tissues. NP delivery enhances drug potency and has important implications for new therapeutic modalities in which alternative nanoparticle delivery routes will better target infected tissues.

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Rapid Determination Technique to Distinguish between Hospital-Acquired (HA-) and Community-Associated (CA-) Methicillin-Resistant Staphylococcus aureus (MRSA)

Waybrant, Tara N, Michigan Technological University, Houghton, MI, 49931 (in collaboration with the Michigan Department of Community Health (MDCH)– Upper Peninsula Lab, Houghton, MI, 49931)

Antibiotic resistance has been an issue since the inception of antibiotic agents in the 1940s.  Staphylococcus aureus has been a particular concern to healthcare providers due to its genome’s ability to more easily adapt than other bacteria.  Antibiotic resistance in Staphylococcus aureus is of great distress in Michigan because, not only was the first reported case of Community-Associated MRSA (CA-MRSA) discovered in urban Detroit, but also seven of the nine Vancomycin-Resistant Staphylococcus aureus (VRSA) isolates were found in Michigan. Currently in a clinical setting it can take several days to a week to determine the exact strain of MRSA in a patient case.  Developing a rapid determination technique to establish the strain can aid in treatment, as well as prevention of spreading the infection.  Real-Time Polymerase Chain Reaction (qPCR) is being utilized because it is a rapid technique that is already in place throughout the United States for other infections, such as Norovirus.  The gene that is being utilized to create this test is part of the SauHsd gene family, which has been previously identified as having a link to strain lineage. Research is still in the preliminary stages, however, detection of primer and probe sequences for the USA300, the most common strain of CA-MRSA, have been identified.  The next phase will be to sequence for the USA100 strain, the most common HA-MRSA strain.  Followed by testing verification utilizing known strains from MDCH. It is hoped that this technique can be utilized in a clinical setting to aid in treatment of patients, prevention of spreading of antibiotic resistant bacteria, and also to aid in identification, and eradication, of bacteria with an increased potential for gained resistance to Vancomycin.

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Optimization of Ethanol Production by Pichia stipitis CBS 6054 in the Presence of Furfural through Adaptation

Jessica L. Beck, Stephanie L. Groves, and Susan T. Bagley, Michigan Technological University, Houghton, MI 49931

The production of lignocellulosic-based biofuels is a growing industry and it is important to find ways to produce it efficiently and inexpensively. The purpose of this study was to increase ethanol yield by using the xylose-fermenting Pichia stipitis CBS 6054 as efficiently as possible. The main goal was to overcome the inhibition that furfural, a byproduct from the degradation of pentose sugars, had on Pichia stipitis CBS 6054. P. stipitis CBS 6054 was adapted to higher concentrations of furfural in order to overcome the inhibitory effect of furfural. The yeast was grown in Yeast-Peptone media containing increasing concentrations of furfural. Every 72 hours the culture was transferred to the next higher concentration of furfural until a concentration of 4.5g/L was reached and no growth inhibition was observed. Using this technique Pichia stipitis CBS 6054 was able togrow and produce ethanol in concentrations that were previous inhibitory. High Performance Liquid Chromatography (HPLC) was used to compare the ethanol yield before and after adaptation. Using this technique, it was confirmed that more ethanol was produced by the adapted yeast than with the original P. stipitis CBS 6054.  The results that come from this experiment will provide biofuel companies with a more cost-effective and efficient option to overcoming the toxicity of lignocellulose.

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Adaptation of Pichia stipitis CBS 6054 to Dilute Acid Pretreated Aspen Hydrolysate for Improved Ethanol Production

Stephanie L. Groves and Susan T. Bagley, Michigan Technological University, Houghton, MI 49931

Lignocellulosic material offers a viable alternative to corn for fuel ethanol production. The fermentable sugars in lignocellulose are tightly bound within the structure and must undergo chemical pretreatment to be liberated and available for use by microorganisms. The pretreatments produce a range of toxins that can inhibit ethanol production.  In addition to the toxins, the sugars are not the ideal sugars for ethanol production by many organisms. Adaptation was used to produce a mutant of Pichia stipitis CBS 6054 for increased ethanol yields from dilute acid pretreated Aspen hydrolysate. The culture was grown in increasing concentrations of hydrolysate until the hydrolysate encompassed 80% of the total media volume. Biomass production, substrate utilization, and ethanol production were all used to evaluate the success of the mutant. Each sample was analyzed using High Performance Liquid Chromatography (HPLC) to determine ethanol and substrate concentrations. All fermentations were carried out at the bench-scale level in 125 mL Erlenmeyer flasks, with a working volume of 50 mL, sealed with aluminum foil and parafilm. The fermentations were carried out over a 72 hour period. The mutant showed a reduction of the inhibitory effects of the pretreated Aspen hydrolysate compared to the wild type. Increased ethanol yields and biomass production on Aspen lignocellulose were also observed. Adaptation is a useful and inexpensive means of optimizing the organism for the production of ethanol from lignocellulose.

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Protozoan parasites and Health Risks in Combined Sewer overflow receiving waters

Rebecca R. Ives and Joan B. Rose,  Michigan State University, East Lansing, MI

The pathogens Cryptosporidium and Giardia have a history as etiologic agents in many recreational water outbreaks in the United States.  Discharges of untreated sewage via Combined Sewer Overflows (CSOs) may contribute to Cryptosporidium and Giardia oo(cyst) contamination of the natural water environment, leading to recreationally acquired disease. To evaluate the impact of CSOs,  Giardia spp. and Cryptosporidium spp. analyses were conducted on water samples from publicly maintained recreational areas within the lower Grand River watershed, MI and from  combined stormwater / sewage  at the Grand Rapids Market Avenue Retention Basin (MARB).  Between April 2005 and August 2006, surface water grab samples were collected from Deer Creek Park (n=21), Riverside Park (n=19), and from North Beach Park( n=19).  North Beach Park is located at the river mouth along the shore of Lake Michigan, the other facilities are located along the river banks.  Composite samples (10 L) were collected from MARB influent during nine rainfall events.   Samples were concentrated and processed using US EPA Method 1623.  A 10,000 trial Monte Carlo analysis was conducted to evaluate risk of gastrointestinal illness in children under 16 due to Giardia spp. and Cryptosporidium spp. exposure while swimming. The median daily risk at the recreational sites ranged from 4.9x10-7 to 9.8x10-7 for cryptosporidiosis and 6.3x10-7 to 1.4x10-7 for giardiasis.  The median daily risks at the CSO discharge point were 4.4x10-5 and 2.5x10-2  for cryptosporidiosis and giardiasis, respectively.  Seasonal risk of illness was also evaluated using an assumption of daily recreational activity over 90 days. The median seasonal risk at the recreational sites ranged from 4.4x10-5 to 8.8x10-5  for cryptosporidiosis and 5.7x10-5 to 1.3x10-4 for giardiasis.  Median seasonal risks at the CSO discharge point were 4.0x10-3 and 4.5x10-1 for cryptosporidiosis and giardiasis, respectively. This study concludes that the daily risk of illness at recreational sites meets the EPA recommended freshwater recreational water quality standard of 8 illnesses in 1000 swimmers at least 99.99% of the time.

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Chemical and Biological Sensing Utilizing Fused Bacteriorhodopsin Protein Hybrids

Eric M. Winder1, Mark H. Griep2, Donald R. Lueking1, Craig R. Friedrich2
1Dept. of Biological Sciences 2Dept. of Mechanical Engineering-Engineering Mechanics 1,2Multi-Scale Technologies Institute, Michigan Technological University, Houghton, MI, 49931 USA

Current chemical sensing systems are cumbersome, not readily deployable, time consuming and generally inadequate for providing the sensitivity required for saving soldiers’ lives.  The best commercial sensors are currently only able to measure toxin concentrations at the micromolar level, while many biological and chemical agents are lethal at the nanomolar level.  The evolutionary development of biological systems however, has created an array of natural nanoscale materials with capabilities beyond that of current technology that can be utilized for development of new sensor systems.  Bacteriorhodopsin (bR), an optoelectric protein found in the cytoplasmic membrane of Halobacterium salinarum, is one such material and has been intensely studied over the years due to its intrinsic ability to function as a light-driven proton pump.  Coupling the bR protein with an electrically conductive allows the efficient quantum conversion of light energy into an electric potential.  As a proof of concept, a maltose binding protein (MBP)-bR gene construct is being used to produce a fused, hybrid protein with transducing and sensing properties. MBP binds the disaccharide maltose with high affinity and undergoes a significant conformational change upon substrate binding.  It is proposed that the energy associated with target molecule binding to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform.  As previously presented at the 2008 Army Science Conference, the gene encoding for bacteriorhodopsin has been successfully isolated from H. salinarum strain S9P using a colony-level PCR approach.  Utilizing this purified DNA and a plasmid expression vector system, a fused protein hybrid consisting of maltose binding protein and apo-(bR)-protein has been expressed in transformed Escherichia coli.  The fusion hybrid has been purified in soluble form from E. coli cell-free extracts at up to 70mg/L.  Renaturation studies to incorporate all-trans retinal within the apo-(bR)-protein are currently underway.

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Transcriptional Control of the Enterohemolysin Operon by H-NS in Shiga Toxin-Producing E. coli Serotypes Which Lack the Locus of Enterocyte Effacement

M. T. Rogers, R. N. Zimmerman, and M. E. Scott, Western Michigan University, Kalamazoo, MI

Shiga toxin-producing E. coli serotypes are divided into two subgroups: enterohemorrhagic E. coli (EHEC) and Shiga toxin-producing E. coli (STEC). The distinction between the two groups lies in a pathogenicity island known as the locus of enterocyte effacement (LEE), which is found on the chromosome of EHEC but is lacking in STEC strains. Both EHEC and STEC strains possess a large virulence plasmid that contains the enterohemolysin (ehx) operon. This operon encodes four genes, ehxC, ehxA, ehxB, and ehxD. Enterohemolysin is coded by the ehxA gene, while the gene products of ehxB and ehxD combine with TolC to form a secretion pathway. The putative promoter region is located upstream of ehxC. Expression of the operon in EHEC strains is thought to be controlled by regulatory proteins located on the LEE locus, while the mechanism of regulation within STEC strains is unknown. A mutant O91:H21 strain was created with a mutation in the gene encoding the histone-like nucleoid structuring (H-NS) protein which was unable to bind human colonic epithelial cells, was non-motile, and expressed a hyperhemolytic phenotype. SDS-PAGE analysis and Western blotting revealed that the level of production and secretion of enterohemolysin was increased in the mutant strain.

To determine if H-NS regulated the expression of the ehx operon by a direct binding mechanism, the promoter region of ehx was cloned and used in an electrophoretic mobility shift assay. In addition, the promoter was used to create a transcriptional fusion with a promoterless lacZ gene. The fusion vector was then placed into an H-NS null background to further test the effects of H-NS regulation on ehx expression. We discovered that H-NS binds to a region that includes the DNA sequence between -100 and -1 bp of the translational start site of ehxC. When this same region was fused to a promoterless lacZ gene, a significant increase in beta-galactosidase activity was observed when the vector was placed into a strain lacking H-NS. The evidence suggests that the DNA region between -100 and -1 bp of ehxC contains the cis element required for negative regulation of the enterohemolysin operon by H-NS.

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Spatial and Temporal Comparison of Bacterial Community Composition in Selected Adult Oral Cavities

Sachi Vyas and Ola Olapade, Albion College, Albion, MI 49224

The oral cavity of the human mouth has a myriad of microbes present that are typically involved with and influenced by individual health and hygiene conditions. This study particularly examined bacterial populations in three selected adult oral cavities with varying hygiene practices. The impacts of the various oral hygiene techniques used by these individuals (e.g., teeth brushing at least twice a day, regular flossing and using of mouthwash) were quantified using standard microbiological techniques.  The bacterial populations, specifically of Streptococcus and Staphylococcus, in the oral cavities were enumerated using various techniques, including nucleic acid staining and viable plate counts.  In summary, the bacterial numbers were observed to be highly elevated in the oral cavities of the adults that failed to practice proper hygiene techniques.  The results from this study appeared to indicate that each of the three adult oral cavities examined appeared to have unique patterns of bacterial growths that are attributable to their specific oral hygiene techniques and dental anatomy.  Overall, this study strongly suggests that an individual’s dental anatomy and types oral hygiene practices could potentially influence overall health status and specifically aid in the prevention of bacterial-induced periodontal diseases such as dental caries in humans.

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