Bioscreen C Hot Topics

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.

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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.

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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.

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