Hot Topics

Current Article
"All I Need is the Air That I Breathe” - studying anerobic bacteria with Bioscreen-C

Earlier Hot Topic Articles
Getting Buggy About Ethanol - production of ethanol and other valuable products from lignocellulosic feedstocks.
One Man’s Trash…May Be a Microbe’s Treasure - microbial remediation of environmental contaminants
“Finding the Antimicrobial Sweet Spot” - Screening and Discovery of Sugar-Based Antimicrobials
"Visualize Whirled Yeast” - Quantifying yeast chronological life span by outgrowth of aged cells
"Pickled In Their Own Juices" - understanding the aging process through yeast research
“Artisanal Cheeses: A Highly Cultural Experience” - growth rates of desireable and potentially pathogenic organisms in food.
“Teeth, Tongue and Beyond” - Applications of Bioscreen-C in Oral Microbiology
“Got Spores?” - measuring bacterial spore germination with Bioscreen-C
Antibiotic Resistant Bacteria - Use of the Bioscreen-C for the study of antimicrobial resistance in clinical settings
Natural Anti-Microbials - Use of Bioscreen-C to evaluate the activities of plant essential oils against foodborne pathogens

Oxygen – the stuff of life. As oxygen-breathers ourselves, we can’t imagine a world without it. However, things weren’t always this way. In his book, “A Short History of Nearly Everything”, author Bill Bryson writes that in the Archaen Eon of Earth’s early history, our planet’s atmosphere consisted mostly of ammonia, methane and carbon dioxide. Then something happened. The advent of cyanobacteria around 2.4 billion years ago marked the beginning of the end of the Archaen Eon. As they multiplied and successfully colonized the oceans, the cyanobacteria spewed out huge volumes of a highly reactive, corrosive and toxic gaseous byproduct into Earth’s ancient atmosphere – enough to eventually “poison” the entire planet and bring about drastic global climate change. What was this poison gas? Oxygen. “That oxygen is fundamentally toxic…”, Bryson states, “…often comes a surprise to those of us who find it so convivial to our well-being, but that is only because we have evolved to exploit it. To other things, it is a terror.”

Today, the Earth we know is an aerobic world, with much of the planet’s habitable space bathed in air containing oxygen at a final concentration of about 10%. Many forms of life, including higher land animals, insects and even marine life have evolved to exploit oxygen, and green algae, cyanobacteria and terrestrial plants continue to pump out untold tons of this gas each year. However, there are still many places on Earth where life teems and thrives in total absence of that “terror” known as oxygen. These anaerobic environments include terrestrial and marine sediments, anoxic deep-sea brines, and possibly even beneath pristine Antarctic subglacial lakes that have been cut off from the surface for millions of years.

Apart from peculiar anaerobic animals such as those belonging to the phylum Loricifera, most anaerobic organisms belong to the domains Bacteria and Archaea, and although we inhabit worlds apart, these anaerobic organisms can have a substantial impact on human life, with some of them playing a large role in our wellbeing. Notable anaerobes include toxigenic pathogens such as Clostridium botulinum, maker of nature’s most potent toxin (see “Got Spores?” Hot Topics entry) or clinically important bacteria. Bacteria in this latter category include either obligate or “part-time” (facultative) anaerobes that may be involved in anaerobic abscesses, colonization of indwelling medical devices, or periodontal disease. As with microbes in general, though, the vast majority of anaerobes are not harmful to us. Conversely, many are vital to human life as we know it. Beneficial anaerobes include those that carry out fermentation of foods, such as fish sauce or soy sauce, from which the aptly-named facultative anaerobes Staphylococcus piscifermentans and Staphylococcus condimenti were isolated, respectively.  Other beneficial anaerobes include the probiotic Bifidobacteria, which aid digestion, enhance the nutrient profile of fermented foods and may also prevent colonization of the GI tract by pathogens. Animal foods, such as corn and hay, have long been preserved through the process of ensiling, which involves storage in an anaerobic environment, where they are partially fermented by lactic acid bacteria such as Lactobacillus, Pediococcus and Leuconostoc. This anaerobic fermentation preserves the fresh quality of the ensiled material, and provides a fresh and nutritive food for animals year-round, not just at harvest time. The biotechnology sector is another key area where anaerobic bacteria play an essential role, fermenting agricultural feedstocks into valuable end products such as ethanol and butanol. Examples include the obligate anaerobe Clostridium acetobutylicum, used to produce butanol, and the facultative anaerobe Zymomonas mobilis, used to produce ethanol industrially (and recreationally, as the primary fermenter of hard apple cider, also known as “apple jack”). Finally, of essential importance to Earth’s chemistry are the methanogens, whose name literally means “methane producer”. Methanogens are important degraders of organic material and are one of the key “microbial engines that drive Earth’s biogeochemical cycles” (Falkowski et al., 2008).

One of the key ways we can learn more about microorganisms, including their properties, processes and if need be, how to suppress their growth, is to culture them under controlled conditions in the lab. The Bioscreen C instrument is a versatile platform for growth-based microbial assays. The instrument enables high throughput microplate-based evaluation of microbial growth by tracking increases in the turbidity of liquid media inoculated with pure cultures or unknown samples. The Bioscreen is a self-contained incubation and analysis unit capable of evaluating up to 200 sample wells over growth periods varying from several hours to several days. Incubation temperatures range from ambient to 60°C, facilitating growth of both mesophilic and thermophilic microorganisms. The instrument is also compatible with coldroom operations, allowing evaluation of psychrotrophic organisms (Plowman and Peck, 2002). Although partially anaerobic conditions may develop in Bioscreen wells during microbial growth, especially for statically grown cultures, a greater degree of control is needed if anaerobic bacteria, especially obligate anaerobes (those that cannot tolerate any amount of oxygen) are to be cultured.

One area where access to anaerobic conditions is critical is in antimicrobial susceptibility testing. In tests performed with facultative anaerobes, it has been noted that some antimicrobials, such as quaternary ammonium compounds, are more effective under anaerobic conditions (Bjergbaek et al., 2008), whereas others, such as the aminoglycosides kanamycin and gentamycin, are less effective under anaerobic conditions (Harrel and Evans, 1977). In their fundamental study, Kohanski et al. (2007) showed that regardless of the actual target within the cell, bactericidal antibiotics like kanamycin stimulate the production of toxic hydroxyl radicals, suggesting a possible mechanism for the lower efficacy of certain antibiotics in the absence of oxygen. When working with obligate anaerobes, there is no choice – growth-based testing must be done under conditions that will support growth of the test organism. In biotechnological applications, maintenance of anaerobic conditions can be critical to product yield, as is the case with fuel ethanol production in Z. mobilis. Yang et al. found that Z. mobilis growing aerobically produced only 1.7% the ethanol that the same culture produced anaerobically (Yang et al., 2009).  In order to investigate the utility of antimicrobials for treatment of abscesses or other anaerobic infections, or to unravel the physiological and genetic events responsible for differences in product yield under various levels of oxygenation, researchers need access to tools like the Bioscreen under both aerobic and anaerobic conditions.

Thanks to the ingenuity of the instrument’s end users, the Bioscreen has been successfully adapted for the study of anaerobic microbes, further underscoring the instrument’s flexibility. Two basic strategies for achieving anaerobic conditions in the Bioscreen are typically used. The first is to overlay individual wells with sterile paraffin (mineral) oil, which provides an optically clear, oxygen-impermeable barrier covering the aqueous growth medium (Aroutcheva et al., 2001; Simoes et al., 2001). The second approach is to place the entire instrument in an anaerobic or modified atmosphere cabinet, where all operations, including inoculation and subsequent growth, can be done under strictly controlled conditions (Plowman and Peck, 2002; Stringer et al., 2005; Yang et al., 2010). In either application, one possible way to verify that anaerobic conditions are achieved and maintained is the use of an oxygen-sensitive, color-changing indicator dye such as Alamar Blue (resazurin), which can be placed in uninoculated wells. Because reduction of resazurin causes the dye to turn from blue to pink, this technique might provide a simple visual control for establishment and maintenance of anaerobic conditions.

An excellent example of how simply a Bioscreen can be installed in an anaerobic chamber is the instrument belonging to the High Throughput Natural Antimicrobial and Prebiotic Discovery facility at Iowa State University (Figure 1), which is contained inside a Bactron Anaerobic/Environmental chamber (Sheldon Manufacturing, Cornelius, OR). Samples can be introduced through the airlock to the left and all sample manipulations can be done under anaerobic conditions using the chamber’s flexible, high-dexterity gloves. Experimental setups such as this enable facile Bioscreen-based determination of microbial performance characteristics under anaerobic conditions.

In summary, adaptation of the Bioscreen for anaerobic analyses can be accomplished through use of simple mineral oil overlays or the installation of a Bioscreen in an anaerobic cabinet. Use of a cabinet enables precise control over gas mixtures and final conditions occurring in each well. Whether the goal is to characterize the response of test organisms to antimicrobials or antibiotics under anaerobic conditions, or to explore microbial growth and metabolism under various levels of oxygen, adaptation of the Bioscreen C instrument for anaerobic analyses leverages the power of this versatile instrument to provide a unique window into the anaerobic world. These applications provide additional examples of how this flexible, powerful microbial analysis system can be used to help answer key questions of interest to microbiologists today.

Figure 1. A Bioscreen-C instrument installed in a Bactron Anaerobic/Environmental Chamber. Courtesy of Dr. A. Mendonca, Iowa State University's High Throughput Natural Antimicrobial and Prebiotic Discovery facility.

References

Aroutcheva, A., J.A. Simoes, S. Shott, and S. Faro. 2001. The inhibitory effect of clindamycin on Lactobacillus in vitro. Infect. Dis. Obstet. Gynecol. 9: 239-244.

Bjergbaek, LA., Haagensen, J.A., Molin, S. and P. Roslev. 2008. Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli. J. Appl. Microbiol. 105: 1310-1317.

Bryson, B. 2003. A Short History of Nearly Everything. Broadway Books, New York, NY.

Falkowski, P.G., Fenchel, T., and E.F. Delong. 2008. The microbial engines that drive Earth’s biogeochemical cycles. Science 320: 1034-1039.

Harrel, L.J., and J.B. Evans. 1977. Effect of anaerobiosis on antimicrobial susceptibility of Staphylococci. Antimicrob. Agents Chemother. 11: 1077-1078.

Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., and J.J. Collins. 2007. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130: 797-810.

Plowman, J. and M.W. Peck. 2002. Use of a novel method to characterize the response of spore of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions. J. Appl. Microbiol. 92: 681-694.

Simoes, J.A., Aroutcheva, A.A., Shott, S., and S. Faro. 2001. Effect of metronidazole on the growth of vaginal lactobacilli in vitro. Infect. Dis. Obstet. Gynecol. 9: 41-45.

Stringer, S.C., Webb, M.D, George, S.M., Pin, C., and M.W. Peck. 2005. Heterogeneity of times required for germination and outgrowth from single spores of nonproteolytic Clostridium botulinum. Appl. Environ. Microbiol. 71: 4998-5003.

Yang, S., Land, M.L., Klingeman, D.M., Pelletier, D.A.,  Lu, T.-Y. S., Martin, S.L., Guo, H.-B., Smith, J.C., and S.D. Brown. 2010.  Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA.

Yang, S., Tschaplinski, T.J., Engle, N.L., Carrol, S.L., Martin, S.L., Davison, B.H., Palumbo, A.V., Rodriguez, M. Jr. and S.D. Brown. 2009. Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations. BMC Genomics 10:34.

For more information, clickhere and enter Anerobes in the subject line of the line of the email or call 732-457-9070.

Three hundred and sixty to two hundred and ninety-nine million years ago - the Carboniferous period - a time that was home to vast forests of tree-like club mosses belonging to the genus Lepidodendron that were able to grow to heights of fifty meters (164 feet) in only fifteen years! Because Lepidodendron did not form branches or a canopy until the end of its life, it is estimated that up to 2,000 of these gigantic trees could grow per hectare, forming the densest forests the Earth has ever seen (Lloyd, 2009). In his book "What on Earth Evolved: 100 Species That Changed the World", author Christopher Lloyd places Lepidodendron near the top of the list. Because Lepidodendron's rapid, dense and prolific growth piled the ancient forest floors deep with fallen timber, much was buried before microbial degradation could begin. The result: The prodigious stores of coal that fueled the Industrial Revolution, altering the course of human history.

Why is coal such an excellent energy source? Lepidodendron and other Carboniferous plants collected the Sun's energy and locked it away in their tissues. After millions of years of heat and pressure, these tissues were transformed into coal and other fossil fuels - the condensed forms of plant carbon that we burn today, releasing that ancient solar energy. Deposits of plant-based fossil fuels can be found across the globe -- even in the Arctic and in Antarctica. However, the world's supply of these fossil fuels is finite. We are running out.

Today, as in Lepidodendron's time, the plant polymers cellulose and lignin are still the most abundant polymers in nature (Frazzetto, 2003; Logan and Thomas, 1987). However, even if there were globe-spanning forests of giant club mosses around today, we can't wait around for another 300 million years until the next batch of coal or oil is ready to be consumed. How can we tap into the stores of latent solar energy bound up in plant biomass now to solve today's energy problems? One way to do this is to ferment renewable plant biomass with the help of bacteria, yeast or filamentous fungi as a means for producing fuel ethanol and other valuable fermentation end products. The most direct route to ethanol is to feed these microbes easily digestible plant starches from corn, potato or wheat, or simple sugars such as sucrose from sugar cane. Microbial enzymes cleave plant starches into glucose monomers or sucrose into fructose and glucose monomers; these can then be fermented into ethanol. However, according to the United Nations, 1.02 billion people are starving worldwide (UN FAO, 2009). As it is, there is not enough food to go around. Does it make economic or moral sense to divert our limited food resources to provide fuel for the richest nations?

What about using non-food plant materials? At the molecular level, plant starch and cellulose are both comprised of glucose monomers. The key difference is in how these monomers are linked together to form each polymer. The alpha linkages in starch are readily cleaved by enzymes expressed by fermentative microbes, but the beta linkages in cellulose are not. Also, apart from notable exceptions, such as cotton, cellulose in plant tissues is not present in pure form. Instead, nanoscale fibrils of crystalline cellulose are bound together with polysaccharides and lignin, a polyphenolic macromolecule. The resulting complex is referred to as "lignocellulose". These tight lignocellulosic complexes provide great structural integrity to plant tissues and are responsible for wood's suitability as a building material. However, the recalcitrance of lignocellulosic materials to digestion by fermentative microbes is a major drawback to the use of these materials as substrates for the production of ethanol. As usual, nature has some answers - saprophytic organisms such as the white rot fungus Phanerochaete chrysosporium have evolved enzymes capable of digesting lignocellulosic materials. In addition to whole-organism or enzymatic approaches for releasing fermentable sugars from lignocellulose, thermal or chemical approaches can also be used (Rumbold et al., 2009). However, large-scale enzymatic, chemical or thermal pre-treatment of lignocellulosic wastes is expensive and some processes may not be environmentally friendly. Apart from fermentable sugars, thermal pretreatments may also release toxic chemicals, such as furfural or hydroxymethylfurfural that can inhibit the organisms used to make ethanol (Rumbold et al., 2009.

Before a process can be scaled up to industrially relevant levels, how can researchers best determine which agricultural waste products might serve as good substrates for ethanol production? What if different organisms need to be compared side-by-side on the same substrate or the same organism tested using multiple substrates? How can the impact of potential fermentation inhibitors be assessed? Several lignocellulosic biofuel researchers have found the Bioscreen-C Microbiology Reader (Growth Curves USA, Piscataway, N.J.) to be an excellent tool for answering these and other questions. For example, Rumbold et al., (2009) took advantage of the Bioscreen's flexibility and high throughput to perform a comprehensive study in which they evaluated four organisms (the bacteria Escherichia coli and Corynebacterium glutamicum; the yeasts Saccharomyces cerevisiae and Pichia stipitis) on thermally or chemically-generated feedstocks derived from four different agricultural sources (corn stover, wheat straw, sugar cane bagasse and willow wood). They also evaluated the capacity of a subset of their test organisms to grow on waste glycerol from biodiesel production. These authors noted that although the capacity of host strains to produce ethanol under ideal conditions has been extensively studied, very little attention has focused on the abilities of common host strains to utilize value-added feedstocks derived from agricultural or other waste streams. In this study, these authors were able to leverage the power of the Bioscreen to help demonstrate the value of this substrate-focused approach to process optimization.

We all know that if apple cider is left out at room temperature, it will eventually ferment into an alcoholic beverage. Zymomonas mobilis, the bacterium responsible for this natural fermentation, can also be used for industrial-scale production of ethanol. As with other hosts, the productivity of Z. mobilis-based fermentations can be affected by inhibitors present in complex lignocellulosic hydrolysates. With this in mind, Franden and colleagues used the Bioscreen to develop a quantitative, high-throughput growth assay for inhibitor activity and kinetics. Critical information provided by their assay included information on lag times and final cell densities. In their conclusions, these authors noted the utility of their approach, stating that "applying this high-throughput technology to quantitatively characterize hydrolysate toxicity under a variety of pretreatment and conditioning processes will eventually help to identify those conditions that are favorable for fermentation organisms and provide critical feedback for selecting and optimizing the pretreatment process for biomass to ethanol conversion".

In another study, Albers and Larsson (2009) also took advantage of the Bioscreen's ability to readily provide critical information such as specific growth rate and population lag time. They used these data to compare stress tolerance and fermentation performance for six yeast strains in synthetic media and in a lignocellulose-based medium. They concluded that although a wild-type industrial isolate performed best in the lignocellulosic medium, genetically well-characterized laboratory strains also were able to use this substrate, suggesting their possible use as parent strains in a genetics-based strain improvement program.

Together, these studies clearly highlight the power of the Bioscreen for studying and improving microbial processes critical to the production of ethanol and other valuable products from lignocellulosic feedstocks. Apart from the Bioscreen's capacity to follow and quantify the responses of natural isolates to different feedstocks, it is expected that this instrument will also be useful for the selection and characterization of versatile new strains developed through the genetic or metabolic engineering approaches that are now poised to revolutionize the biofuels industry (Liu et al., 2009).

References

Albers, E., Larsson, C. 2009. A comparison of stress tolerance in YPD and industrial lignocellulose-based medium among industrial and laboratory yeast strains. J. Ind. Microbiol. Biotechnol. 36: 1085-1091.

Food and Agriculture Organization of the United Nations (UN FAO). 2009. http://www.fao.org/news/story/en/item/20568/icode/

Franden, M.A., Pienkos, P.T. and M. Zhang. 2009. Development of a high-throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis. J. Biotechnol., doi: 10.1016/j.biotec.2009.08.006.

Frazzetto, G. 2003. White biotechnology. EMBO Rep. 4: 835-837.

Liu, X. and R. Curtiss III. 2009. Nickel-inducible lysis system in Synechocystis sp. PCC 6803. Proc. Natl. Acad. Sci. U.S.A. 106: 21550-21554.

What on Earth Evolved: 100 Species That Changed the World, Bloomsbury USA, New York, NY. http://www.whatonearthevolved.com.>

Logan, K.J. and B.A. Thomas. 1987. The distribution of lignin derivatives in fossil plants. New Phytol. 105: 157-173.

Rumbold, K., Van Buijsen, H.J.J., Overkamp, K.M., van Groenestijn, J.W., Punt, P.J., and M. J. van der Werf. 2009. Microbial production host selection for converting second-generation feedstocks into bioproducts. Microb. Cell Fact. 8: 64 http://www.microbialcellfactories.com/content/8/1/64

For more information, clickhere and enter Biofuels in the subject line of the line of the email or call 732-457-9070.

The level of comfort, convenience and quality that we experience in our modern lifestyle is unparalleled in human history. However, as we know, everything must come at a cost. The same global industrial engine responsible for the processes, materials and products that make our modern lives possible is also an extraordinary source of environmental pollutants. These pollutants find their way into our soil, water and air from multiple sources. Examples include fertilizers and pesticides used in agriculture, heavy metals and polychlorinated compounds resulting from leather tanning or paper milling operations, and a litany of compounds flowing into the environment from high tech sources, such as the plastics and electronics industries.

Microorganisms are extremely adaptive and can evolve quickly to take advantage of various materials present in their environments, even pollutant compounds. Examples of toxic compounds or pollutants that are degraded naturally by microbes include formaldehyde or toluene (Marx et al., 2005; Pareles et al., 2008; Sardessai and Bhosle, 2002). The idea that microbes are able to degrade or mineralize (break down to individual atoms) almost anything put to them forms the basis of ‘Principle of Microbial Infallibility’. Proponents of this principle argue that for every pollutant, natural or manmade, there is an organism or group of organisms capable of breaking it down. Although this principle should not be considered as inviolable, the number and range of environmental pollutants known to be degraded or modified by microbes is impressive and includes crude oil, nitroglycerin and even plutonium (Blehert et al., 1997; Icopini et al., 2009).

The term ‘bioremediation’ refers to any process that utilizes living organisms (bacteria, fungi, plants) or their enzymes to degrade toxic substances down into less toxic components. Bioremediation can be performed on material removed from a site (ex situ remediation) or in-place, at the site of contamination (in situ remediation). In situ remediation can be accomplished by pumping nutrients into contaminant-saturated soils or groundwater in an effort to promote the growth and desired activities of endogenous microbes. In addition, microbes having the desired degradative abilities can be added. If microorganisms are to be added, they must first be characterized for their degradative activities and the conditions under which they perform optimally must also be determined.

Methyl tert-butyl ether (MTBE) is an organic compound used as an additive to gasoline to increase the octane rating and prevent knocking or pinging, which can cause engine wear or damage. Although MTBE was originally introduced in the late 1970’s as a safer alternative to tetra-ethyl lead, it can become an environmentally pervasive contaminant of groundwater itself when MTBE-containing gasoline is released into the environment from leaky underground storage tanks. (Vosahlíková-Kolárová et al., 2008) used the Bioscreen C Microbiology Reader to identify strains or consortia capable of growing in the presence of and degrading MTBE under aerobic conditions. Microbes that had been isolated from contaminated soil or groundwater were incubated for one week at 28°C in the Bioscreen in wells containing media supplemented with 0.1% MTBE and various accessory nutrients, such as yeast extract or casamino acids. The specific growth rate, generation time and lag phase were determined using the Bioscreen’s absorbance data and residual MTBE in the spent media after growth was quantified via gas chromatography-mass spectrometry (GC-MS). Two pure bacterial isolates were found to be capable of growing on and degrading MTBE and were identified via 16S rDNA sequencing as Rhodococcus pyridinivorans and Achromobacter xylosoxidans. The identification of pure isolates capable of degrading MTBE, rather than complex microbial consortia, will be an important step toward determining the mechanisms behind contaminant degradation and may further the development of microbial biocatalysts for in situ degradation of MTBE (Vosahlíková-Kolárová et al., 2008).

The enhanced absorption caused by the inherent coloration of certain substrates may also be used to quantitatively follow the degradation of environmental pollutants by bioremediative strains. For example, Tvrzová et al. (2006) were able to use the Bioscreen C to monitor the degradation of yellow nitrophenolic compounds based on the reduction in their absorbance over time. Several nitrophenol compounds are listed on the U.S. Environmental Protection Agency’s "Priority Pollutants” list (http://www.epa.gov/waterscience/methods/pollutants.htm). The Bioscreen method was comparable to a more labor-intensive HPLC for determining nitrophenol concentrations and authors concluded that could enable efficient high-throughput screening of degradation where many samples must be examined.

Together, these studies highlight the efficacy and flexibility of the Bioscreen C Microbiology Reader in studies focused on microbial remediation of environmental contaminants. These studies show that the Bioscreen can be leveraged as a powerful tool for identification and characterization of microbial strains that can be used to help clean up our planet, making it a safer and healthier place to live.

References:

Blehert, D.S., Knoke, K.L., Fox, B.G., and G.H. Chambliss. 1997. Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species. J. Bacteriol. 179: 6912-6920.

Icopini, G.A., Lack, J.G., Hersman, L.E., Neu, M.P., and H. Boukhalfa. 2009. Plutonium (V/VI) reduction by the metal-reducing bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 75: 3641-3647.

Marx, C.J., Van Dien, S.J., Lidstrom, M.E. (2005) Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism. DOI: 10.1371/journal.pbio.0030016

Parales, R.E., Parales, J.V., Pelletier, D.A. and J.L. Ditty. 2008. Diversity of microbial toluene degradation pathways. Adv. Appl. Microbiol. 64: 1-73.

Sardessai, Y. and S. Bhosle. 2002. Tolerance of bacteria to organic solvents. Res. Microbiol. 153: 263-268.

Tvrzová, L., Prokop, Z., Navrátilová, J., Müllerová, R., and J. Neca. 2006. Development of a microtiter plate-based method for determination of degradation profile of nitrophenolic compounds. J. Microbiol. Methods 65: 551-556.

Vosahlíková-Kolárová, M., Krejcík, Z., Cajthaml, T., Demnerová, K., and J. Pazlarová. 2008. Biodegradation of methyl tert-butyl ether using bacterial strains. Folia Microbiol. 53: 411-416.

We all know that a spoonful of sugar helps the medicine go down, but what about the sugar itself -- could it have intrinsic medicinal value? The answer to this question may eventually come from the new field of glycomics, which takes its name from the "glycome" the diverse array of sugar molecules found inside or on cells of all types. Our emerging understanding of the glycome underscores the observation that sugars are multifaceted molecules, with a variety of biological activities distinct from their basic role in energy storage. For example, glycosylation (decoration with sugars) of bacterial surfaces is now recognized as a key factor mediating processes as diverse as pathogenesis, symbiosis and immune evasion (Hsu et al., 2006). Interestingly, a wide range of naturally occurring or synthetic sugars or their derivatives are also garnering increased attention for use as antimicrobials. For instance, xylitol, a low-calorie sugar alcohol derived from plant fibers, including corncobs and husks, has been found to suppress the growth of Streptcococcus mutans, the causative agent of dental caries (cavities) (Söderling, et al., 2008). Xylitol may therefore play a dual role in reduced calorie/sugar-free chewing gums and candies as both a sweetener and as a means for reducing the incidence of cavities. Similarly, anhydrofructose (AHF) is a naturally occurring, low calorie sugar found in morel mushrooms and in seaweed (Danisco, Inc.). It is also a component of green tea, where it acts as an antioxidant to prevent browning of the tea. Like xylitol, AHF has antimicrobial properties, preventing growth and spore germination in Gram-positive bacteria (Danisco, Inc.). Alternatively, chemically modified sugars, such as alkylated or acylated mono- or oligosaccharides, which are industrially useful as surfactants, have also been explored for antimicrobial use (Bunger et al., 2002).

The Bioscreen C instrument from Growth Curves, U.S.A. (http://www.growthcurvesusa.com) is a workhorse tool for generating the data needed to build and support intellectual property claims in the field of industrial microbiology. A recent search of the US Patent & Trademark Office's website (http://patft.uspto.gov/) for the term "Bioscreen" yielded 36 patents and 84 patent applications in areas as diverse as biofuel or solvent production, vaccine research, pharmacological drug discovery and development of antimicrobials for food or clinical applications. This widespread use of the Bioscreen highlights the recognized utility of this instrument among industrial researchers.

As with many other types of molecules, the Bioscreen has been an indispensible tool for the screening of sugar-based compounds for their antimicrobial activities. For example, Fiskesund and colleagues evaluated 1,5-anhydro-D-fructose (AHF) and 10 enzymatic or chemical AHF derivatives for their antimicrobial activities against a broad selection of 13 Gram-negative bacteria, Gram-positive bacteria, yeasts and molds (Fiskesund et al., 2008). Although this represents a daunting number of analyses for traditional cultural methods, use of the Bioscreen enabled these authors to rapidly and effectively assess the antimicrobial potential of AHF and its 10 derivatives against this panel of microbes. Similarly, Thomas and colleagues used the Bioscreen to characterize the antimicrobial effects of ascopyrone P (APP), a fungal secondary metabolite derived from the AHF pathway, against a selection of 12 microbes; Elsser and colleagues conducted an expanded screen of APP against a larger set of organisms. These publications prominently feature the informationally-dense, high-resolution growth curves that can be obtained using the Bioscreen instrument (Thomas et al., 2002; Elsser et al., 2003). Other researchers have leveraged the Bioscreen for examining additional sugar-derived compounds, namely furanose-based bicyclic molecules, for their antimicrobial activities (Sas et al, 2008). Finally, although not a sugar, the amino acid glycine is used as a sweetener or as a flavor-rounding component in sweetener compositions. The activities of glycine-based antimicrobials have been determined using Bioscreen-based analyses, with some of these compounds being shown to have efficacy in foods (Bontenbal and De Bert, 2006). Together, these papers, patents and patent applications highlight the vital benefits of the Bioscreen in today's industrial antimicrobial discovery programs.

Although we have focused here primarily on direct use of sugars and sugar-derived compounds as antimicrobials, simple sugars may also be useful as molecular "switches"for controlling gene activity in support of screening other compounds for biological activities. For example, in the pharmaceutical industry, screening of large compound libraries for specific activities is problematic, as some compounds may show  "off target", non-specific antimicrobial activities stemming from physicochemical properties such as detergency. To address this, DeVito et al. (2002) engineered a set of bacterial strains in which the intracellular levels of individual target proteins could be altered. In this way, these strains were made to act as cellular "canaries", reporting the inhibition of specific essential enzymes within the cell when exposed to library compounds targeting these enzymes. In each strain, target genes were placed under the control of an arabinose promoter, so that by adjusting the extracellular concentration of this simple sugar, the levels of the target protein could be adjusted. The Bioscreen played a critical role in this work by enabling these researchers to select the optimal level of arabinose to use for each gene-specific library screening (DeVito et al., 2002).

In sum, the Bioscreen C is a powerful and flexible screening tool that has the capacity to accelerate the pace and throughput of antimicrobial and drug discovery, as highlighted here for sugar-based antimicrobial compounds. From this work, it's clear that the Bioscreen has played a vital supporting role in helping us obtain our "sweet revenge" against disease-causing microbes.

References:

Bontenbal, E.E.W., T.V. De Bert. 2006. Use of glycine and/or glycine derivatives as antibacterial agent against gram negative bacterial pathogens in foods and/or drinks. U.S. Patent Application US 2006/0127546 A1.

Bunger, J., Schneider, G., Schreiber, J., Teichmann, S., and F. Wolf. 2002. Use of sugar derivatives as antimicrobial, antimycotic and/or antiviral active substances. U.S. Patent Application US 2002/0165168 A1.

Danisco, Inc. Frequently Asked Questions: Anhydrofructose (AHF). 06 September 2009.

DeVito, J.A., Mills, J.A., Liu, V.G., Agarwal, A., Sizemore, C.F., Yao, Z., Stoughton, D.M., Cappielo, M.G., Barbosa, M.D.F.S., Foster, L.A., and D.L. Pompliano. 2002. An array of target-specific screening strains for antibacterial discovery. Nature Biotechnology 20: 478-483.

Elsser, D., Morgan, A.J., Thomas, V.T. and S. Yu. 2003. Antimicrobial agent. U.S. Patent Application US 2003/0203963 A1.

Fiskesund, R., Thomas, L.V., Schobert, M., Ernberg, I., Lundt, I. and S. Yu. 2009. Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and Ðnegative bacteria, yeasts and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines. Journal of Applied Microbiology 106: 624-633.

Hsu, K.L., Pilobello, K.T., and L.K. Mahal. 2006. Analyzing the dynamic bacterial glycome with a lectin microarray approach. Natural Chemical Biology 2: 1537-157.

Sas, B., Van hemel, J., Vandenkerckhove, J., Peys, E., Van der Eycken, J., and S. Van Hoof. 2008. Furanose-type bicyclic carbohydrates with biological activity. U.S. Patent 7,368,475 B2.

Sõderling, E.M., Ekman, T., and T.J. Taipale. 2008. Growth inhibition of Streptococcus mutans with low xylitol concentrations. Current Microbiology 56: 382-385.

Thomas, L.V., Yu, S., Ingram, R.E., Refdahl, C., Elsser, D., and J. Delves-Broughton. 2002. Ascopyrone P, a novel antibacterial derived from fungi. Journal of Applied Microbiology 93: 679-705.

The Journal of Visualized Experiments (JoVE) is a new peer-reviewed "online research journal employing visualization to increase reproducibility and transparency in biological sciences" http://www.jove.com. JoVE Video-Articles may be in the form of original research articles, or tutorial-like visualizations of experimental methods and techniques. This journal's YouTube-meets-periodical format opens an entirely new dimension in scientific reporting, providing visual learners with access to material not readily available through the written word alone.

An excellent example of the JoVE format is a recently published tutorial on "Quantifying yeast chronological life span by outgrowth of aged cells" (Murakami and Kaeberlein, 2009) This tutorial describes the use of the Bioscreen-C Microbiological Reader, a versatile, automated, self-contained incubation and analysis unit capable of evaluating up to 200 samples over growth periods varying from several hours to several days. With the Bioscreen, increases in optical density can be followed at discrete intervals and plotted against time, allowing the generation of rich and informative data sets. In this work, Murakami and Kaeberlein leveraged the capabilities of the Bioscreen-C to evaluate the effects of different growth conditions on yeast chronological aging. Briefly, S. cerevisiae cells were aged under defined conditions, with constant agitation. At various time points, a subpopulation of cells was removed and inoculated into a rich medium. High-resolution growth curves were generated using the Bioscreen-C and an algorithm was used to determine the relative proportion of viable cells in each sampled subpopulation, yielding a measurement of yeast chronological aging. Apart from serving as a model for aging in higher organisms, understanding and control of cell aging in yeasts also has tremendous potential for maximizing the efficiency and productivity of industrial fermentations such as in the brewing of beer (Powell et al., 2000).

Murakami and Kaeberlein's JoVE protocol provides step-by-step instructions on their process, ranging from preparation of aging cultures to sampling cells at each age-point, and loading the Bioscreen-C MBR instrument to analysis of the resulting data. The protocol is provided as a YouTube-like instructional video with accompanying PDF directions. The viewer's ability to watch each hands-on step of the reported work provides instant and invaluable insight into the procedures used and facilitates reproduction of the results. Although JoVE is a subscription-based journal, articles can be accessed on a trial basis after registering as a JoVE user. Describing their Bioscreen-based visual protocol for yeast cell aging, Murakami and Kaeberlein state that "...In direct comparison with low-throughput chronological life span assays, this method has been shown to achieve comparable (or better) precision, while substantially increasing the number of samples that can be analyzed…". Readers can access this protocol at the following address: http://www.jove.com/index/details.stp?ID=1156 A picture is worth a thousand words. An instructional video protocol is priceless.

References:

Murakami, C., and M. Kaeberlein. 2009. Quantifying yeast chronological life span by outgrowth of aged cells. J. Vis. Exp. doi: 10.3791/1156.

Powell, C.D., Van Zandycke, S.M., Quain, D.E., and K.A. Smart. 2000. Replicative ageing and senescence in Saccharomyces cerevisiae and the impact on brewing fermentations. Microbiology 146: 1023-1034.

Whether you’re a person, a rat, a yeast or a bacterium, aging and death are inescapable facts of life. Yet although it is such a fundamental process, the molecular mechanisms behind aging are still poorly understood. Many factors have been suggested as mechanisms for the aging process, including telomere shortening, oxidative damage caused by free radicals, DNA mutation and others (Burtner et al., 2009). Still, the mystery remains and scientists continue to search for the “key” to this process. If aging can someday be ascribed to a defined set of biochemical events or molecular mechanisms, would this knowledge allow us to someday control (or stop or reverse) the aging process? Beyond simply satisfying the strong human drive to explain the natural world, discovery of this long sought after “fountain of youth” could have almost unimaginable consequences on society and on the globe. Even though the thought is tantalizing, a “cure” for death will probably (hopefully?) remain in the realm of science fiction. Still, discoveries about the molecular mechanisms of cellular aging could hold the keys to curing cancer and other diseases, and this is a very active area of research.

Several lines of experimentation have provided glimpses into possible mechanisms behind the aging process. For example, it has long been known that the life span of the nematode and model organism Caenorhabditis elegans can be extended significantly under low-oxygen conditions. Honda et al., (1993) reported that the mean life span of worms cultivated in an atmosphere containing only 1% oxygen was extended by 21%, while that of worms grown under 60% oxygen was reduced by more than half (Honda et al., 1993). Alternatively, complex polyphenols found in red wine are known to promote cardiovascular health in humans. If drinking red wine is so good for people, how does it affect yeasts, which, after all, are steeped in the stuff? Interestingly, resveratrol, the same red wine polyphenol found to be beneficial to humans has also been shown to extend the life span of the yeast Saccharomyces cerevisiae by up to 70% (Howitz et al., 2003). Finally, in rodents, worms and yeasts, caloric restriction, where the diet provides only 30-40% of the normal level of calories, is also known to extend life span (Guarente and Kenyon, 2000). How do these very different factors lead to such similar outcomes? Is it possible that reduced oxygen, resveratrol and calorie restriction all act on similar pathways at the molecular level?

In a recent study, Burtner et al., 2009 Burtner et al., 2009 sought to learn more about how dietary restriction might affect yeast chronological aging - defined as the length of time yeast cells can survive in a non-dividing, quiescent state. A key tool used in this work was a method for high-throughput quantitative analysis of yeast chronological life span developed earlier by this group (Murakami et al., 2008). Briefly, S. cerevisiae cells are aged in a defined growth medium, with constant agitation. At various time points in the aging process, a subpopulation of cells is removed, inoculated into a rich medium and high-resolution growth curves are generated using a Bioscreen-C Microbiology Reader (Growth Curves, USA). An algorithm is then used to determine the relative proportion of viable cells in each sampled subpopulation based on growth kinetics. This provides a measurement of chronological aging. Unlike traditional plating-based assays, the Bioscreen method saves time and increases throughput while still ensuring both reproducibility and precision.

Using this technique, Burtner et al. (2009) investigated several environmental conditions known to extend yeast life span. These included use of synthetic complete (SC) medium with four- to forty-fold less glucose than the control (dietary restriction), growth in 3% glycerol (a non-fermentable carbon source) and transfer of glucose-grown cells to water (dilution) after 2 days of culture in SC + 2% glucose. From these experiments, evidence for the role of an extrinsic or environmental factor in yeast chronological aging began to emerge. One clear difference between control and restricted diet treatments was the lower final pH of the controls. HPLC analysis of spent media and additional experimentation indicated that the key factor responsible for chronological aging in control treatments was the buildup of the toxic fermentation end product acetic acid in the medium. Essentially, the more rapidly aging yeasts had been pickled in their own juices.

Yeasts have provided a valuable, experimentally useful model for genetic and physiological mechanisms of aging in eukaryotic cells. It is remarkable that such a simple mechanism can explain the connections between dietary restriction and life span extension in yeasts. The relevance of these findings to the mechanisms governing aging in more complex organisms is not immediately apparent, but these findings suggest that care is needed when extrapolating observations made across such large evolutionary distances. This work underscores the utility of the Bioscreen-C Microbiology Reader in helping to answer some of biology’s most fundamental questions. As is often the case in science, it has uncovered an entirely new area of inquiry. This, too, can undoubtedly benefit from use of the Bioscreen.

References:

Burtner, C.R., Murakami, C.J., Kennedy, B.K., and M. Kaeberlein. 2009. A molecular mechanism of chronological aging in yeast. Cell Cycle 8: 1-15.

Guarente, L., and C. Kenyon. 2000. Genetic pathways that regulate ageing in model organisms. Nature 408: 255-262.

Honda, S., Ishii, N., Suzuki, K., and M. Matsuo. 1993. Oxygen-dependent perturbation of life span and aging rate in the nematode. Journal of Gerontology: Biological Sciences 2: B57-B61.

Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., Scherer, B., and D.A. Sinclair. 2003. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425:191-196.

Murakami, C.J., Burtner, C.R., Kennedy, B.K., and M. Kaeberlein. 2008. A method for high-throughput quantitative analysis of yeast chronological life span. Journal of Gerontology A 63:113-21.

You eat “bad” food. You get sick. You go to the doctor. We know from recent recalls (Salmonella on jalapeño peppers or in peanut butter, E. coli O157:H7 in bagged lettuce, etc.) that food can be an important vector for clinically important pathogens that are present as contaminants.

But what about the natural flora of foods that are not technically considered to be contaminated or adulterated? Can these be the next clinical threat? Although some processed foods are either commercially sterile or have very low microbial counts, most foods carry some level of natural flora. Some foods, like alfalfa sprouts, artisanal cheeses and fermented products like sausages or kimchi are self-contained and microbiologically complex mini-ecosystems (Kim and Chun, 2005; Loui et al., 2008; Mounier et al., 2008).

The natural flora of such foods may contain not only starter cultures, but also many other types of organisms important to the development of flavor, aroma, color and other desirable characteristics of the food. Some of these microorganisms may even be closely related to known pathogens. For example, the sausage starter Staphylococcus carnosus belongs to the same genus as the foodborne pathogen Staphylococcus aureus (Rosenstein et al., 2009).

Sometimes, these “kissing cousin” relationships between desirable food flora and undesirable clinical pathogens can cause problems. Food-to-clinical connections are a part of life that we are becoming increasingly aware of, after people become ill.

How can we make these connections between our foods and the clinic before we have a problem? Carlos, et al. (2009) studied enterococci from ewe’s milk and artisanal cheeses made from this milk. The genus Enterococcus is a double-edged microbial sword – some enterococci are aesthetically valuable as cheese ripening flora or have important health-promoting, probiotic activities. Others are emerging as an important branch of antibiotic-resistant clinical pathogens.

Concerned by upward trends in clinical infections involving E. faecalis and E. faecium, two of several enterococcal species routinely found in artisanal cheeses, Carlos, et al. (2009) used a Bioscreen-C automated microbiology growth curve analysis system for side-by-side comparisons of reference strain enterococci, enterococci isolated from artisanal cheeses and those isolated from clinical infections.

They used media that simulated the nutritional conditions found in environmental colonization and infection sites (brain-heart infusion broth, skim milk, urine, rabbit serum, etc.) and varied conditions such as pH, salt concentration and temperature. Leveraging Bioscreen-C’s unique ability to generate and record detailed and precise growth curve data for each condition, Carlos, et al. (2009) calculated “relative indices” or RIs for these isolates, based on each strain’s ability to grow in a given environment.

Disturbingly, similar or higher RIs were calculated for food-derived strains grown under infection-simulating conditions than for the clinical isolates themselves. These results underscore the adaptability of this genus to new environments and suggest the potential for ready migration of food-derived enterococci into new infectious niches within the human body.

This work provides a clear example of how Bioscreen-C can be applied for simultaneous, high-throughput phenotypic comparison of multiple microbial strains, allowing these researchers to make valuable connections between the kitchen and the clinic – an increasingly well-traveled microbial thoroughfare.

References:

Carlos, A.R., Santos, J., Semedo-Lemsaddek, T., Barreto-Crespo, M.T., and R. Tenreiro. 2009. Enterococci from artisanal dairy products show high levels of adaptability. Int. J. Food Microbiol. 129: 194-199.

Kim, M. and J. Chun. 2005. Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis. Int. J. Food Microbiol. 103: 91-96.

Loui, C., Grigoryan, G., Huang, H., Riley, L.W. and S. Lu. 2008. Bacterial communities associated with retail alfalfa sprouts. J. Food Prot. 71: 200-204.

Mounier, J., Monnet, C., Vallaeys, T., Arditi, R., Sarthou, A.S., Hélias, A., and F. Irlinger. 2007. Microbial interactions within a cheese microbial community. Appl. Environ. Microbiol. 74: 172-181.

Rosenstein, R., Nerz, C., Biswas, L., Resch, A., Raddatz., G., Schuster, S.C. and F. Götz. 2009. Genome analysis of the meat starter culture bacterium Staphylococcus carnosus TM300.

For more information, clickhere and enter Cheese in the subject line of the line of the email or call 732-457-9070.

The human mouth is a rich and microbiologically diverse natural environment, playing host to over six hundred different types of bacteria alone. The various surfaces in the oral cavity (floor of the mouth, bottom of the tongue, roof of the mouth and the teeth), are selectively colonized by different types of microbes (Mager et al., 2003). The ecology of the human mouth changes over time, with events such as the eruption of a child’s first teeth providing new habitat for hard surface-colonizing species such as S. mutans. It is the chief organism responsible for cavities - an age-old problem for mankind (Sealy et al., 1992), and marks the end for sweet-smelling “babies breath”.

The composition of the oral flora can also be affected by external factors, including diet, smoking, antibiotic therapy, drug use or systemic disease. It’s been suggested that changes in the composition of the salivary microflora can be used as an early indicator for oral cancer (Mager et al., 2005). Apart from the “above ground” portion of the tooth, the gingival crevice (the space between the surface of the tooth and the surrounding gum) supports the growth of a number of anaerobic genera, including Actinomyces, Bacteroides, Propionibacterium and spirochetes such as Treponema (Sutter et al., 1984).

To study this complex microbial habitat, oral microbiologists need powerful tools. Some researchers want to quantify the in vitro activities of promising agents for control of periodontal disease, including chlorhexidine, triclosan, antimicrobial peptides, and other novel biocides. Others seek to correlate changes in key genes belonging to oral pathogens with the impact these changes have on cellular fitness and growth.

One instrument that has been used widely in the study of human oral microflora is the Bioscreen-C Automated Growth Curve Analysis System. Bioscreen-C is a versatile automated system that enables relevant studies to be carried out on microbes of concern to oral health. These studies include antimicrobial development and biocide testing, modeling of pathogen growth, and basic physiological characterization of mutants.

Fraud et al. (2005) used Bioscreen-C to determine the minimum amount of amine oxide (C10-C16-alkyldimethyl N-oxides) needed to inhibit the growth of S. mutans NCTC 10449 in laboratory media. These experiments showed that a low amount (0.006% v/v) of amine oxide was bacteriostatic against S. mutans, highlighting the potential of this agent for use in combating dental caries (cavities) and other forms of periodontal disease.

Writing in the prestigious journal Proceedings of the National Academies of Science (PNAS), Hasona et al. (2005) unlocked the power of the Bioscreen-C to help probe the genetic bases for key physiological properties that make S. mutans so problematic as an oral pathogen, namely resistance to stresses brought on by rapid changes in environmental conditions, such as pH.

These studies and others demonstrate the utility of Bioscreen-C for probing the secrets of the human oral microbiome and suggesting new opportunities for understanding and controlling periodontal disease caused by the microbes that live among (and on) us.

Reference:

Fraud, S., J.-Y. Maillard, M.A., Kaminski, and G. W. Hanlon. 2005. Activity of amine oxide against biofilms of Streptococcus mutans: a potential biocide for oral care formulations. J. Antimicrob. Chemother. 56: 672-677.

Hasona, A., Crowley, P.J., Levesque, C.M., Mair, R.W., Cvitkovitch, D.G., and A.S. Bleiweis. 2005. Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2. Proc. Natl. Acad. Sci. U.S.A. 48: 17466-17471.

Levesque, C. M., Mair, R.W., Perry, J.A., Lau, P.C.Y., Cvitkovitch, D.G. 2007 Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties. Letters in Applied Microbiology 45: 398-404.

Mager, D.L., Ximenez-Fyvie, L.A., Haffajee, A.D., and S.S. Socransky. 2003. Distribution of selected bacterial species on intraoral surfaces. J. Clin. Periodontol. 30: 644-654.

Mager, D.L., Haffajee, A.D., Devlin, P.M., Norris, C.M., Posner, M.R., and J.M. Goodson. 2005. The salivary microbiota as a diagnostic indicator of oral cancer: a descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects. J. Transl. Med. 3: 27.

Sealy, J.C., Patrick, M.K., Morris, A.G., and D. Alder. 1992. Diet and dental caries among later stone age inhabitants of the Cape Province, South Africa. Am. J. Phys. Anthropol. 88: 123-134.

Sutter, V.L. 1984. Anaerobes as normal oral flora. Rev. Infect. Dis. 6 Suppl. 1: S62-66

For more information, clickhere and enter Teeth, Tongue and Beyond in the subject line of the line of the email or call 732-457-9070.

Bacterial spores are marvels of suspended animation. Under the correct conditions, they can remain viable, able to germinate after thousands of years or longer. Viable spores have even been isolated from the gut contents of ancient insects trapped in 25-40 million year old amber! The ability to enter into such an inherently rugged dormant form is an excellent survival mechanism, enabling spore-forming bacteria to endure harsh or unfavorable environmental conditions that are lethal to other forms of microbial life. Unfortunately, their durability and persistence also makes them problematic to humans, especially from a food safety perspective.

One spore-forming species, Clostridium botulinum, makes a toxin (botulinum toxin) that is considered the most poisonous natural substance known. It is so deadly that less than a pound of pure toxin would be enough to kill every person on the planet. Botulism, the disease associated with ingestion of this toxin, has been linked primarily to improperly canned foods, but can also occur in less processed foods such as sausages, uneviscerated salted fish, garlic-infused olive oil and unpasteurized carrot juice. However, only the cellular form of C. botulinum produces botulinum toxin – the spores themselves are relatively inert. Therefore, control of spore germination and subsequent outgrowth of vegetative cells may be an important means for preventing the production of botulinum toxin in at-risk foods.

Certain biochemicals, including the amino acid L-alanine (and even some synthetic compounds), have been shown to acts as triggers for spore germination. For some types of C. botulinum, though, little is known about which of these “germinants” are most potent or under which conditions (pH, temperature, oxygen tension) they work best. Typically, phase contrast microscopy is used to test for and visually score spore germination in the presence of potential germinants. In this approach, spore germination is detected when the spore undergoes a “phase bright” to “phase dark” transition. Alternatively, a spectrophotometer may be used to follow this transition. Unfortunately, these methods are tedious, time-consuming and not suited for high-throughput studies where testing of multiple germinants, bacterial strains, or environmental conditions is desired.

In a novel adaptation of technology designed for measuring the growth of vegetative microbial cells, researchers at the Institute for Food Research in Norwich, England used the Bioscreen-C Microbiological Reader for measuring bacterial spore germination (Plowman and Peck, 2002). While cell growth is characterized by increases in optical density (OD), spore germination results in a decrease in the OD of a bacterial spore suspension – both of which are measurable by the Bioscreen instrument. The Bioscreen is a self-contained incubation and analysis unit capable of evaluating up to 200 sample wells over growth periods varying from several hours to several days. Changes in optical density can be followed at discrete intervals and plotted against time, allowing the generation of rich and informative data sets. The high-throughput format of the Bioscreen instrument enabled these researchers to screen the impact of several variables and their combinations on the germination of three strains of non-proteolytic C. botulinum. Variables tested included eleven potential germinants and appropriate non-germinant controls, the use of heat-activated and unheated spores, aerobic vs. anaerobic conditions and the effect of temperature on germinant efficacy. Because the Bioscreen provides time-resolved readings, it was also possible to follow the kinetics of spore germination as a function of temperature. To achieve anaerobic conditions or refrigeration temperatures, these workers found that the entire instrument could be placed inside an anaerobic tent or in a refrigerated workspace, highlighting not only the versatility of this instrument, but also its mechanical stability.

Plowman and Peck concluded that “…The experimental procedure, using the Bioscreen system, proved to be highly efficient in determining the effect of a large number of potential germinants…” and that “…Spore germination measured using the Bioscreen system correlated well with direct counts of phase-dark spores by phase-contrast microscopy…”. The information gained from this novel application of the Bioscreen-C instrument has resulted in an increase in our understanding of factors contributing to the germination of C. botulinum spores. Ultimately, this information may lead to new and effective measures for improved food safety through detection and control of these spores in at-risk foods.

Reference:

For more information, clickhere and enter Got Spores in the subject line of the line of the email or call 732-457-9070.

With their short generation times and malleable genomes, bacteria are nature’s ultimate “adaptability machines” - able to evolve quickly to take advantage of prevailing environmental conditions and to adjust to selective pressures that would quickly dispatch less flexible life forms. Because of this trait, bacterial resistance to antibiotics is a continual concern in the healthcare and medical fields. Group B streptococci (GBS), part of the normal gut and urogenital flora of many women, can be passed to an infant during birth and are therefore an important cause of neonatal sepsis, sometimes leading to death. Because of this risk, women are commonly tested for GBS carriage prior to the onset of labor. If GBS are detected, a common practice is to administer intravenous penicillin, or other antibiotics, to prevent mother-to-child transmission. However, bacterial resistance to antibiotics can eliminate the protective effects of this practice and put these infants back at risk for infection. Additionally, some patients are allergic to penicillin, and alternative antibiotics are needed. In clinical practice, rapid methods for identifying microbial resistance patterns have the potential to not only enhance patient outcome, but also to reduce the length of hospitalization and costs associated with ineffective or misdirected antimicrobial therapies.

The Bioscreen-C automated turbidimeter is a versatile microbiological testing platform capable of analyzing up to 200 samples at a time, and is therefore ideal for high-throughput determinations of microbial resistance, using existing Clinical and Laboratory Standards Institute (CLSI) broth microdilution protocols. Simoes et al., used the Bioscreen-C instrument and the CLSI broth microdilution assay to determine the in vitro resistance profiles of 52 clinical GBS isolates to 12 different antibiotics, including penicillin. Thirty five percent of the clinical isolates examined were found to be resistant to half of the antibiotics tested. These authors were able to effectively use the Bioscreen-C instrument to survey the susceptibility patterns of several patient GBS isolates to clinically available antibiotics. These data will be helpful in determining suitable alternatives to penicillin and in identifying antibiotic treatments that do not select for resistance among resident urogenital microflora. The outcome of this work is expected to enable effective chemoprophylaxis against GBS, even in patients with allergies to penicillin, and may lead to reduced infant mortality from GBS infection.

Reference:

For more information, clickhere and enter HT-ARB in the subject line of the line of the email or call 732-457-9070.

Throughout human history, plants have played an important role not only as a key source of nutrients, but also as a source of biologically active substances offering relief and therapy for a wide range of conditions. The discovery of penicillin early in the last century – an event that inaugurated the “Age of Antibiotics” - shifted humankind’s attention from naturally derived drugs and therapeutics to an ever-expanding library of synthetic and semi-synthetic agents. Although these “wonder drugs” have since saved millions of lives and substantially reduced human suffering, their overuse or misuse over the past 50 years has led to the emergence of antibiotic and antimicrobial-resistant bacteria. This development has impacted both the healthcare and food production industries and has led to a resurgence of interest in non-antibiotic alternatives for control of bacterial infection and contamination of foods.

Plants have co-evolved with bacteria for millions of years and have developed a number of remarkably effective strategies for dealing with bacterial infection. In addition to protective coverings, such as the waxy cuticle of leaves, plants also produce a number of antimicrobial compounds known collectively as “phytoalexins”. Compounds in the phytoalexin family include alkaloids, phenolics, coumarins, organic acids and terpenes. Terpenes are the principal component of plant “essential oils” – aromatic oils derived from plant material via steam distillation. Because they have pleasing aromas, essential oils have traditionally been used in perfumery applications. However, in recent years, their potent antimicrobial activities have elicited keen interest from microbiologists, including food microbiologists.

The strong aromas and flavors of essential oils preclude their use in some food applications, but may be complementary in others. For example, Knight and McKellar examined the use of clove or cinnamon essential oils and related food-grade flavorants for their effects against Escherichia coli O157:H7 in apple cider, where cinnamon and clove are traditionally used as mulling spices. The Bioscreen-C Microbiological Reader played a central role in this work, facilitating the screening of a large number of different treatments and variables. Briefly, duplicate concentrations of cinnamon, clove or lemon oils, or the essential oil-derived flavorants geraniol, methyl jasmonate, p-anisaldehyde, (R)-(-)-carvone or (S)-(-)-perillaldehyde were prepared in rich growth media (tryptic soy broth, or TSB). Oil or flavorant concentrations ranged from 0.001% to 0.11% (vol/vol). A standardized inoculum of E. coli O157:H7 was added to each well and the plates were incubated at 37ºC for 5 days. Optical density measurements were taken every 20 min, yielding high-resolution growth curves for each set of duplicate treatments. As additional variables, each reading was carried out in “standard” (pH 7.2) or acidified (pH 4.5) TSB and the effects of a stabilizing agent (0.15% agar) on essential oil or flavorant activities were examined.

These authors reported strong inhibition of E. coli O157:H7 by cinnamon and clove oils under both neutral and acidic conditions, moderate inhibition by (R)-(-)-carvone and (S)-(-)-perillaldehyde under both conditions, moderate inhibition by citral and geraniol only under acidic conditions and little or no inhibition by lemon oil, methyl jasmonate or p-anisaldehyde under either condition. No statistically significant impact was observed for 0.15% agar used as a stabilizing agent. This initial screen was followed by plate count-based studies on the effects of the two top oils (cinnamon and clove) combined with a mild heating process. It was discovered that low concentrations of these oils (0.01%) resulted in enhanced killing of E. coli O157:H7 after mild heat heating.

These results are of great practical impact to cider producers, who must demonstrate a 5-log reduction process for E. coli O157:H7 in this product. However, the number of experimental variables needed to directly compare the various treatments and determine which oils were most effective would be daunting if only manual methods for microbial evaluation were available. These authors noted that the “…Bioscreen Microbiological Growth Analyzer provides an efficient, nondestructive, reproducible, and rapid method for screening large numbers of antimicrobial compounds…”. Used as described here, the Bioscreen-C provides and effective solution for speeding the development of new natural antimicrobial treatments for use in both the food and healthcare fields and can serve as a key platform in accelerated formulation and preservation studies.

Reference:

For more information, clickhere and enter HT-NAM in the subject line of the line of the email or call 732-457-9070.