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44 Emerging Bacterial Food-Borne Pathogens and Methods of Detection 841been identified as a potential zoonoses following detection of the
pathogen in a range of animal hosts. MRSA strain types have
been isolated from pigs (De Boer et al. 2009) and a prevalence of
MRSA in retail meats of 11.9% was also observed with highest
prevalence in poultry meats.
Resistance of food-borne pathogens is currently monitored in
the United States through the National Antimicrobial Resistance
Monitoring System. There are two branches that follow and
assess drug resistance in animal production and foods of animals
and also the clinical side of disease. Trends in resistance are
released annually and indicate emerging resistance types and
changing profiles forSalmonella,E. coli,Campylobacter,and
some gram-positive organisms (see http://www.cdc.gov/narms/
and http://www.fda.gov/AnimalVeterinary/SafetyHealth/Anti
microbialResistance/NationalAntimicrobialResistanceMonitor
ingSystem/default.htm). For further discussion on antimicrobial
resistance in relation to food-borne pathogens the authors
recommend the review by Cleveland McEntire and Montville
(2007).METHODS FOR THE DETECTION OF
FOOD-BORNE PATHOGENSIn this section, we discuss some of the approaches used in de-
tection of food-borne pathogens. The review discusses methods
from the classical to the rapid to the next generation of tech-
nologies. While this section provides an overview of the most
common methods used it is not comprehensive, as the field of
rapid technologies is changing fast and newer technologies have
taken advantage of nano sciences for their applications. This sec-
tion covers detection methods; for detail on typing (see Logue
and Nolan (2009)).Culture-Dependent MethodsDetection of food-borne pathogens in a food sample usually
involves increasing the population of the target organism
present to levels that can be detected by conventional or other
means. Typically, culture-dependent methods involve a series
of enrichment phases designed to increase the population of the
pathogen present, which can be followed by a second selective
enrichment designed to selectively enrich the target pathogen,
while inhibiting or suppressing the growth of contaminant flora
that may also be present. The method for culture enrichment
is relatively straightforward and for the primary enrichment
usually involves volumes about 25 g of the suspect food in 225
mL of a nonselective enrichment broth (a 1:10 ratio of sample
to enrichment broth is typically recommended). The sample is
homogenized and then incubated at a temperature optimum for
the target pathogen with an incubation time in the region of
18–24 hours. Following the primary enrichment, a secondary
enrichment designed to select the pathogen and suppress
contaminants is usually carried out. In this case, the selective
enrichment phase, media, and ratios of sample to selective
broth are significantly reduced and volumes of 0.1–0.5 mL (or
at slightly higher ratios if needed) of the primary enrichment
broth are transferred to 9.9–9.5 mL volumes of the secondarybroth. Secondary enrichment broths are usually incubated
for a further 18–24 hours at the optimum temperature of the
pathogen. Incubation temperature for the selective broth can be
the same or different from the temperature used for the primary
enrichment broth. Following secondary enrichment, the sample
is typically struck to a selective or differential agar designed for
specific isolation of the target pathogen. Incubation of selective
agar plates to recover the target can take an additional 24–48
hours (or longer for some pathogens) to determine presence
of a suspect pathogen. Further confirmation and identification
of a suspect pathogen from selective plates to the genus or
species level may take an additional 4–5 days to complete using
conventional biochemical methods, morphological analysis,
serological analysis, or other such tests. Some of the post
isolation analysis can be considerably shortened with the appli-
cation of automated or molecular testing. While the methods
such as those described here are considered the standard for
detection of a target pathogen, they are recognized by federal
agencies as the gold standard for the detection of bacterial
contamination. Some examples of agencies who provide stan-
dardized methods online for use in the detection of pathogens in
foodstuffs include the FDA Bacteriological Analytical Manual
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/
BacteriologicalAnalyticalManualBAM/default.htm; the USDA
FSIS Microbiology Laboratory Guidebook http://www.fsis.
usda.gov/science/microbiological_Lab_Guidebook/; other
guides of importance include the American Association of
Analytical Chemists (AOAC) and the International Standards
Organization (ISO); additional standards and protocols are
recommended on a national level for individual countries and
the reader is advised to refer to the protocols recommended
on a national level. While the use of standard methods is of
value in pathogen detection, they have considerable drawbacks
in terms of time and labor required to obtain a result and cost
issues associated with isolation and detection; some of these
methods also suffer in their detection capabilities (sensitivity
and specificity) and may fail to provide adequate detection
of the target pathogen if the medium is too selective for a
particular strain type or the state of the organism may not allow
its detection prior to application of the protocol. Other problems
also complicating pathogen detection include nonuniform
distribution of the pathogen in a sample, which can mean
that dependent on where the sample is collected it may not
be representative of the whole sample. Also, a low level of
pathogen may be present in the sample, which may complicate
its detection in the presence of high levels of natural microflora.
The heterogeneous nature of the food matrix can also interfere
with the growth or detection of the target pathogen. Finally,
injured cells as a result of processing may result in inability to
detect target pathogens using selective media.
Culture methods for the detection of a pathogen are therefore
subject to limitations such as the initial number of organisms in
a sample: (1) if the number of organisms in a sample is very low
and the sample is enriched, competition from other naturally
occurring microflora present in the sample may outcompete
the target organism, other issues associated with competition
may not only be competition for space and nutrients but also