IFT's Inaugural Research Summit explored research needs for the physiology of bacterial spores and other dormant microbes, rapid quantitative measurement methods, and advanced techniques for evaluating microbial viability.
June 1, 2003
The Institute of Food Technologists convened its first Research
Summit on January 12–14 in Orlando, Fla., to facilitate an indepth
interchange of information among world-renowned
investigators and other scientists conducting basic and applied research
on the physiology of bacterial spores and other dormant microbes, rapid
quantitative measurement methods, and advanced techniques for
evaluating microbial viability. The main goal of the interchange was to
ultimately identify the needs for further research in these areas.
During the summit, “Rapid Measurement of Bacterial Spores and Other
Dormant, Difficult-to-Measure Microorganisms—Emphasizing Quantitative
Methods and Nanotechnology,” 35 scientists from six countries
and multiple areas of research interests participated in dynamic exchanges
and reached consensus on specific research needs.
State of the Science
Two keynote addresses, one by Grahame Gould, formerly of Unilever
Research, Bedford, UK, and the other by Peter Setlow of the University of
Connecticut Health Center, delved into what is known and what remains
elusive in our understanding of spore physiology and the underlying system
of dormancy and resistance, sporulation, initiation, germination, outgrowth,
and mechanisms of inactivation.
“The spore itself has a preservation system that is par excellence, with a
shelf life of thousands to millions of years,” Gould said. “If we understood
more about the way a spore is put together, then maybe we would better
understand how to deal with it. Maybe we could even copy some of the
mechanisms employed by the spore to better preserve ambient stable
foods.” More specifically, he said, the barrier to further progress is our inadequate
understanding of the basic mechanisms of spore resistance, dormancy,
germination, extreme dormancy (viable but nonculturable state,
VBNC), and heterogeneity. Setlow said this same barrier existed 30 years
ago. While considerable progress has been made in understanding the
modes of action for some bacterial inactivation methods, such as irradiation,
we still do not know how heat kills spores, “which is disgraceful,”
Gould opined.
Key areas needing investigation, Gould said, are: (1) How does the cortex
peptidoglycan act at the architectural/molecular/material science level?
(2) What is the physical status of the protoplast; e.g., is it in
glassy state? (3) How does calcium dipicolinate (CaDPA) contribute
to dormancy and resistance? (4) How do alkyl amines
and CaDPA act in germination? (5) What are the exploitable
new hurdle techniques (e.g., nisin in new combinations)? (6)
What are the most profitable approaches for food science/technology/
industry/consumers? For most of these topics, well-integrated,
multidisciplinary research collaborations offer the
best chance of success.
Regarding spore killing, Setlow said it is essential to study
the initial event; and when investigating the kinetics of killing
in particular, it is crucial to evaluate the inactivation of 90–
95% of the total population so that killing events can be related
to the specific chemical changes in the spore. Setlow also expressed
the concern that studies on spore killing, particularly
with Bacillus subtilis, often use mutants. While mutants can be
useful, they can be misleading, since there is evidence that
some mutants exhibit altered spore resistance because of alterations
in global gene expression caused by the mutation and
not because of direct, specific effects on spore resistance.
Other principles that Setlow highlighted and concerns he
expressed are: (1) Because sporulation conditions can alter
spore resistance, might this alter the mechanism of spore killing?
(2) Most work on killing mechanisms has been done with
B. subtilis; do all species, in particular ones of applied interest,
behave similarly? (3) Can multiple killing mechanisms contribute
to inactivation of a spore population? (4) Are spores dead
or just “superdormant?”
Several additional presentations followed the keynote addresses
before participants broke into smaller groups to reflect
on what is known vs unknown about spores and the implications
for future research. Highlights of the presentations are
briefly captured below.
Microbial Detection and
Measurement Technologies
Various methods are being applied or developed to detect
microorganisms and measure their activities:
•
Traditional recovery methods were discussed in the context
of their application to mesophilic anaerobes to investigate
a recall of infant formula that was presumptively implicated in
a case of infant botulism in the United Kingdom. Eric Johnson
of the University of Wisconsin’s Food Research Institute provided
the presentation for Scott Donnelly of Wyeth Nutritionals,
Burlington, Vt., who was unable to be present.
The recovery methods were used to (1) determine bioload
of the raw material, (2) test air, water, ingredients, and product
contact surfaces for Clostridium botulinum, and (3) propose
specifications for mesophilic anaerobes. Existing methods used
during the investigation included standard plate counts and established
procedures delineated in the fourth edition of the
Compendium of Methods for the Microbiological Examination of
Foods (American Public Health Association, Washington,
D.C.). In addition, a new method was developed specifically for
this investigation.
Many of the media for mesophilic anaerobic sporeformers
listed in the Compendium result in considerable variability
and false positives, Johnson said. A major challenge that had to
be overcome in the investigation was the lack of methods for
enumerating spores in air. The formula manufacturer teamed
up with Paddy O’Reilly of the University County Cork to develop
a method involving modification of an SAS air sampler
and gelatin and agar plates.
Another challenge that had to be overcome was the selection
of a method for testing the water because an official, validated
method does not exist. A filtration-based method described
in the Official Journal of the European Communities was
selected. Johnson’s review of the methods employed for the
outbreak investigation showed the extraordinary measures to
which a single case of botulism can lead and the considerable
problems, even with traditional methods such as plating, that
investigators can face.
•
Measuring lag time, particularly from single spores of C.
botulinum, and studying steps within the lag phase is where the
biggest contribution can be made to better predicting growth
in minimally heat processed (e.g., sous vide) foods. When a
food safety problem involving minimally processed foods occurs,
it will likely result from just a single spore, according to
Mike Peck of the Institute of Food Research, Norwich, UK. If
chances for microbial growth are low, as is often the case for C.
botulinum, lag times and the resultant time to a 1-log increase
can vary significantly, thus challenging food manufacturers’
abilities to ensure appropriate safety margins.
Peck described an image analysis system that he and colleagues developed for measuring time to lag steps (e.g., germination,
emergence, time to one cell) and for quantifying the effect
of stress conditions (pH, temperature, pretreatments) on
lag. The system met several needs, including allowing identification
of the same spore/cell at different developmental steps
under automated, anaerobic, and temperature-controlled conditions.
Peck and colleagues concluded from their work to date that:
(1) considerable variability exists in times to the different steps
in the lag phase; (2) for individual spores there is poor correlation
between times to the different lag steps; and (3) time to
two cells cannot be predicted from time to germination or
emergence. With further development of the system, Peck
plans to examine the effect of environmental factors on each
step in the lag phase, develop predictive models of the effect of
food environment on each of the steps, establish which step is
rate limiting, and establish why lag is so variable.
•
Peptide nucleic acid (PNA)-based probes offer advantages
over DNA-based based probes, particularly for hard-to-permeabilize
microbial forms such as spores, encysted parasites,
and Gram-positive bacteria. PNA is a pseudopeptide DNA
mimic having an uncharged, achiral backbone. The unique
chemical makeup of PNA confers a number of advantageous
properties, including rapid hybridization kinetics, resistance to
nucleases, and the ability to hybridize to positions on the ribosome
that are inaccessible to DNA-based probes. PNA probes
are also able to penetrate recalcitrant biological structures such
as parasite cysts and Gram-positive bacterial cell walls. For this
reason, PNA probes are especially useful with fluorescence insitu
hybridization (FISH), a rapid microbial identification
method which uses fluorescently labeled nucleic acid probes to
target variable regions of ribosomal RNA inside whole cells.
Byron Brehm-Stecher of the University of Wisconsin-Madison’s
Food Research Institute described the development of
PNA-FISH probes and their combination with flow cytometry
for the detection of Listeria spp.
•
Flow cytometry systems are composed of fluidic, optic,
and electronic components. Flow cytometry involves laserbased
irradiation of single cells containing bound fluorochromes;
with the resultant light scattering providing cell size
information. Systems allow rapid and quantitative analysis of
individual cells. The technology was developed in late 1950 and
is used primarily in clinical settings. Kristi Harkins of Advanced
Analytical Technologies, Inc. (AATI), Ames, Iowa, described
the design and applications of an analyzer, the
RBD2100, developed by AATI and its successful application to
B. subtilis spores and vegetative cells, Cryptosporidium parvum
oocysts, Listeria spp., and Escherichia coli O157:H7.
•
Fluidized bed capture uses molecules covalently bound
via patented surface chemistry to glass beads to bind and hold
target organisms and spores. Liquification of the sample before
running it through the device at flow rates greater than 100
mL/min leads to fluidization of the beads and capture of microorganisms
onto bead surfaces. The organisms are then detected
via immunological or genetic testing. The procedure
eliminates the need to pre-enrich a food sample and can be
done in 30 min from the time of sample collection to final
result. The technology may be able to detect as few as 100 cfu/
mL, in an optimized format and depending on the capture
ligand. This technology has been successfully used with Bacillus
spores, E. coli O157, Listeria spp., and Salmonella.
Fluidized bed capture is the antithesis to flow cytometry,
said Bart Weimer of Utah State University. Microbe detection
with fluidized bed capture uses large volumes and enables alternatives
to chemiluminescence. Weimer described the development
of a number of rapid “capture technology” sensors—
ImmunoFlow™, ImmunoDNA®, GlycoBind®, and TissueTag
®—for detecting spores and microbes in food and the
environment.
•
Multiplexing allows testing of many molecules simultaneously.
Microarrays (spatially encoded micro-sized spots, typically
on a slide) and microbeads (magnetic system of microsized
encoded beads) are common multiplexing systems. Joydeep
Lahiri of Corning, Corning, N.Y., provided insight into
different types of multiplexing systems that Corning is working
with as part of its research program aimed at development
of advanced tools for biotechnology. He detailed state-of-theart
cDNA (long oligo), protein, and membrane protein and
lipid microarrays.
Lahiri also described achievements made in their work to
overcome issues associated with microbead assays. For example,
to address the problem of the autofluorescence that is associated
with current polymeric materials, Corning is using microbarcodes
that are made of pristine glass, which is chemically
inert, and contain rare earths, which emit only narrow fluorescence
bands.
He also described work in assay detection systems ranging
from label displacement via fluorescence resonance energy
transfer (FRET) or quenching to “true” label-free detection
(without fluorescent, radioactive, or other labels) via optical
biosensors, electrochemistry, and mass spectroscopy. A microplate-
based label independent detection system that he described
is based on changes in refractive index and has a detection
goal of about 1 × 10–6 refractive index units (molecular
weight <500).>
Corning’s applications of microarrays and microbeads
build on the organization’s strengths in photonics. To date, applications
have been in organic and biochemical technologies,
rather than food.
•
Micro/nano bioanalytical systems allow detection of
pathogenic bacteria and viruses at the level of the single cell.
Antje Baeumner of Cornell University described these systems
and their advantages over traditional methods. Microanalytical
systems offer speed (e.g., 10–15 min), sensitivity, reliability, inexpensive
cost, simplicity for the user, portability, multi-analyte
high-throughput capability, and new application areas.
Based on specific optical RNA biosensors for E. coli and C. parvum,
microbioanalytical systems are being developed for the
Dengue virus and Bacillus anthracis; their detection is combined
with sample preparation. The systems use the same biological
principles as in simple biosensors; however, novel sample-
preparation systems, novel molecular biology amplification
(nucleic acid sequence–based amplification, NASBA)
chambers, and intricate hybridization channel patterns are being
developed.
The principles and techniques of the system’s modules are a
laser-induced cell lysis system, interdigitated ultramicroelectrodes
as transducers, NASBA amplification on the chip, and
modeling and fabrication of optimized micromixers. Systems
are specifically designed for RNA, basically providing amplification
of mRNA as PCR does for DNA; however, the system
does not produce false positives from dead microorganisms
and does not require thermal cycling. Detection limits as low
as femtomolar and single-cell range were demonstrated. System challenges include macroworld integration, analysis of appreciable
volumes (from 1 mL to 100 L), and miniaturization
of sample preparation.
•
The GeneExpert instrument platform developed by
Cepheid, Sunnyvale, Calif., and described by Kurt Petersen performs
all the steps, from sample preparation to final validated
result, of DNA and RNA molecular detection technologies. The
platform is being used in a U.S. Postal Service pilot program to
detect biothreat agents. Other applications being developed include
studying human infectious diseases, antibiotic resistance,
foodborne pathogens, agricultural and veterinary diseases, and
cancer.
The platform provides proper sample preparation, internal
controls, simultaneous monitoring of multiple DNA targets,
and a supply of single-dose reagents. It also overcomes the
false-positive and false-negative problems associated with PCR,
enabling an investigator to look for the
needle in the haystack (e.g., one Listeria
monocytogenes cell in a 300-g sample).
•
Ganglioside-liposome nanovesicle
immunoassay has been newly developed
and shown to detect botulinum
and cholera toxins at femto/attomolar
concentrations. Described by Richard
Durst of Cornell University, this
nanovesicle immunoassay offers extremely
good sensitivity and speed (15–
20 min from the point of mixing the
sample with liposome solution and test
strip application). The assay can detect
toxins in food and water with only
moderate loss of sensitivity. Bioassays
such as this use antigen–antibody interactions,
nucleic acid hybridization, or
natural receptor binding for recognition;
liposomes or NASBA for signal enhancement;
optical or electrochemical methods for detection;
and flow-injection liposome analysis or migration strip, fluorometric
tube, or microfluidic assays as formats. Gangliosides
are useful in such bioassays because they can serve as toxin receptors
(e.g., GM1 is the receptor ganglioside for the toxin produced
by Vibrio cholera). Liposomes are useful because their
composition can be controlled and tailored, their surface modified
by conjugation or insertion, and their size and surface-tag
concentration controlled; a variety of detection methods are
possible; and lysis, when needed, gives instantaneous amplification.
Consensus on Research Needs
The outcomes of the discussions among the small groups
were presented in a final plenary session that was immediately
followed by development of consensus on research needs for addressing
the unresolved questions and concerns in order to advance
food quality and safety.
Three primary areas were identified as key for future investigations:
(1) fundamental knowledge of spores, i.e., why spores
are resistant and dormant; (2) sampling issues; and (3) microbial
detection and measurement technology. Research in any of these
areas will benefit the other areas. Furthermore, because many
types of microorganisms could be considered difficult to measure,
i.e., dormant, the conclusions of this forum may have
broad application to mold and yeast spores, parasitic cysts, viruses, and VBNC microorganisms. A fourth item—the need to
integrate the various pertinent disciplines (e.g., engineering, materials
science, physical chemistry, microbiology) for a multidisciplinary
approach—was also identified as important for future
work.
•
Fundamental Knowledge of Spores. There are extraordinary
opportunities to ultimately understand dormancy and extreme
resistance of bacterial spores and similar dormant microbial
systems (e.g., VBNC microorganisms). Spore components
(e.g., core, cortex, and inner membrane) must be studied at the
architectural/molecular/material science level. Study at this level
will contribute to the understanding of the basic mechanisms of
spore resistance, dormancy, germination, and heterogeneity. Further,
the outcome of such research will elucidate the physical
state of the cortex and how its peptidoglycan acts at that level,
clarify the status of the protoplast, enable understanding of the role
of CaDPA in dormancy and resistance,
and show how CaDPA and other
agents (e.g., alkyl amines) act in germination.
Such information could lead to the
development of specific targets for spore
inhibition and detection. One participant
said it would be great to be able to discover
a generic component on the spore
surface to which molecules could then be
attached to induce superdormancy (thus
completely inactivating a spore via a
coating technique). Alternatively, with
new information on how spores may be
induced out of dormancy, the cells could
be more easily inactivated.
•
Sampling/Capture Issues. As a reflection
of the difficulty of overcoming
sampling/capture issues, one participant
said, “If we could solve the capture problem,
detection would be a piece of cake.” Extracting or removing
spores from their food matrices presents substantial challenges.
However, results of some of the ongoing research offer the prospect
that microorganisms may not always have to be extracted
from the food matrix in the future. Although technologies allowing
identification of microorganisms and their toxins continue
to advance, sample preparation challenges remain because of
the varied food matrices and, hence, different sample classes
(e.g., grains, raw meat and poultry, spray-dried ingredients,
powders, flavors). Furthermore, not only are mechanisms needed
for overcoming the challenges presented by different food
matrices, but tools or methods for validating the mechanisms
are also needed.
•
Microbial Detection and Measurement Technology. To
conduct these much-needed investigations into fundamental
spore physiology, nanotechnology and other contemporary
techniques are crucial. As highlighted by several participants,
considerable advances are rapidly being made in many areas.
Techniques such as nanotechnology, PNA probes, flow cytometry,
multiplexing, RNA biosensors, NASBA, and the GeneXpert
platform hold tremendous potential for being effectively employed
in this fundamental research. Why nanotechnology? Because
genetic identification, automation, and use of lower concentrations
of cells and materials can permit faster and more accurate,
precise, and economical research into these dormant cell
systems.
These and other advanced techniques and systems will enable new insight
into the fundamental aspects of spore physiology and dormant systems,
and the understanding and detection of whole cells, cellular constituents,
and metabolic products. With this insight, food manufacturers
would then be better able to solve the challenges and problems associated
with spores by being able to determine and apply practical, appropriate
rapid “corrections” of processing parameters in various food matrixes/
environments. Further, qualitative and quantitative nondestructive
testing could be a great advantage for quality assurance systems, including
off-line and in-line measurements that could be continuous.
The impressive capabilities of nano- and other advanced technologies
raise several questions, however. What ultimate lower limits of detection
will be attainable and meaningful? What will be the ultimate roles and
advantages of these techniques in food safety and quality, whether in the
field or the laboratory? Can these advanced techniques improve our understanding
of dose response? Where will all the opportunities lie in
food safety epidemiology? What are the many roles these advanced techniques
might play in intentional contamination of food or the food environment?
One participant pointed out that nanotechnology will not be “the
holy grail,” however. He said application of nanotechnology will bring
about many more issues for research, which should be addressed at the
outset by the food industry directing, via funding, specific development
of biosensors with the food sector in mind. Elaborating, he said that
nanotechnology presents pitfalls when trying to apply it to particulatecontaining
samples, as is the case of food. Thus, work in surface chemistry,
definition of surface interactions, and basic microbiology is needed
to effectively use the technology for food matrices.
Although the advancing work in functional genomics was not a focus
of the meeting, it is understood that genomics and expression arrays also
hold considerable promise for fundamental research and detection of
dormant organisms.
•
Disciplinary Integration/Multidisciplinary Approach. Ongoing
and new technological developments must move from basic to applied
research and meld together to effect enhanced food quality, safety, and
public health. Thus, different disciplines having a bearing on or potentially
contributing to the effective connection of basic and applied research
must be integrated for a multidisciplinary approach to research.
Further cross-disciplinary education and training will contribute immensely
to the positive impact and outcome of basic and applied research.
•
Research Benefits. Who needs the outcomes of the research? Who
will benefit from application of resources to the areas described above?
Regulatory agencies could use this knowledge for process validation,
foodborne disease surveillance, risk assessment, criteria for standards,
and outbreak investigations. Homeland Security and Defense could use
the information and findings in military situations as well as in terrorism
control, effective anti-terrorism measures, and general quality assurance.
The food industry could obviously benefit from programs aimed at
food safety, spoilage control, and quality management related to bacterial
spores, yeast spores, mold spores, and cysts. New research and development
opportunities, such as sporeforming probiotics and new preservation
techniques could result as well. Environmental control efforts also
could benefit from this research, to design enhanced integrated pest
management systems and to help monitor and control contamination of
air, hospitals, public buildings, and strategic facilities by these resistant
spores. In fact, these research outcomes could prove fundamental for
many biological systems other than spores and other dormant microorganisms.
Thus, the findings in this area will greatly impact not only the economics
of the food industry but also public health and public confidence
in the food supply.