Green Nanosilver - Worth Its Weight in Gold
Earlier Hot Topic Articles
The Incredible Antimicrobial Egg - Applications of the Bioscreen C in Formulation of Natural Antimicrobial Systems and in Discovery of New Bacterial Defense Strategies
Stuck On You - Applications of the Bioscreen C Microbiology Reader in Parasitology
Sick Economy: The Impact of Infectious Disease - Effectiveness of antimicrobials as measured with Bioscreen C.
Rugged Individualism: The Impact of Strain or Cell Variability on Food Safety - measuring variability of spore generation of foodborne pathogens.
"All I Need is the Air That I Breathe” - studying anerobic bacteria with Bioscreen-C
Nanotechnology – the science of things on the order of one-billionth of a meter in size – is coming to a neighborhood near you and promises to make a BIG impact on just about every aspect of your life. The list of consumer products containing nanoscale materials is surprisingly large and growing, containing everything from sunscreen and house paint to antimicrobial toothbrushes and odor-fighting socks. The Project on Emerging Nanotechnologies (PEN; http://www.nanotechproject.org/cpi/), a “living” catalog of nano-based consumer products now contains over 1,600 entries, up from just 54 in 2005. These include products in categories ranging from “Health and Fitness” to “Home and Garden” and from “Food and Beverage” to “Goods for Children” (!). Although the site lists a variety of active nano components, including carbon nanotubes, nanoclays and titanium dioxide, the majority of the products for which the active technology is divulged contain silver nanoparticles (“nanosilver”) primarily for it’s antimicrobial properties. Nanosilver is a highly effective antimicrobial, active against a broad range of microorganisms, including foodborne pathogens such as Salmonella and clinically important pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). The ultimate safety of nanosilver particles and products containing them is a topic of hot debate on it’s own, but some of the processes for their manufacture – high-vacuum deposition of silver vaporized by a plasma torch at temperatures as high as 17,500 degrees Fahrenheit, or electrical explosion of silver wire, for example – may represent acute environmental hazards.
The emerging field of “Green” chemistry is focused on developing new synthesis techniques that minimize the negative environmental impacts often characteristic of traditional methods, many of which actually generate more waste material than product. Green chemical processes are therefore seen as sustainable alternatives to current wasteful and environmentally unfriendly approaches for manufacture of chemical intermediates or finished materials. Green chemical methods may use natural raw materials, generate valuable end products from low-value waste materials and/or replace toxic organic solvents with water-based systems containing biodegradable active agents such as enzymes and zero-residue physical processes such as ultrasound or high pressure.
Apart from high-temperature plasma vaporization and underwater electrical explosion of silver wire, nanosilver particles may be made by combining a silver salt, such as silver nitrate, with a chemical reducing agent, such as sodium borohydride. Unfortunately, the Material Safety Data Sheet (MSDS) for sodium borohydride lists the following safety warnings: “very toxic by inhalation”, “causes severe burns”, “risk of serious damage to eyes” and “reacts violently with water liberating extremely flammable gasses”. Alternatively, bio-derived materials can be used as sources of natural reducing power and can provide surfaces in colloidal suspension that can act as “seeds” for nucleation and growth (a very biological term!) of silver nanoparticles. In fact, there has been so much interest in this approach that a practically cornucopic selection of esoteric biological extracts, infusions, broths, decoctions or tinctures has been examined. For example, resourceful scientists have successfully grown “green” silver (or gold) particles using spent liquids left over from mushroom production, bacterial or fungal lysates, aqueous earthworm extracts (a fancy name for “worm water”), marine sponges and extracts of water hyacinth, Madagascar rosy periwinkle, honey, tea and herbs (sage) or spices (clove and cinnamon). Bioreduction of metal ions into metallic particles using bacteriophage - viruses that infect bacteria - has even been suggested as a green alternative to cyanide leaching or mercury extraction for reclamation of precious metals such as gold from mining effluents.
Depending on how they are generated, silver nanoparticles can vary widely in size and shape, with possible shapes including spheres, rods and even triangles. Because particle size and shape govern both available surface area and physical interactions with microorganisms, these parameters also affect nanosilver efficacy. Therefore, not all silver nanoparticles are created equal, and approaches for measuring and comparing the biological activities of particles generated using different bioreduction methods are needed.
Okafor et al., (2013) described the use of boiled leaf extracts from various plants to generate silver nanoparticles - a truly green process! Leaves used included Black Cohosh, Magnolia, Aloe, Eucalyptus and Geranium. These researchers then used a Bioscreen C Microbiological Reader (Growth Curves USA, Piscataway, NJ) to evaluate the antimicrobial effects of these particles against Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Kocuria rhizophila and Bacillus thuringiensis. Briefly, suspensions of bacterial cells and the various leaf-derived nanosilver particles were made in nutrient broth and samples were incubated for 24 h at 37°C, with optical density measurements taken at 15 min intervals. The Bioscreen instrument is a high-throughput, precision-incubating automated turbidimeter that enables researchers to evaluate up to 200 sample wells over time periods ranging from several hours to several days. Optical density readings are made at discrete intervals and are plotted against time, generating detailed growth curves that can enable quantitative comparisons among antimicrobial treatments or dosages and provide new insights into antimicrobial efficacy and mode of action.
Okafor and colleagues found that although nanosilver particles grown using various leaf types appeared physically similar (i.e. spherical, average size 3-15 nm), they exhibited different biological activities against the various test organisms. For example, Aloe-based particles were the most effective, followed by Black Cohosh. The Geranium-based particles had the lowest antimicrobial activity. Because the particles where physically similar and equal concentrations of each particle type were compared, the observed biological differences could not be attributed to size- or shape-based effects alone, suggesting that other factors affecting antimicrobial efficacy were in play.
These researchers theorized that particles grown using different leaf extracts may contain bioactive molecules unique to the species of plant examined, such as quinones and other aromatic compounds, which have their own antimicrobial properties. The resulting hybrid bio-metallic particles could therefore have unique activities not found in chemically reduced or physically produced nanosilver particles. Further, the antimicrobial spectrum of such particles may vary according to the type of plant extract used to produce them. The ability of the Bioscreen C to quantify differences in antimicrobial activity among these different “green” nanoparticles allowed this team to speculate on why such differences may occur, potentially opening up a new area of research into the use of different biological extracts to grow silver nanoparticles having controllable antimicrobial spectra.
A long-standing drawback of nanosilver has been its broad and indiscriminate action against all types of microbes and even human cells. Nanosilver particles having narrower, more targeted spectra might allow selective killing of pathogens without destroying a person’s normal microbial flora. Combined with minimal toxicity towards mammalian cells, as demonstrated by this group, this new category of green nanoparticle could be just the “silver bullet” needed for control of emerging antibiotic-resistant pathogens. In other words, green silver nanoparticles that are worth their weight in gold.
Kim, H.K, Choi, M.-J. Cha, S.-H. Koo, Y.K., Jun, S.H, Cho, S. and Y. Park. 2013. Earthworm extracts utilized in the green synthesis of gold nanoparticles capable of reinforcing the anticoagulant activities of heparin. Nanoscale Res. Lett. 8: 542.
Nune, S.K., Chanda, N., Shukla, R., Katti, K., Kulkami, R.R., Thilakavathi, S., Mekapothula, S., Kannan, R., and K.V. Katti. 2009. Green nanotechnology from tea: phytochemicals in tea as building blocks for production of biocompatible gold nanoparticles. J. Mater. Chem. 19(19): 2912-2920.
Phillip, D. 2009. Honey mediated green synthesis of gold nanoparticles. Spectrochim. Acta A Mol. Biomol. Spectrosc. 73(4): 650-653.
Setyawati, M.I, Xie, J., and D.T. Leong. 2013. Phage based green chemistry for gold ions reduction and gold retrieval. ACS Appl. Mater. Interfaces doi: 10.1021/am404193j
Vigneshwaran, N., Kathe, A.A., Varadarajan, P.V., Nachane, R.P., and R.H. Balasubramanya. 2007. Silver-protein (core-shell) nanoparticle production using spent mushroom substrate. Langmuir 23: 7113-7117.
Eggs. Small oval marvels. Tight little packages themed in white, brown, turquoise, speckle or pastel, depending on which of the 10,000 species of birds they come from. Even the lowly chicken egg is a masterpiece of millions of years of evolution, a compact and efficient package containing everything needed to support the growth and development of Gallus gallus domesticus, the common chicken. Chicken and other birds’ eggs are an excellent source of protein, vitamins, minerals, carotenoids and other goodies that the developing chick needs to survive and thrive. Unfortunately, what’s good for the developing embryo will also support the growth of other organisms, including bacteria. In the United States, eggs are kept refrigerated from truck to grocery to home. Yet, in other countries or at farmer’s markets, eggs are routinely kept at ambient temperatures without going bad. How is this possible? In short, eggs are self-preserving foods – they possess a combination of physical barriers and biological systems capable of keeping spoilage organisms and pathogens at bay. The first barrier to microbial entry is, of course, the shell. Every savvy shopper knows to inspect their eggs before purchase and if any are cracked, to return the carton to the shelf. Although the shell contains very small pores that allow the egg to “breathe”, these are too small to admit bacteria, and therefore, an intact shell is an excellent barrier to microbial entry.
But like a tiny fortress safeguarding the yolk within, the egg also has secondary lines of defense, in case the outer emplacements are breached. The egg white (albumen) is almost 90% water – but water alone is not much of an obstacle, since bacteria can easily swim a moat. It’s the other 10% dissolved in this water that matters – a suite of more than a dozen antimicrobial proteins and enzymes that work together to directly or indirectly challenge and inhibit microbial invaders. These include lysozyme (an enzyme that degrades the bacterial cell wall), ovotransferrin (also known as conalbumin, an iron-binding protein that scavenges the iron needed for bacterial growth), ovoinhibitor (a protein that inhibits the microbial proteases expressed during infection) and avidin (a protein that strongly binds biotin, also an important growth factor for bacteria) (Feeney et al., 1963; Gould, 2000). Lysozyme is one of the more familiar proteins in this set and is widely distributed throughout the animal kingdom. Lysozyme is found not only in avian eggs, but also in milk, tears, saliva and on termite eggs, where it serves double duty as an antimicrobial and as a signaling pheromone, enabling termites to locate and recognize their own eggs (Matsuura et al., 2007). The fact that termites also respond to hens egg lysozyme suggests its possible use for termite control – potentially enabling us to fool termites into “recognizing” poisonous baits as eggs and taking them back to the colony (Matsuura et al., 2007). Food applications of hens egg lysozyme as a natural antimicrobial include its longstanding use in semi-hard cheeses like Gouda and Gruyère to prevent the “late blowing” defect, which results from gas formation by Clostridium species. Annually, over 100 tons of lysozyme are used for this alone and it has recently been investigated for the preservation of unpasteurized beer (Callewaert et al., 2011; Gould, 2000).
The success of hens egg lysozyme as a natural antimicrobial for the preservation of foods stems from at least three factors. Firstly, it is an effective antimicrobial, particularly against Gram-positive bacteria. Secondly, it is derived from eggs, a common food source, and can therefore be used in any food system where eggs or egg products are allowable. Finally, hens egg lysozyme is present at relatively high levels in eggs (~3.5% of egg white) and it is economical to extract, allowing its cost-effective use at the levels demanded by the food industry. What about the other dozen or so naturally antimicrobial proteins present in egg whites - why aren’t they used as routinely as lysozyme? Ovotransferrin (OTF), for example, is present in egg white at nearly 12% by weight. While OTF is effective against both bacteria and viruses, food-derived and abundant, cost-effective methods for its extraction have traditionally not been available, making it prohibitively expensive for use in foods. Recently, though, Ko and Ahn (2008) developed a simple and economical approach for large-scale purification of OTF from egg whites, potentially opening the door for new food-based applications of this natural antimicrobial protein.
As in the natural example of the egg, food preservatives are most effective when applied using a systems approach. In such systems, each preservative serves as a unique “hurdle” which microbes must overcome in order to grow. The presence of multiple hurdles, each interacting with and supporting the effects of the others, results in microbial inhibition or death. Listeria monocytogenes is a pathogen of great concern as a post-processing contaminant of foods such as frankfurters and deli meats. Although chemical preservatives have long been used effectively for control of L. monocytogenes in meats, consumer demand for “greener” foods is driving the formulation of products that do not contain such compounds. Because safety is paramount, some sort of preservative must be used, and there is substantial interest in discovery of new natural antimicrobial systems or hybrid systems that incorporate lower levels of chemical preservatives. Using the new, cost-effective method for purification of OTF described by Ko and Ahn (2008), Moon et al. (2011) examined the impact of the natural antimicrobial peptide nisin and commonly used meat additives (salt, lactate, lactate-diacetate blend and polyphosphates) on the antimicrobial efficacy of OTF. The overall goal of this work was to identify OTF-based systems using novel combinations of natural or chemical preservatives as supporting hurdles.
The first step in formulation of such systems is to perform in vitro screening in liquid growth media of individual components at various concentrations against the target pathogen. The most inhibitory levels of each component can then be combined in the food and the system monitored for effects on pathogen growth. A major limitation is that this type of screening involves many variables – for example, different antimicrobials, at different concentrations and in different combinations. As a result, comprehensive evaluation of these systems can be daunting and labor intensive when traditional methods such as plating are used. Optical density-based approaches provide a convenient way to monitor cell growth and inhibition, but these can also be difficult to manage if regular test tube-based measurements are used. The Bioscreen C Microbiological Reader is a versatile automated turbidimeter useful in a wide variety of microbiological testing applications. The Bioscreen enables high throughput, microplate-based evaluation of microbial growth in broth culture by tracking increases in optical density. 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. Increases in optical density can be followed at discrete intervals and plotted against time, allowing the generation of rich and informative data sets.
Moon et al. (2011) leveraged the screening power of the Bioscreen to investigate the interactions between OTF, nisin and traditional chemical preservatives in Brain Heart Infusion (BHI) broth. Moon and colleagues found that while 40 mg/ml OTF strongly inhibited the growth of L. monocytogenes in broth, a combination of OTF at this level and 1,000 IU nisin completely inhibited growth in both broth and in frankfurters held at 25°C for 12 h. Importantly, this group determined that 2% salt or 0.05% polyphosphate interfered with the inhibitory effects of OTF, providing critical information regarding product formulation needs if OTF-based preservative systems are to be used in this food. This work highlights the ability of the Bioscreen to efficiently screen for both positive and negative antimicrobial interactions, guiding researchers toward effective combinations and away from antagonistic preservative pairings that could have disastrous implications for food safety.
Because antimicrobial proteins such as lysozyme or OTF are such ancient and widespread antimicrobials, it stands to reason that some bacteria may have developed approaches to circumvent their activities. After all, host-pathogen interactions are marked by a tit-for-tat, measure-countermeasure balancing act of weapons and defenses. Regarding lysozyme, Gram-negative bacteria are intrinsically more resistant to this enzyme than Gram-positive bacteria because they possess an outer membrane, which prevents the enzyme from reaching its cell wall target. Recently, though, Callewaert et al., (2008) have also discovered a new type of proteinaceous periplasmic lysozyme inhibitor that contributes to the lysozyme resistance of Salmonella Enteritidis, a major foodborne pathogen transmitted through consumption of contaminated eggs. Because treatment with lysozyme esults in the lytic destruction of bacterial cells, the activity of this antimicrobial can be followed over time as a function of decreased optical density of a test culture.
In a novel non-growth-based application of the Bioscreen C instrument, Callewaert et al. (2008) reasoned that the presence of lysozyme inhibitors can also be examined using this approach. By combining crude periplasmic extracts or purified proteins, hens egg lysozyme, and the lysozyme indicator organism Micrococcus lysodeikticus, the presence and activity of lysozyme inhibitors could be determined in potassium phosphate buffer over a period of 2 h. This original approach for use of the Bioscreen C enabled this group to compare the lysozyme-inhibiting activities of periplasmic extracts of wild type and strains in which the lysozyme inhibitor was overexpressed. Additionally, the method facilitated the screening of crude periplasmic extracts from a wide variety of bacteria for lysozyme inhibitors. Their preliminary results suggest a wide distribution for this newly discovered virulence factor in bacteria, a fertile topic for future study (Callewaert et al., 2008). Discovery of this new mode for bacterial resistance to lysozyme may enable development of new drugs targeting these inhibitors. Co-application of these drugs with lysozyme may open new avenues for use of hens egg lysozyme for clinical treatments in humans or to address the resistance of S. Enteritidis to lysozyme in layer hens, and ultimately, in eggs. It is expected that the Bioscreen-based method developed by Callewaert and colleagues for quantification of lysozyme inhibition will play a critical role in the screening and development of these new drugs.
The work done by these two groups demonstrates both the remarkable antimicrobial power of egg proteins and how pathogens have evolved effective strategies for addressing the egg’s natural defenses. Both groups applied the Bioscreen instrument advantageously to forward their research in ways that could not have been easily accomplished using traditional microbiological screening methods. As a result, we now have new information about effective combinations of natural egg-based and other antimicrobials for use in food preservation, as well as a glimpse into how we might act to circumvent bacterial resistance to hens egg lysozyme, once again rendering these organisms sensitive to this powerful weapon in nature’s defensive arsenal.
Callewaert, L., Aertsen, A., Deckers, D., Vanoirbeek, K.G.A., Vanderkelen, L., Van Herreweghe, J.M., Masschalck, B., Nakimbugwe, D., Robben, J. and C.W. Michiels. 2008. A new family of lysozyme inhibitors contributing to lysozyme tolerance in gram-negative bacteria. PLoS Pathogens 4(3): e1000019. doi:10.1371/journal.ppat.1000019
Callewaert, L., Walmagh, M., Michiels, C.W., and R. Lavigne. 2011. Food applications of bacterial cell wall hydrolases. Current Opinion in Biotechnology. 22: 164-171.
Feeney, R.E., Stevens, F.C., and D.T. Osuga. 1963. The specificities of chicken ovomucoid and ovoinhibitor. Journal of Biological Chemistry 238: 1415-1418.
Gould, G.W. 2000. Preservation: past, present and future. British Medical Bulletin 56: 84-96.
Ko, K.Y and D.U. Ahn. 2008. An economic and simple purification procedure for the large-scale production of ovotransferrin from egg white. Poultry Science 87: 1440-1450.
Matsuura, K., Tamura, T., Kobayashi, N., Yashiro, T. and S. Tatsumi. 2007. The antibacterial protein lysozyme identified as the termite egg recognition pheromone. PLoS ONE 2(8): e813. doi:10.1371/journal.pone.0000813
Moon, S.H., Paik, H.-D., White, S., Daraba, A., Mendonca, A.F., and D.U. Ahn. 2011. Influence o nisin and selected meat additives on the antimicrobial e ffect of ovotransferrin against Listeria monocytogenes.
Applications of the Bioscreen C Microbiology Reader
In his book “Parasite Rex”, Carl Zimmer states that “Every living thing has at least one parasite that lives inside it or on it. Many, like leopard frogs and humans, have many more…According to one estimate, parasites may outnumber free-living species four to one. In other words, the study of life is, for the most part, parasitology” (Zimmer, 2000). Although this may seem like an zealous overestimation of the importance of parasites, we now know that even bacteria are plagued by parasites - consider the case of the parasitic bacterium Bdellovibrio (literal translation “curved leach”). This organism burrows into the periplasmic space of other Gram-negative bacteria and eats them alive, converting the host cell’s biomass into 20-30 new Bdellovibrio cells, which then burst out of host’s dead husk, looking for fresh prey. New research suggests that this virus-like behavior may be promising for the control of human pathogens, including both free-living cells or biofilms of Salmonella, Pseudomonas, Yersinia and others (Dashiff et al., 2011). Microscopic hindsight is 1,000x/1,000x, as the following poem, written by the satirist and wit Jonathan Swift almost 230 years prior to the discovery of Bdellovibrio and its relatives attests:
“So nat’ralists observe, a flea
Parasitism has been with us since the beginning of time, and as a class of microbes, protozoa (single-celled eukaryotes) have survived multiple geological epochs relatively unchanged, as fossilized examples trapped in 220-million-year-old droplets of amber have shown (Schmidt et al., 2006). In fact, it has recently been suggested that lesions on the mandibles of Tyrannosaurus rex were caused by infection with the protozoan parasite Trichomonas gallinae, an infection that still plagues modern birds, especially raptors (Wolf et al., 2009). Today, members of the genus Trichomonas also infect humans, with T. vaginalis being the leading cause of sexually transmitted disease in industrialized nations. Trichomonas and the related genera Giardia and Spironucleus are also associated with intestinal or topical infections in man and in other animals, including those that humans depend on for sustenance, such as fish. For example, Spironucleus vortens is suspected as the causative agent of hole-in-the-head disease (HITH) of cichlids, a family of fish that includes both ornamental aquarium fish and important food fishes such as tilapia (Millet et al., 2011a). A problem among captive fishes, HITH is a major issue in aquaculture, especially since effective chemotherapeutic agents such as metronidazole have been banned for use in farmed fish destined for human consumption (Millet et al., 2011b).
The ability to grow a pathogen in the lab is prerequisite for studies in metabolism or for screening and development of antimicrobial treatments. However, unlike most bacteria, which are readily culturable, protozoa such as S. vortens can be difficult to grow in the lab. A common practice is to expose protozoa to potentially useful substrates or inhibitory agents in liquid media and to monitor increases or decreases in cell growth or viability directly using an Improved Neubauer haemocytometer (Millet et al., 2011a; Wilkinson, 2006). Unfortunately, this manual approach is time-consuming and is not amenable to high-throughput screening of multiple compounds or even various concentrations of a single compound. Therefore, alternate methods for monitoring the growth or inhibition of S. vortens and other protozoa would be a welcome development. Such methods could significantly improve our abilities to understand the metabolism of poorly understood organisms or to search for and identify alternative antimicrobial agents useful for control of these organisms.
Optical density (OD)-based methods have been used advantageously to follow the growth of bacteria in liquid media, or to detect changes in absorbance associated with germination of bacterial spores. OD-based methods are less labor intensive than plate counting or direct microscopic observation and are non-invasive – measurements can be carried out on closed culture tubes, with the response collected as a function of incubation time. OD-based approaches are therefore very useful for examining the impact of added substrates on growth or the efficacy of antimicrobial agents. However, little has been reported on the use of OD-based methods for the study of protozoa. Recently, though, a group from Cardiff University in Wales has developed an approach for automated OD-based growth monitoring of S. vortens using the Bioscreen C Microbiology Reader (Growth Curves, LLC, Piscataway, NJ). These authors grew S. vortens at 25°C for 120 h, with readings taken every 20 minutes. Maximal yield was achieved after 60 h and a very high cell count was achieved, even though a dilute and simplified medium not containing expensive vitamin supplements was used (Millet et al., 2011a). Although there was excellent correlation between the automated method and direct microscopic counts taken at multiple discrete time points, the Bioscreen C assay was found to be superior, as it revealed a biphasic growth pattern that was not detectable using the traditional approach.
In a separate study, the same group leveraged this new Bioscreen-based approach for growth and monitoring of S. vortens to examine the nutrient requirements of this organism in detail (Millet et al., 2011c). Diluted or full-strength culture media and culture media supplemented with eight other substrates, including glucose, maltose or various amino acids were examined and the impact of supplementation on the first and second exponential growth rates observed during biphasic growth, as well as on total cell yield was determined. The ability to grow S. vortens in the Bioscreen enabled this group to examine multiple treatments in parallel and to perform the replicates needed to determine statistical significance – feats that would not have been possible using the traditional Improved Neubauer direct count method (Millet et al., 2011c).
Finally, in a third study, this group used the Bioscreen to examine the potential use of garlic and garlic-derived compounds as antimicrobials against S. vortens, as a common folk remedy among aquarists for treatment parasitic fish diseases is the feeding of 1 – 2% garlic (Millet et al., 2011b). While garlic compounds have been shown to be effective against a wide variety of bacteria, fungi, viruses and other protozoa, the minimum inhibitory concentrations (MICs) determined here indicated that S. vortens may have an unusually high tolerance to these compounds. Although these results suggest that use of garlic compounds may not represent an effective means for control of this organism, generation of the MICs required to compare these compounds and make this determination would have been difficult or impossible to do without the use of the Bioscreen C method.
The success of the Bioscreen approach for investigating the growth properties, metabolism and antimicrobial susceptibility of S. vortens suggests that similar methods could be used to examine the growth or inhibition of related protozoan parasites involved in human disease, such as Trichomonas or Giardia. A common limitation of microwell-based culture of protozoa is the uneven distribution of gases in different wells, which can affect the growth of organisms that have strict requirements for gas composition, including anaerobiosis (Wilkinson, 2006). The ability of the Bioscreen to operate in an anaerobic chamber for the cultivation of such organisms has been covered in a previous “Hot Topics” entry, suggesting that this approach could be used effectively for the study of anaerobic protozoa involved in human disease.
Leveraging the power of the Bioscreen C for the study of additional protozoan parasites could have wide impact in clinical microbiology as well as in food or environmental microbiology, where the importance of free-living protozoa as potential vectors for bacterial pathogens is only now becoming more widely recognized (Baré et al., 2011).
Baré, J., Houf, K., Verstraete, T., Vaerewijck, M., and K. Sabbe. 2011. Persistence of free-living protozoan communities across rearing cycles in commercial poultry houses. Applied and Environmental Microbiology 77: 1763-1769.
Dashiff, A., Junka, R.A., Libera, M. and D.E. Kadouri. 2011. Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. Journal of Applied Microbiology 110: 431-434.
Millet, C.O.M., Lloyd, D., Williams, C. and J. Cable. 2011a. In vitro culture of the diplomonad fish parasite Spironucelus vortens reveals unusually fast doubling time and atypical biphasic growth. Journal of Fish Diseases 34: 71-73.
Millet, C.O.M., Lloyd, D., Williams, C., Williams, D., Evans, G., Saunders, R.A., and J. Cable. 2011b. Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens. Experimental Parasitology 127: 490-499.
Millet, C.O.M., Lloyd, D., Coogan, M.P., Rumsey, J. and J. Cable. 2011c. Carbohydrate and amino acid metabolism of Spironucleus vortens. Experimental Parasitology (in press) doi: 10.1016/j.exppara.2011.05.025.
Schmidt., A.R., Ragazzi, E., Coppellotti, O., and G. Roghi. 2006. A microworld in Triassic amber. Nature 444: 835.
Swift, J. 1733. On Poetry: A Rhapsody.
Wilkinson, J.M. 2006. Methods for testing the antimicrobial activity of extracts. In Modern Phytomedicine: Turning Medicinal Plants into Drugs, I. Ahmad, F., Aqil and M. Owais, eds). Wiley-VCH, Weinheim, Germany.
Wolf, E.D.S., Salisbury, S.W., Horner, J.R., and D.J. Varricchio. 2009. Common avian infection plagued the tyrant dinosaurs. PLoS ONE 4(9): 37288. doi:10.1371/journal.pone.0007288.
Zimmer, C. 2000. Parasite Rex: Inside the Bizarre World of Nature’s Most Dangerous Creatures. Touchstone Books, New York, NY.
For more information, clickhere and enter Stuck on You in the subject line of the line of the email or call 732-457-9070.
Effectiveness of antimicrobials as measured with Bioscreen C.
Although they are small, pathogenic microorganisms affect our lives in a very big way, causing a huge burden of disease. Statistics on just how big the problem is are available for foodborne illnesses. The most recent estimates are that 9.4 million cases of illness caused by consumption of tainted food occur in the United States each year, resulting in more than 55,000 hospitalizations and almost 1,500 deaths. Although viruses play the largest role, pathogenic bacteria are no slouches when it comes to making people sick.
While we usually think of such illnesses in human or emotional terms, they also have an economic impact. For example, the economic burden of bacterial disease may take the form of direct health care costs (emergency room visits, hospitalization, antimicrobial therapy, etc.) or as indirect costs such as lost wages, reduction in overall productivity due to missed work, reduction in lifetime employment, premature death, cost of litigation, etc. With these factors in mind, the annual cost in the U.S. alone of just one foodborne pathogen – Escherichia coli O157 - has been estimated at over $400 million (Frenzen et al., 2005) ! With a roster of 31 major foodborne pathogens (Scallan et al., 2011a) as well as many others whose impact is not yet fully understood (Scallan et al., 2011b), the cumulative cost of human disease due to foodborne microorganisms is staggering. Compound this further with the costs of infections caused by non-foodborne microorganisms, such as Pseudomonas aeruginosa, which infects patients with cystic fibrosis, and the size of the problem is nothing short of astronomical.
Luckily, humans are not simply a reactive species, but a proactive one as well. We have developed the ability to bend the strengths of the natural world to our favor, harnessing the power of chemical and biochemical tools that we can use to defend ourselves against pathogenic bacteria. This ability gave rise to the antibiotic “renaissance” that lasted roughly from the 1940’s to the 1980’s, where it seemed as though we were on course toward ultimately conquering pathogenic bacteria. However, every story has a twist, and it eventually became clear that the bacteria were also resourceful, and given their short generation times, were able to quickly evolve resistance toward even our best weapons. We now recognize that a multi-level strategy of new antimicrobial development and appropriate, measured use of these antimicrobials is required for their sustained use. However, a key problem is that there are still relatively few classes of compounds currently in the antimicrobial development pipeline. In order to minimize the human and economic impacts of infectious disease, we need new ways to help quickly identify new antimicrobial compounds or treatments and to characterize their effects on pathogens of interest.
The Bioscreen C is an automated microbiology growth curves analysis system capable of measuring up to 200 individual wells simultaneously. Plates can be incubated at temperatures ranging from ambient to 60°C, and optical density readings can be taken using eight different wavelengths at intervals as closely spaced as 5 min. These capabilities allow the Bioscreen C to generate high-throughput growth curves useful for detailed description of microbial responses to test treatments, including antimicrobials, antibiotics or other inhibitory agents. The Bioscreen C can also be used for basic physiological or genetic studies of resistant organisms (Sun et al, 2010). Together, these capabilities make it an excellent platform for the development of the new anti-infectives needed today (and tomorrow).
The most common use of the Bioscreen C is as a tool for direct assessment of chemical toxicity to microbial test strains, as described in other sections of this blog. However, a few resourceful scientists have devised ingenious approaches for alternate tests using the Bioscreen C. An example is its use to monitor the impact of amino acid germinants on Clostridium botulinum spores (see “Got Spores?” entry for further details). However, not all things worth measuring can fit into a test tube, let alone a microplate well. For example, the disease-preventing action of sanitizers or hand rubs is based on their abilities to reduce the load of pathogenic organisms present on hands or other solid surfaces. Adding these antimicrobials directly to liquid microbial growth media is not likely to be a useful predictor of their real-world efficacy. In order to address these apparent limitations and to leverage the power of the Bioscreen C for testing the effectiveness of antimicrobial rubs applied to contaminated surfaces, Cheeseman et al., (2010) used a “carrier test” approach. This involved applying a known concentration of test bacteria to the surface of 2 cm diameter stainless steel discs. To simulate the presence of surface soil, the bacterial suspensions were mixed with bovine serum albumin (3 g/L) prior to inoculation of the discs. Portions of the antimicrobial hand rubs (AHRs) to be tested were added to the surfaces of the inoculated discs and AHRs were allowed to react with the test inoculum for either 10 s, 30 s, 1 min, 2 min or 5 min. Once the contact time had been reached, test discs were placed face down into a 10 ml portion of neutralization broth and surviving bacteria were removed with agitation in the presence of sterile glass beads, then enumerated using traditional pour plating. Alternatively, survival was also quantified using the Bioscreen C instrument by inoculating a portion of neutralized bacterial suspension into Bioscreen C wells containing trypticase soy broth and incubating for 14 h at 37° C (Cheeseman et al., 2010) . Cell numbers were calculated from optical density readings using a linear equation and log reduction values vs. exposure time were determined for the AHRs tested. Using this approach, these authors were able to compare the efficacy of three different antimicrobial hand rubs against a panel of 57 clinical isolates of Staphylococcus aureus, a feat that would have been challenging if traditional plating were used. Interestingly, the efficacy of some AHRs was found to be variable when tested against “real world” isolates of S. aureus, suggesting that their effective use in clinical settings might be limited. Additionally, comparison of traditional plating with the Bioscreen C approach indicated that cell recovery might be possible after as long as 2 min exposure to one of the AHRs, underlining the importance of contact time on AHR efficacy and providing valuable lessons on how this AHR should be applied.
Apart from its use in testing chemical compounds, the Bioscreen C may also be used to provide useful insight into the action of “non-traditional” antimicrobials. For example, bacteriophage are bacteria-infecting viruses (the name literally means “bacteria-eater”). Although bacteriophage (or simply, “phage”) were considered for use as potential antibacterial agents soon after their discovery in the early 1900’s, effective chemical antimicrobials such as petroleum-based dyes were by that time readily available, and the use of phage fell out of favor. In the age of waning antibiotics, however, some investigators are revisiting the concept of phage as therapeutic agents. Like all viruses, phage are peculiar in that they are not really living at all – they have no metabolic activities outside of their bacterial hosts, but like a phalanx of sub-micron zombies, they can invade bacterial defenses and subvert their host’s metabolic machinery to do nothing more than make copy upon copy of themselves. This is done at the expense of the host, which at some point breaks open to spew forth thousands of copies of new phage, each seeking a new host to infect and multiply within. Optical density-based platforms such as the Bioscreen C have clear advantages for characterizing phage-bacteria interactions, as changes in optical density can be used to follow both lysis of infected cells and regrowth of resistant bacteria. Cooper et al., (2011) used the Bioscreen C to quantitate increases in infectivity of phage subjected to a processes intended to select for more virulent phage. Phage (1011 plaque forming units) were mixed with test bacteria (108 colony forming units) in Bioscreen C wells (400 μl total volume each well). The high-throughput capacity of the Bioscreen C enabled the use of 10 replicate wells per phage within each experiment. With each experiment carried out 3 times, 30 different readings were collected per data point, enhancing the reproducibility of the results. In all, 4 phage were screened against 14 isolates of Pseudomonas aeruginosa, including clinical isolates from cystic fibrosis patients. Data were also collected using traditional streak testing, which is a qualitative test. Phage efficacy was measured in the Bioscreen C using two key parameters: bactericidal effect (≥ 2-log decrease in bacterial number after 8 and 20 h) and bacterial regrowth after infection (growth to 0.1 above the original optical density after ≥ 480 min). The Bioscreen C approach developed here was advantageous in several ways: it provided a rapid and quantitative assessment of phage efficacy (extent and rate of bacterial lysis), facilitated the collection of multiple replicates for each phage-bacteria combination, and provided data on bacterial regrowth that were not available using the conventional (and more time-consuming) streak test. Although this study did confirm the utility of the phage-enhancement treatment, these phage failed to suppress regrowth in approximately 50% of the P. aeruginosa strains tested, suggesting that these phage are not yet suitable for therapeutic use. Alternatively, the criteria used by the authors to judge acceptable levels of activity may have been too stringent. Further in vivo efficacy testing of the most effective phage could shed further light on the questions raised in this work.
These two studies highlight the use of the Bioscreen C Microbiological Reader in applications designed to meet disease-causing microbes head-on and develop new anti-infective approaches that may eventually help alleviate the human and economic costs of infectious disease. In both cases, the Bioscreen C was able to detect potential limitations of the treatments tested, limitations that could not be detected using conventional analyses. Realistically, the humans vs. microbes arms race will be an ongoing fight, but instruments such as the Bioscreen C can help provide the edge we need to promote both medical and economic recovery.
Cheeseman, K.E., Denyer, S.P., Hosein, I.K.,
Williams, G.J., and J.-Y. Maillard. 2009. Evaluation of the bactericidal
efficacy of three different alcohol hand rubs against 57 clinical isolates of S. aureus. Journal of Hospital Infection
Cooper, C.J., Denyer, S.P., and J.-Y. Maillard.
2011. Rapid and quantitative automated measurement of bacteriophage activity
against cystic fibrosis isolates of Pseudomonas aeruginosa. Journal of Applied
Microbiology 110: 631-640. Frenzen, P.D., Drake, A., Angulo, F.J., and The
Emerging Infections Program Foodnet Working Group. 2005. Economic cost of
illness due to Escherichia coli O157
infections in the United States. Journal of Food Protection 68: 2623-2630. Scallan, E., Hoekstra, R.M., Tauxe, R.V., Widdowson,
M.-A., Roy, S.L., Jones, J.L., and P.M. Griffin. 2011a. Foodborne illness
acquired in the United States – Major Pathogens. Emerging Infectious Diseases
17: 7-12. Sun, F., Cho, H., Jeong, D.-W.,
Li, C., He, C. Bae, T. 2010. Aureusimines in Staphylococcus aureus are not involved in virulence. PLoS One 12: e15703 For more information, clickhere and enter Infectious Diseases in the subject line of the line of the email or
Cooper, C.J., Denyer, S.P., and J.-Y. Maillard. 2011. Rapid and quantitative automated measurement of bacteriophage activity against cystic fibrosis isolates of Pseudomonas aeruginosa. Journal of Applied Microbiology 110: 631-640.
Frenzen, P.D., Drake, A., Angulo, F.J., and The Emerging Infections Program Foodnet Working Group. 2005. Economic cost of illness due to Escherichia coli O157 infections in the United States. Journal of Food Protection 68: 2623-2630.
Scallan, E., Hoekstra, R.M., Tauxe, R.V., Widdowson, M.-A., Roy, S.L., Jones, J.L., and P.M. Griffin. 2011a. Foodborne illness acquired in the United States – Major Pathogens. Emerging Infectious Diseases 17: 7-12.
Sun, F., Cho, H., Jeong, D.-W., Li, C., He, C. Bae, T. 2010. Aureusimines in Staphylococcus aureus are not involved in virulence. PLoS One 12: e15703
For more information, clickhere and enter Infectious Diseases in the subject line of the line of the email or call 732-457-9070.
Measuring variability of spore generation of foodborne pathogens
“Biologically the species is the accumulation of the experiments of all its successful individuals since the beginning” – H.G. Wells
“Four short words sum up what has lifted most successful individuals
“I just gotta be me!” – Sammy Davis, Jr.
It’s a jungle out there in the food industry. Between heating, freezing, dessication, extremes of pH, and regular cleaning or chemical sanitization of food processing equipment and production environments, it’s hard for the average foodborne bacterial pathogen to get ahead. For a few select individuals, though, the world is their oyster.
A recent sampling of recalls and outbreaks due to contaminated foods reads like a complete grocery list: milk, eggs, ice cream, peanut butter, ground beef, salami, celery, alfalfa sprouts, carrot juice, tomatoes, peppers, spinach and lettuce, just to name a few. And at this time of year, when our minds travel to thoughts of candies and other autumnal delights, we must not forget to also add chocolate, apple cider and pumpkin seeds to this list. So, with all the efforts focused on controlling or eradicating bacterial pathogens in foods, why do we still have a problem with foodborne illness? For some foods, the answer is fairly straightforward. For example, if alfalfa is contaminated in the field with animal feces (from birds, cattle, rodents, etc.), Salmonella spp. or Escherichia coli O157:H7 can end up as contaminants on the seed’s surface. The wet and warm conditions used to sprout the seeds for our salads and sandwiches are also ideal for the growth of these pathogens, allowing them to quickly multiply to very high levels in the product – sometimes even as high as several billion cells per gram of sprouts.
Other foods, however, are not such ideal playgrounds for microbes, and in fact, may even be considered to be downright inhospitable. For example, peanut butter has a very high level of fat (~50%) and very little available water. Sufficient available water is an absolute requirement for microbial growth, and is measured in terms of water activity (abbreviated as “aw”). While an aw of around 0.91 is needed for bacterial growth, the water activity of peanut butter is much lower - around 0.20 – 0.33 (Burnett et al., 2000). Although pathogens such as Salmonella cannot grow in peanut butter, they have been known to survive for long periods of time in peanut butter under the normal conditions of storage for this food (Burnett et al., 2000; Shachar and Yaron, 2006). Two multistate outbreaks of Salmonella in peanut butter or peanut paste, first in 2007, then again in 2009, ultimately sickened nearly 1,000 people in almost all 50 states. Peanut Corporation of America, the company at the center of the 2009 outbreak, supplied contaminated products to much of the food industry. Because peanut butter and peanut paste are base ingredients used in many different foods (sauces, toppings, seasonings, snack crackers, cookies, candies, ice cream, etc.), almost 4,000 products from various manufacturers have been recalled to date. As Salmonella is known to survive in peanut butter for months or longer, it has been speculated that the 2009 outbreak might drag on for years as recalled products stored in the nation’s pantries continue to be eaten by unsuspecting consumers.
The ability of certain strains of a given pathogen to persist in inhospitable foods, or their ability to survive the physical or chemical stresses routinely encountered in the food processing environment is of key interest to food microbiologists. These robust, rugged or tenacious strains are often very different than the “domesticated” strains typically studied in the lab. For example, peanut butter outbreak-related strains of Salmonella show greater heat resistance than other strains (Ma et al., 2009). So how are these strains able to withstand the hardships of life in today’s food processing facilities or in high-stress food environments? Microarray-based genetic analysis of epidemic strains of Salmonella enterica reveals that many of these otherwise unrelated strains share genes associated with enhanced fitness, including genes needed for survival of starvation, or genes involved in respiration and processing of key cellular metabolites (Kang et al., 2006).
In addition to such genetic approaches, growth-based methods can also be used to explore variability between multiple strains of a given pathogen (Lianou and Koutsoumanis, 2010) or even between individual cells or spores of a single strain (Robinson et al., 2001; Stringer et al., 2005). Why is variability among different strains or between individuals within a single strain important to food microbiologists? Accurate knowledge of how a pathogen behaves in a food system has direct bearing on the safety of that food. Food microbiologists routinely perform basic research to determine the thermal processing steps needed to inactivate pathogens of concern, or to build in antimicrobial hurdles that will prevent the growth of these pathogens in the food. Knowledge of how a pathogen behaves in a food also enables accurate modeling, quantitative risk assessment and setting of food safety objectives. However, a pathogen intervention process is only as good as the data used to design it – underestimation of a pathogen’s thermal resistance, nutritional needs or growth rate can have potentially catastrophic results. While much is known about the handful of strains that are typically studied in academic and government labs, variability among wild type strains is still poorly characterized.
Variability is a hallmark of biological systems – the ability to diversify through mutation and take advantage of prevailing environmental conditions is an essential survival mechanism for bacteria and one that has made them arguably the most successful life form on the planet. It is this same variability among strains and among individuals within a strain that remains as the potential Achilles’ heel of our efforts at food safety. The Bioscreen C Microbiology Reader has emerged as an indispensible tool for the characterization of variability among foodborne pathogens at both the strain and individual levels. The Bioscreen C is a self-contained incubator and automated turbidimeter used to monitor growth of microbes via changes in optical density. With the ability to take automated measurements at close intervals, and to evaluate up to 200 samples per experiment, the instrument generates detailed microbial growth curves, allowing highly parallel comparisons across multiple microbial strains and/or growth conditions. These capabilities have enabled investigators to begin addressing the dearth of knowledge on variability among foodborne pathogens. The recent study by Lianou and Koutsoumanis is an excellent example of this. These authors examined the response of 60 strains of Salmonella enterica to growth conditions that varied according to pH (pH 4.3-7.0) and/or NaCl concentration (0.5-6.0%). In total, these authors generated 9,600 growth curves – a feat that would simply be unattainable using a traditional test tube approach. Because the Bioscreen measures growth as a function of time, kinetic parameters such as specific growth rate can easily be calculated and compared across different strains and growth conditions. Using this approach, these authors were able to directly observe and measure heterogeneity of growth responses among these different Salmonella isolates. Interestingly, variability of growth responses within each strain increased substantially as environmental conditions became more stressful (i.e. lower pH, higher NaCl concentration). This observation may be cause for concern, as current approaches for pathogen modeling do not take into account the potential impact of stress on growth variation (Lianou and Koutsoumanis, 2010). This work highlights the importance of using more than one strain of any given pathogen in food safety challenge testing or for data collection in support of growth model development. The ability to accurately characterize a breadth of strains for their growth responses under different environmental conditions also provides a means for selecting strains for use in future testing applications (Lianou and Koutsoumanis, 2010).
In separate work, Stringer et al. (2005) used a combination of direct microscopic observation and Bioscreen-based turbidimetry to measure the variability in germination of spores of Clostridium botulinum. Early events in spore germination and outgrowth were observed via microscopy and distributions in the lag phase of cell growth were obtained via time-to-detection measurements with the Bioscreen. This novel study revealed substantial variability among individual spores along the germination-to-outgrowth-to-toxin production pathway. Although a commonly accepted maxim is that the first spore to germinate in a food system is the one to worry about, this study raised questions about the predictability of germination for individual spores and suggested that “late bloomer” spores may be of equal concern in terms of toxin production. Importantly, evaluation of data obtained using either the highly specialized and technically demanding microscopy approach or the simpler Bioscreen assay showed that the two methods provided comparable information. This observation underscores the flexibility of the Bioscreen system and how it can be leveraged to solve important questions in microbiology that may otherwise require specialized instrumentation. A similar degree of variability in lag times for individual cells was also seen in the study of Robinson et al., (2001), who evaluated the effect of inoculum size on the lag phase for Listeria monocytogenes using the Bioscreen instrument. These authors found that the ability of this pathogen to grow under conditions of severe salt stress was dependent on the presence of a resistant subpopulation of cells within the inoculum. As in the work of Stringer et al., (2005), these results underscore the importance of cell-to-cell variation to risk estimation and for calculation of safe storage times for foods containing low inocula of L. monocytogenes.
In summary, change is good, and variability is the secret to the success of the microbial lifestyle. Foodborne pathogens are creative at expressing their individuality, displaying many different “variations on a theme” at both the strain and individual cellular levels. This variation can manifest itself as differences in growth rate, resistance to antibiotics, dessication tolerance, or other important cellular properties that may provide a competitive or survival advantage to a pathogen, enabling it to persist in foods and cause disease. Just how adept microbes are at change can sometimes be a little unsettling, especially when we think of our food as the laboratory for their ongoing “experiments”. However, at the end of the day, in spite of the seemingly endless stream of bad news from the food sector ratcheting up our collective level of anxiety about the potential dangers lurking in everything we eat, we must remember the importance of stepping back, putting things in perspective, and enjoying the small pleasures in life…in other words don’t forget to stop and smell the roses!
Burnett, S.L., Gehm, E.R., Weissinger, W.R., and L.R. Beuchat. 2000. Survival of Salmonella in peanut butter and peanut butter spread. Journal of Applied Microbiology 89: 472-477.
Kang, M.S., Besser, T.E., Hancock, D.D., Porwollik, D., McClelland, M., and D.R. Call. 2006. Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica. Applied and Environmental Microbiology 72: 6938-6947.
Ma, L., Guodong, Z., Gerner-Smidt, Mantripragada, V., Ezeoke, I. and M.P. Doyle. 2009. Thermal inactivation of Salmonella in peanut butter. Journal of Food Protection 72: 1596-1601.
Robinson, T.P., Aboaba, O.O., Kaloti, A., Ocio, M.J., Baranyi, J., and B.M. Mackey. 2001. The effect of inoculum size on the lag phase of Listeria monocytogenes. International Journal of Food Microbiology 70: 163-173.
Shachar, D., and S. Yaron. 2006. Heat tolerance of Salmonella enterica serovars Agona, Enteritidis and Typhimurium in peanut butter. Journal of Food Protection 69: 2687-2691.
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. Applied and Environmental Microbiology 71: 4998-5003.
“All I Need is the Air That I Breathe”
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.
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.
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.
Getting Buggy About Ethanol
|The Ferment Over Lignocellulosic Biofuels|
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).
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.
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.
One Man’s Trash…May Be a Microbe’s Treasure
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.
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.
“Finding the Antimicrobial
Screening and Discovery of Sugar-Based Antimicrobials
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.
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.
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.
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.
“Visualize Whirled Yeast”
Applications of Bioscreen-C in Yeast Research
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.
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.
"Pickled In Their Own Juices”
Applications of Bioscreen-C in Yeast Research
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.
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.
“Artisanal Cheeses: A Highly Cultural Experience”
Applications of Bioscreen-C in Food Research
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.
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.
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“Teeth, Tongue and Beyond”
Applications of Bioscreen-C in Oral Microbiology
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.
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
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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: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. Journal of Applied Microbiology 92: 681-694.
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Antibiotic Resistant Bacteria
|Use of the Bioscreen-C Automated Microbiology Growth Curve System for the Study of Antimicrobial Resistance in Clinical Settings|
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:Simoes, J.A., Aroutcheva, A.A., Heimler, I., and S. Faro. 2004. Antimicrobial resistance patterns of group B streptococcal clinical isolates. Infect. Dis. Obstet. Gynecol. 12: 1-8.
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|Use of Bioscreen-C Automated Growth Curve Analysis System to Evaluate the Activities of Plant Essential Oils Against Foodborne Pathogens|
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:Knight, K.P. and R.C. McKellar. 2007. Influence of cinnamon and clove essential oils on the D-and z-values of Escherichia coli O157:H7 in apple cider. Journal of Food Protection 70: 2089-2094.
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