916 Caruso: Journal of AOAC International Vol. 101, No.

4, 2018

Special Guest Editor Section

Antibiotic Resistance in Escherichia coli from Farm

Livestock and Related Analytical Methods: A Review
Giorgia Caruso
Enbiotech S.r.l., Via Aquileia, 34, 90144, Palermo, Italy

The indiscriminate use of antibiotics for the a reliance on antibiotics and, consequently, to the emergence
treatment of human and animal infections has of resistant strains. It was observed that the predictable amount
led to the rise of resistance in pathogens and in of these antimicrobial agents for human use is limited when
commensal bacteria. In particular, farm animals compared with the massive consumption of antibiotics in

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may act as vectors for the dissemination of agricultural sectors (3). In addition, large amounts of antibiotics
drug-resistant genes because of the intensive are not metabolized, retaining their activities even after renal
use of antibiotics in animal production, enabling excretion (4). Hence, the propagation of active antibiotics and
resistance to a wide range of antimicrobial metabolites represents another route of transmission from farms
agents, including those normally used in human to the environment.
medicine. Escherichia coli, being a widespread Although nontherapeutic use in Europe and North America
commensal, is considered a good indicator of was forbidden, in many countries, the “antibiotic growth
antibiotic use. Ultimately, it is emerging as a global promoters” phenomenon (i.e., the addition of antibiotics to
threat, developing dramatically high levels of subtherapeutic concentrations in order to increase the growth
antibiotic resistance to multiple classes of drugs. rate, favoring spread and persistence of resistant bacteria and their
Its prevalence in food animals is hence alarming, genesis) has been spreading since 1950. In fact, the chronic use
and more studies are needed in order to ascertain of a single antibiotic can enable resistance to more structurally
the spread dynamics between the food chain and unrelated antibiotics, linked on plasmid and transposon genes (5).
humans. In this context, great attention should Antimicrobial substances are used in breeding animals for a
be paid to the accurate detection of resistance by variety of reasons: they are administered for therapeutic use in
conventional and molecular methods. In this review, sick animals, for metaphylaxis in healthy animals to minimize
a comprehensive list of the most widely used an expected disease outbreak, or for prevention when it is
testing methods is also addressed. highly probable for healthy animals to develop diseases (6). For
example, preventive use of antibacterial drugs often depends on
management practices, such as the introduction of new animals

T
within a group, stressful events such as castration and weaning,
he introduction of antibiotics into clinical practice was
and viral epidemics with potential bacterial over-infection.
a major achievement in the medical field, contributing
In addition, incorrect standards in procedures in food
substantially to the reduction of morbidity and mortality
processing industries, such as slaughtering and processing
associated with infectious diseases.
plants, where productive performances may be notable in terms
Nonetheless, the excessive and thoughtless use of these
of number of processed animals (particularly for poultry),
molecules in the last decades has led to the global threat of
increase the risk of antibiotic resistance in the food chain.
a potential postantibiotic era (1), in which drug efficacy is
Incorrect design and insufficient standard operative procedures
compromised by resistance mechanisms to all existing antibiotic
may also increase this risk.
classes. This phenomenon is the multifactorial result of bacterial
Particulates (fine and solid materials) are included in
genome plasticity, characterized by several mobile genetic
aerosolized dispersions and are consequently diffused by air
elements delivering resistance determinants, as well as of the
movement. These particulates may be both organic compounds
anthropic role, which has exerted a sudden and impressive
and living microorganisms, with safety implications for animals
selective force against bacterial populations.
and for human workers. Another problem in food processing
Even though environmental reservoirs of resistance genes
plants is related to the geometry of working areas and the
are ancient, existing prior to the anthropogenic introduction
possibility of bioaerosolized dispersions, especially with regard
of antibiotics (i.e., resistome) (2), the veterinary sector, above
to fungal spores (7). These plants should be equipped with
all, was certainly also responsible for accelerating the spread of
adequate ventilation (negative-pressure) systems to continuously
antibiotic resistance. In fact, the increase in intensive livestock
move contaminated air and bioaerosols (potential sources of
practices, both terrestrial and aquaculture, has inevitably led to
pathogens and spoilage microorganisms) toward exhaust fans
with the aim of purifying processing and packaging areas.
However, several failures of the ventilating system and other
problems such as condensation phenomena in some points of
Guest edited as a special report on “Analytical Approaches and
Safety Evaluation Strategies for Antibiotics and Antimicrobial Agents in
the plant (multipurpose areas, etc.) can compromise the efficient
Food Products: Chemical and Biological Solutions” by Salvatore Parisi. dispersion and elimination of bioaerosols from the inner
Corresponding author’s e-mail: giorgia.cars@gmail.com atmosphere. For these reasons, notable amounts of potentially
DOI: https://doi.org/10.5740/jaoacint.17-0445 resistant pathogens and commensal bacteria can be found on
 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018 917

working surfaces, including packaging equipment (8, 9). For damage in cells, or even in death, with liberation of DNA due to
instance, antibiotics or resistant bacteria have been detected in lysis, including eventual gene coding for antibiotic resistance.
dust (10) and in air streams from pig-feeding operations (11, 12). These factors may have an impact on the microbial resistance
As for food, the greatest risk of contamination occurs during phenomenon, as observed by Cirz et al. (27), who detected
slaughtering, when resistant strains from the intestinal tract an association between the SOS response activity following
often come into contact with carcasses during evisceration, stressful conditions and increased antibiotic resistance in
posing a potential danger. Escherichia coli pathogenic strains. More specifically, the
In fact, there is unquestionable evidence that foods from many conjugation rate of plasmids comprising resistance determinants
animal sources and from all processing stages contain abundant in E. coli was noted to rise more in strains that had undergone
amounts of resistant bacteria and their resistance genes (13, 14). the application of sublethal preservation conditions than in
Large proportions of seemingly innocuous commensal nonstressed strains (28). According to this study, the impact on
bacteria continually exchange genes, completely unnoticed (15), phenotypic antimicrobial resistance persisted even when the
mobilizing resistance genes. These bacteria are a largely ignored stress source had been removed, having a durable effect.
reservoir of resistance and provide many complex pathways by Therefore, stress conditions may trigger many adaptation
which the resistance genes present in animals may directly or responses and, in some cases, enhanced resistance, suggesting

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more likely indirectly find a way to reach human pathogens sublethal preservation treatments or methods could play a role
through food, water, mud, and manure applied as fertilizers. in the propagation of antibiotic resistance (29).
It was observed that gut microbiota may be considered Antibiotic resistance is hence a challenging subject from
hubs for the transfer of resistance genes (16). In particular, different viewpoints; the above-mentioned points have
even if transformation does not seem to play a significant role, demonstrated that a multidisciplinary approach is needed in the
conjugation and transduction contribute significantly. The ambit of public health and safety on the one hand, and in the
conjugation phenomenon was observed to occur between both food industry on the other.
closely and distantly related microbial species, contributing
to the dissemination of plasmids and conjugative transposons. Antimicrobial-Resistant E. coli
Moreover, there seems to be a correlation between the
inflammation state of the host’s gut and the conjugation rate (17). By the public health viewpoint, it is of the utmost importance
On the other hand, phages have also been increasingly to monitor antibiotic resistance in various contexts and for the
recognized as responsible for shaping the gut resistome; indeed, main representative microorganisms.
being as numerous as bacteria in mammals’ intestinal tracts, E. coli is considered an indicator, being a commensal
they may frequently undergo lytic cycles, thereby spreading bacterium ubiquitous in mammals and capable of providing
insidious genes (18). Experimental evidence has noted that relevant hints on the spread of the antibiotic resistance (30). In
antibiotics in feeds induced prophages in the fecal microbiome of fact, the frequency of resistance in commensal strains of E. coli
pigs, thus promoting transduction-mediated gene transfer (19). is believed to be an effective marker for the selective pressure
In healthy humans, more than 70% of feces samples were found applied by antibiotic use in animals and the future resistance
to be positive for genes coding resistance, mostly β-lactamases that is predicted in pathogens (31).
(blatem and blactx-m) and quinolone resistance genes (qnrA) (20). Recently, antimicrobial-resistant E. coli has emerged as
The lower frequency of transformation is possibly due a global threat, with the development of dramatically high
to DNA vulnerability with regard to nucleases and physical levels of antibiotic resistance to multiple classes of drugs. In
variables such as temperature, chemical degradation, and DNA fact, some strains may cause a wide range of diseases, from
length; however, in certain foods, such as sausages, complex intestinal pathotypes to life-threatening infections, which could
matrices and biofilms occurring in the processing environment be exacerbated by the already common limited therapeutic
may fulfill the protection requirements needed for a successful choice of antibiotics. The strains responsible for extraintestinal
transfer (21). In addition, DNA may also be protected by infections (ExPEC) cause disease in immunocompetent
particular food components (e.g., arginine, maltol) (22). individuals in both nosocomial and community contexts;
Moreover, biofilms—often found in farms and processing they commonly colonize the intestinal tract, establishing a
plants—have also been identified as hot spots for horizontal completely asymptomatic balance until they erupt into other
gene transfer, perhaps because of the proximity of microbial districts (32).
cells and the minimal shear force (23). ExPEC represent one of the most common causes of urinary
Beside food spoilage, biofilms may increase the resistance rate tract infections (UTIs) and bloodstream infections. They play a
among the bacteria present in these environments through both significant role in health, both in the community and in hospital
conjugation and transformation (24, 25). This in turn may enhance settings, with a significant financial impact on the health system.
the transmission to pathogens, constituting a dramatic public In addition, they may cause diseases in farm animals, such as
health risk, with an augmented threat of invasive infections and mastitis in cattle, UTIs in swine, and colibacillosis in poultry,
mortality. Moreover, antimicrobial resistance may pose a higher with severe losses due to mortality and unsuitable quality for
risk of more virulent strains because of a possible co-selection of commercial sale.
resistance and virulence determinants through integration of both ExPEC are characterized by a high number of virulence factors,
types of plasmids (i.e., virulence and resistance types) (26). which allow colonization, avoidance of host immune defenses,
In addition, processing and preservation mechanisms and sequestration of growth factors, stimulating a damaging
may have unpleasant effects on antimicrobial resistance inflammatory response.
dissemination. In fact, these treatments may determine The management of ExPEC as pathogens is increasingly
inhibition of bacterial growth, resulting in stress or sublethal complicated by the emergence and propagation of resistance
918 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018

mechanisms, especially for fluoroquinolones, trimethoprim/ dispersion of cefotaxime (CTX)-M enzymes have been observed
sulfametoxazole, and extended spectrum β-lactamases (ESBL) (49), perhaps due to a more efficient way of hydrolyzing some
(33, 34). molecules than the other enzymatic β-lactamase variants. This
According to a 2014 European Surveillance of Veterinary exponential CTX-M spread across the globe has been referred
Antimicrobial Consumption report, 8936 tons of antimicrobial to as the “CTX-M pandemic” (50). Farm environments have
drugs for veterinary use in the 28 European Union countries contributed widely to this phenomenon because of their
have been consumed (35), with tetracyclines being the most intensive use of broad-spectrum cephalosporins, such as
commonly used class among veterinary antibiotics, followed ceftiofur and cefquinome, in animals, which has facilitated the
by penicillins and sulfonamides, and among critically important spread of resistance to this class of antibiotics; this resistance is
antibiotics, mainly colistin. With regard to E. coli, the highest now present with high percentages both in animals and in food
resistance prevalence was found for ampicillin, streptomycin, products (51, 52).
sulfonamide, and tetracycline antibiotics (also combined in The increasing dissemination of multidrug-resistant (MDR)
several multiresistance patterns), and particularly in poultry and enterobacteria has resulted in a renewed attention for colistin
pigs, with values ranging between 29.5 and 54.7%, although as a last therapeutic option. However, colistin (belonging to
values were significantly lower in cattle (24.5–30.6%). High polymyxins) has been widely used in veterinary medicine,

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percentages were also found in resistance to fluoroquinolones, mainly in Asia, where a high prevalence of resistance is
which are extremely important antimicrobials used for the beginning to spread. In particular, colistin resistance was
treatment of human infections, with prevalence up to 57.6 and observed with high frequency in China, with up to 24% in E. coli
47.4%, respectively, for ciprofloxacin and nalidixic acid in isolates from animals, especially pigs and poultry (53), where
chickens. Resistance to third-generation cephalosporins (e.g., it probably originated (54). The lack of effective therapeutic
cefotaxime), another class of crucial importance, was found options against already MDR E. coli has contributed to this
more rarely: 1.4% in pigs and 2.4% in cattle, although up to increased consumption, and piglets were particularly involved
10.2% was observed in poultry. Multidrug resistance has also in colibacillosis treatment until its use was banned. Different
been observed in the last decades in the United States (36), transmissible plasmid mechanisms have been observed for
where, according to the National Antimicrobial Resistance colistin resistance, such as the six variants of the mcr-1 gene,
Monitoring System (NARMS), the highest resistance and new ones are always being described, such as mcr-2 and
observed for E. coli strains in poultry (year 2011) was related mcr-3 (55, 56).
to sulfisoxazole (54.7%), streptomycin (50.8%), gentamicin As colistin is the last resort against MDR Gram-negative
(49.0%), and tetracycline (46.6%). Penicillin resistance was infections, a careful monitoring of the development and
observed in 16% of resistant strains, and cephalosporins, such dissemination of colistin resistance should be carried out.
as cefoxitin and ceftriaxone, exhibited about 9% (37). The diffusion of antimicrobial resistance along the food chain
Particularly concerning is the decline in efficacy of hence represents a major public health issue, with numerous
fluoroquinolones, which are considered among the group studies reporting food animals and products colonized and
of “Critically Important Antimicrobials” according to the contaminated by strains carrying a wide variety of antibiotic
World Health Organization. The impressive increase globally resistance genes (57–59), associated with adaptability to
in resistance to quinolones cannot be explained solely by multiple hosts, even those of animal origin (60).
the phenomenon of spontaneous random mutations at the In many recent studies, isolates from retail meat were compared
chromosomal level. In fact, spreading rapidity is surely with human ExPEC clinical strains, with significant results
attributable to plasmid-mediated quinolone resistance (PMQR) in terms of resemblance (59, 61). Beside meat consumption,
genes. the transmission pathways from animals to humans may also
The emergence of epidemic clones, such as sequence type 131 include environmental contamination (62); for instance, fruits
(ST131), which exhibits various mobile elements conferring and vegetables may come into contact with ExPEC through
PMQR, has undoubtedly contributed to this worldwide spread manure (61) in fields or later on in processing stages. ExPEC
(38). A strong association between ST131 and resistance pathotypes were also found in aquatic environments (63) and in
to fluoroquinolones has been found (39). Fluoroquinolones the feces of wild birds (64). It is now recognized that workers
are frequently used in farm animals, particularly in poultry; and their families are another likely route of entry for resistant
consequently, resistant E. coli strains are commonly found in bacteria and genes into the community and health care settings,
alarming percentages (40). although the observed dispersion may be correlated only in part
The main PMQR genes are qnr types, belonging to the with these causes (65).
pentapeptide repeat family (41). qnr is a variant for the Therefore, this widespread presence in farm animals and the
gene coding aminoglycoside transferase enzyme (aac(6’)- similarities between these E. coli, more specifically the strains
Ib-cr), which is able to inactivate molecules belonging to isolated from chicken, turkey, and pork products, and human
fluoroquinolones and aminoglycosides by the addition of an ExPEC suggest a potential key role as reservoir (66). However,
acetyl group (42) and effluent pumps, or proton-dependent the often long interval between ExPEC consumption through
membrane transporters, such as qepA (43) and oxqAB (44). food and disease development makes a clear understanding
These mobile elements have often been found in E. coli isolated of dissemination pathways difficult to obtain. In a recent
from animal sources, such as poultry and pigs (45–47). study by Depoorter et al. (67), the probability of exposure to
Furthermore, associations between qnr and β-lactamases are 1000 colony-forming units of cephalosporin-resistant E. coli
increasingly noted (42), with E. coli progressively becoming while consuming a meal containing chicken was evaluated as
the protagonist of their dissemination (48). Since the beginning 1.5%, with cross-contamination in the kitchen being the major
of the new century, an accelerated evolution and extensive responsible factor.
 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018 919

Due to the globalization of animal and food trade, antibiotic anaerobes and Helicobacter spp. (74). In addition, it allows for
resistance can easily propagate via the food chain (68), thus the testing of various species on the same agar plate.
requiring integrated, multidisciplinary, and global solutions. Automatization of characterization systems has now increased
E. coli prevalence data in food animals are alarming, and the rapidity of antimicrobial resistance detection, taking
more studies are therefore needed in order to ascertain the spread nearly 12 h following bacterial culturing and isolation. These
dynamics between the food chain and humans. In this context, standardized panels test multiple antibiotics, giving detailed
the choice of an appropriate antibiotic agent is critical, making information on MIC values. The most popular susceptibility
clinicians greatly dependent on resistance testing. Hence, great categorization and MIC guidelines are provided by the clinical
attention should be paid to accurate detection of resistance by breakpoints of the Clinical Laboratory Standard Institute and the
conventional and molecular methods. European Committee on Antimicrobial Susceptibility Testing.
Other assays are less frequently utilized, usually in research
laboratories to test antimicrobial susceptibility to various
Analytical Methods antimicrobial molecules:
(a)  Agar well diffusion method (75).
The assessment of antimicrobial resistance can be carried (b) Time-kill test, which is an accurate technique evaluating

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out in different ways, including traditional and molecular the bactericidal effect that enables comprehension of whether
approaches. The first group allows a phenotypic observation the antimicrobial effect depends on time or on the concentration
of the resistance, whereas the second is more focused on the used (76).
genetic elements resulting in resistance. The most well-known (c)  Flow cytometry, which assesses the efficacy of the
conventional analyses include the following: antibiotic through its effect on cells, detecting viable, injured,
(a) The Kirby–Bauer disk diffusion test, in which agar and dead cells by means of specific dyes staining (77).
plates—inoculated with a standardized inoculum of the Although costly, flow cytometry represents a rapid (2–6 h) and
microorganism in question—are seeded with specific disks reproducible method, providing additional useful information
containing the antibiotic molecule at the desired concentration. on the number of damaged cells, whose potential recovery
The inhibition zone is proportional to the diffusibility and could affect the clinical therapeutic outcome.
concentration of the antibiotic itself and on the bacterial (d) Lastly, high performance liquid chromatography
susceptibility. This test has some advantages, including the low (HPLC) was reported as a very recent alternative method
cost, the ease of interpretation, and the possibility to screen large for antibiotic testing, although it must be tested further with
numbers of molecules and microorganisms. On the other hand, more antimicrobial drugs. The HPLC study was based on the
it is not suitable for certain fastidious or slow-growing species, breakdown of the antibiotic cefotaxime by the enzymatic activity
even if new standardized test conditions have been developed of microbial β-lactamases (78). This method is characterized by
for Haemophilus spp., streptococci, and Neisseria gonorrhoeae a high sensitivity and specificity in the detection of resistance
(69). Moreover, it gives qualitative results. As a consequence, it mediated by ESBL, and it could be useful for rapid answers (up
is not a very informative method, and it may be time-consuming. to 1.5 h) in urgent cases.
(b) Antimicrobial gradient method, combining the disk
diffusion principle and a quantitative value, in order to
establish the minimum inhibitory concentration (MIC), which Molecular Methods
is the lowest concentration of the antibiotic inhibiting the tested
microorganism. The strip is impregnated with a concentration Genotypic methods are increasingly important in assessing
gradient, allowing determination of the MIC at the beginning of antimicrobial resistance, as they are more rapid and allow for
the growth inhibition ellipse. This method, although quite costly discernment of the real resistance potential of microorganisms.
depending on the number of molecules tested, allows for the They provide an indication of the typology of resistance genes,
study of the eventual interaction between two drugs as potential are useful for epidemiological studies, and allow for a better
synergistic effects (70) and has proved to correlate well with comprehension of the microbial communities. In addition, they
other MIC-determining methods (71, 72). may be very useful in certain cases, such as for slow-growing
(c)  Broth and agar dilution, which are the most adequate bacteria or species that cannot be cultured easily or at all. They
techniques for assaying the MIC (usually expressed in μg/mL initially contemplated the amplification of a specific sequence,
or mg/L). These two methods examine the growth of tested defined by one or more pairs of primers (i.e., multiplex
microorganisms in a liquid or agar medium, respectively, at various polymerase chain reaction), thus only targeting a few genes at
antibiotic concentrations, usually differing 2-fold. The broth a time.
dilution involves either tubes (macrodilution) or microtitration Currently, microarrays are improving rapid diagnostics,
plates (microdilution), which are more standardized, whereas agar offering the possibility of simultaneous detection of multiple
dilution varies the antibiotic concentration into the agar medium. classes of resistance genes for a wide range of species (79).
Macrodilution is based on turbidity, indicating bacterial growth; Moreover, microarrays are able to accurately detect the genes
microdilution is less easy to read, and colorimetric systems based of interest in low- or absent-expression cases, and even plasmid
on tetrazolium salts have hence been developed (73), such as fragments lost from bacteria, demonstrating a high sensitivity (80).
3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide As microarrays provide standardized panels, they need regular
and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) updates in order to identify the most significant resistance genes.
carbonyl]-2H-tetrazolium hydroxide. Automated instrumentations However, the real evolution of rapid characterization methods
reading end points are available, enabling results more quickly, for antimicrobial resistance genes was the development of
although at a substantial economic cost. Agar dilution method is next-generation sequencing systems, dramatically increasing
particularly indicated for fastidious microbial species such as sequence-based capabilities. This technique represents a powerful
920 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018

tool, enabling exploration of the bacterial genome in its entirety resistance surveillance system is essential, following the “One
(e.g., whole genome sequencing) (81). Metagenomic reads Health” approach, which takes into account all potential
are compared against sequences present in various databases, reservoirs and diffusion pathways for resistance determinants
such as ARG-ANNOT, ResFinder, and Resfams (82–84). This and also identifies the emergence of new mechanisms that confer
could also represent a helpful tool in clinical diagnostics in the the ability to survive on antimicrobial molecules.
future, determining the great diversity of antibiotic resistance In general, it is crucial to introduce “antimicrobial
genes; however, it has some drawbacks, such as the lack of stewardship” programs in health organizations. These programs
standardization, a high database dependency, and a potential are a set of actions aimed at promoting the selection, dose, and
underestimation of resistance genes, given that new mechanisms optimal duration of antibiotic therapies, both in farms and in
go unnoticed. Reads can be reanalyzed for detection of new hospital settings, in order to obtain the best clinical outcome in
inserted resistance gene sequences after database updates. the treatment and prevention of infections, and the least impact
Another major technique is functional metagenomics, which, on the development of resistance.
interestingly, allows for the detection of novel resistance genes. With regard to animal production systems, a revision in
In fact, it clones DNA fragments into different kinds of vectors farm management is essential, combining good practices,
(e.g., plasmid, or for larger inserts, fosmids and cosmids), animal welfare and applying good hygiene practices, in order

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which are then expressed by a host (85) and studied through to reduce the use of antimicrobials, thus acting on the reservoir
sequencing (86). In this way, both phenotype and genotype of antibiotic resistance genes. In fact, modern breeding systems
can be observed, thus allowing for a deepened investigation. rely on the routine use of antibiotics as a measure of disease
However, this method also has some disadvantages. For prevention or for treatment of avoidable disease outbreaks
instance, it is rather time-consuming, it does not provide very in a nonsustainable way. Therefore, different levels of safety
informative results about the species harboring these genes, and measures are required, including “tertiary prevention.”
it relies on the host’s expression. Lastly, other environmental sources of antibiotics and
Therefore, if molecular methods have revolutionized resistance genes, such as agricultural and human waste, lack an
antimicrobial resistance analysis on the one hand, on the other appropriate risk assessment. Consequently, the idea of carrying
hand they may overestimate the resistance potential, focusing out official controls at various levels should be taken into
on genes that may not be expressed, such as in cases in account; in fact, although there are maximum permitted limits for
which isolates can result occasionally positive for a gene but antibiotic residues in foodstuffs, there are no guidelines regarding
susceptible to the corresponding antimicrobial. any threshold for the presence of resistance determinants in the
Hence, no method is valid for all purposes, because it is various environmental niches, primarily including foods.
important to know the origin of the genes and understand
their dissemination through molecular studies, especially for
nonculturable bacteria, which represent the majority. These
References
microorganisms are integrated into all ecosystems, including
animal and human microbiota; consequently, they may also
(1) World Health Organization (2014) Antimicrobial resistance:
exchange genes, contributing to the phenomenon of antibiotic global report on surveillance. World Health Organization
resistance. On the other hand, there are currently no molecular (WHO), Geneva, Switzerland, http://apps.who.int/iris/
tools that meet all the necessities, as some techniques overlook bitstream/10665/112642/1/9789241564748_eng.pdf (accessed
still-unknown genes, and others depend heavily on gene on October 24, 2017)
expression, such as functional metagenomics. Therefore, it is (2) D᾽Costa, V.M., McGrann, K.M., Hughes, D.W., & Wright, G.D.
necessary to integrate the two types of methods at present in order (2006) Science 311, 374–377. doi:10.1126/science.1120800
to get a complete concept and to gather as much information (3) Livermore, D.M. (2009) J. Antimicrob. Chemother. 64,
as possible about the antibiotic resistance phenomenon. More i29–i36. doi:10.1093/jac/dkp255
research is still needed for the development and implementation (4) Thanner, S., Drissner, D., & Walsh, F. (2016) M Bio 7,
e02227–e02215. doi:10.1128/mbio.02227-15
of rapid methods to immediately and easily identify potentially
(5) Summers, A.O. (2002) Clin. Infect. Dis. 34, S85–S92.
emerging microorganisms and associated resistance genes doi:10.1086/340245
in many ambits, including food production activities such as (6) Hughes, L., Hermans, P., & Morgan, K. (2008) J. Antimicrob.
intensive livestock farms. Chemother. 61, 947–952. doi:10.1093/jac/dkn017
(7) Ottaviani, F. (1996) in Microbiologia dei Prodotti di Origine
Vegetale– Ecologia ed Analisi Microbiologica, Chiriotti Editori,
Conclusions Pinerolo, Italy, pp 347–366
(8) Gilbert, Y., & Duchaine, C. (2009) Can. J. Civ. Eng. 36,
The problem of antibiotic resistance exhibited by E. coli 1873–1886. doi:10.1139/l09-117
and other microorganisms, both commensal and pathogen life (9) Venter, P., Lues, J.F., & Theron, H. (2004) Poultry Sci. 83,
forms, is evident enough in the current world of food industries. 1226–1231. doi:10.1093/ps/83.7.1226
The emergence of antibiotic resistance has limited the number (10) Hamscher, G., Pawelzick, H.T., Sczesny, S, Nau, H., &
of molecules that act against pathogens; on the other hand, there Hartung, J. (2003) Environ. Health Perspect. 111, 1590–1594.
doi:10.1289/ehp.6288
has not been a proportional introduction of new antibiotic classes
(11) Chapin, A., Rule, A., Gibson, K., Buckley, T., & Schwab, K.
in the market, narrowing the number of therapeutic options
(2005) Environ. Health Perspect. 113, 137–142. doi:10.1289/
available and thus amplifying the problem. In order to reduce the ehp.7473
animal reservoir of ExPEC, vaccines are being studied on highly (12) Gibbs, S.G., Green, C.F., Tarwater, P.M., Mota, L.C.,
conserved antigens to replace and reduce the use of antimicrobials Mena, K.D., & Scarpino, P.V. (2006) Environ. Health Perspect.
in animal production (87, 88). The implementation of an integrated 114, 1032–1037. doi:10.1289/ehp.8910
 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018 921

(13) Chang, Q., Wang, W., Regev-Yochay, G., Lipsitch, M., & ARSUserFiles/60401020/NARMS/NARMS2011/NARMS%20
Hanage, W.P. (2015) Evol. Appl. 8, 240–247. USDA%202011%20Report.pdf (accessed on October 21, 2017)
doi:10.1111/eva.12185 (37) Giufrè, M., Graziani, C., Accogli, M., Luzzi, I., Busani, L., &
(14) Witte, W. (2000) Int. J. Antimicrob. Agents 16, S19–S24. Cerquetti, M. (2012) J. Antimicrob. Chemother. 67, 860–867.
doi:10.1016/S0924-8579(00)00301-0 doi:10.1093/jac/dkr565
(15) Marshall, B.M., Ochieng, D.J., & Levy, S.B. (2009) Microbe (38) Cerquetti, M., Giufrè, M., Garcia-Fernandez, A., Accogli, M.,
4, 231–238 Fortini, D., Luzzi, I., & Carattoli, A. (2010) Clin. Microbiol.
(16) Van Schaik, W. (2015) Phil. Trans. R. Soc. B 370, 20140087. Infect. 16, 1555–1558. doi:10.1111/j.1469-0691.2010.03162.x
doi:10.1098/rstb.2014.0087 (39) Johnson, J.R., Kuskowski, M.A., Menard, M., Gajewski, A.,
(17) Stecher, B., Denzler, R., Maier, L., Bernet, F., Sanders, M.J., Xercavins, M., & Garau, J. (2006) J. Infect. Dis. 194, 71–78.
Pickard, D.J., Barthel, M., Westendorf, A.M., Krogfelt, K.A., doi:10.1086/504921
Walker, A.W., Ackermann, M., Dobrindt, U., Thomson, N.R., & (40) Johnson, J.R., Kuskowski, M.A., Smith, K.O᾽Bryan, T.T, &
Hardt, W.D. (2012) Proc. Natl Acad. Sci. USA 109, 1269–1274. Tatini, S. (2005) J. Infect. Dis. 191, 1040–1049.
doi:10.1073/pnas.1113246109 doi:10.1086/428451
(18) Waller, A.S., Yamada, T., Kristensen, D.M., Kultima, J.R., (41) Martinez-Martinez, L., Pascual, A., & Jacoby, G.A. (1998)
Sunagawa, S., Koonin, E.V., & Bork, P. (2014) ISME J. 8, Lancet 351, 797–799. doi:10.1016/S0140-6736(97)07322-4

Downloaded from https://academic.oup.com/jaoac/article/101/4/916/5654005 by guest on 16 November 2023

1391–1402. doi:10.1038/ismej.2014.30 (42) Robicsek, A., Strahilevitz, J., Sahm, D., Jacoby, G.A., &
(19) Allen, H.K., Looft, T., Bayles, D.O., Humphrey, S., Hooper, D.C. (2006) Antimicrob. Agents Chemother. 50,
Levine, U.Y., Alt, D., & Stanton, T.B. (2011) M Bio 2, 2872–2874. doi:10.1128/AAC.01647-05
e00260–11. doi:10.1128/mBio.00260-11 (43) Yamane, K., Wachino, J., Suzuki, S., Kimura, K., Shibata, N.,
(20) Quiros, P., Colomer-Lluch, M., Martinez-Castillo, A., Miro, E., Kato, H., Shibayama, K., Konda, T., & Arakawa, Y. (2007)
Argente, M., Jofre, J., Navarro, F., & Muniesa, M. (2014) Antimicrob. Agents Chemother. 51, 3354–3360. doi:10.1128/
Antimicrob. Agents Chemother. 58, 606–609. doi:10.1128/ AAC.00339-07
AAC.01684-13 (44) Hansen, L.H., Johannesen, E., Burmolle, M., Sorensen, A.H., &
(21) Straub, J.A., Hertel, C., & Hammes, W.P. (1999) J. Food Sorensen, S.J. (2004) Antimicrob. Agents Chemother. 48,
Protect. 62, 1150–1156. doi:10.4315/0362-028X-62.10.1150 3332–3337. doi:10.1128/AAC.48.9.3332-3337.2004
(22) Bauer, T., Hammes, W.P., Haase, N.U., & Hertel, C. (2004) (45) Fortini, D., Fashae, K., García-Fernández, A., Villa, L., &
Environ. Biosaf. Res. 3, 215–223. doi:10.1051/ebr:2005005 Carattoli, A. (2011) J. Antimicrob. Chemother. 66, 1269–1272.
(23) Molin, S., & Tolker-Nielsen, T. (2003) Curr. Opin. Biotechnol. doi:10.1093/jac/dkr085
14, 255–261. doi:10.1016/s0958-1669(03)00036-3 (46) Liu, J.H., Deng, Y.T., Zeng, Z.L., Gao, J.H., Chen, L.,
(24) Król, J.E., Nguyen, H.D., Rogers, L.M., Beyenal, H., Arakawa, Y., & Chen, Z.L. (2008) Antimicrob. Agents
Krone, S.M., & Top, E.M. (2011) Appl. Environ. Microbiol. 77, Chemother. 52, 2992–2993. doi:10.1128/AAC.01686-07
5079–5088. doi:10.1128/aem.00090-11 (47) Soufi, L., Sáenz, Y., Vinué, L., Abbassi, M.S., Ruiz, E.,
(25) Hannan, S., Ready, D., Jasni, A.S., Rogers, M., Pratten, J., & Zarazaga, M., Abbas, A., Dbaya, R., Khanfir, L., Ben Hassen, A.,
Roberts, A.P. (2010) FEMS Immunol. Med. Microbiol. 59, Hammami, S., & Torres, C. (2011) Int. J. Food Microbiol. 144,
345–349. doi:10.1111/j.1574-695x.2010.00661.x 497–502. doi:10.1016/j.ijfoodmicro.2010.11.008
(26) Fluit, A.C. (2005) FEMS Immunol. Med. Microbiol. 43, 1–11. (48) Yano, H., Uemura, M., Endo, S., Kanamori, H., Inomata, S.,
doi:10.1016/j.femsim.2004.10.007 Kakuta, R., Ichimura, S., Ogawa, M., Shimojima, M., Ishibashi, N.,
(27) Cirz, R.T., Chin, J.K., Andes, D.R.de Crecy-Lagard, V., Aoyagi, T., Hatta, M., Gu, Y., Yamada, M., Tokuda, K.,
Craig, W.A., & Romesberg, F.E. (2005) PloS Biol. 3, e176. Kunishima, H., Kitagawa, M., Hirakata, Y., & Kaku, M. (2013)
doi:10.1371/journal.pbio.0030176 PLoS ONE 8, e64359. doi:10.1371/journal.pone.0064359
(28) McMahon, M.A.S., Blair, I.S., Moore, J.E., & McDowell, D.A. (49) Cantón, R. (2008) in Evolutionary Biology of Bacterial and
(2007) J. Appl. Microbiol. 103, 1883–1888. Fungal Pathogens, F., Baquero, C., Nombela, G.H., Casslel,
doi:10.1111/j.1365-2672.2007.03412.x Gutierrez-Fuentes, J.A. (Eds), ASM Press, Washington, DC,
(29) McMahon, M.A.S., Xu, J., Moore, J.E., Blair, I.S., & pp 249–270
McDowell, D.A. (2007) Appl. Environ. Microbiol. 73, 211–217. (50) Cantón, R., & Coque, T.M. (2006) Curr. Opin. Microbiol. 9,
doi:10.1128/AEM.00578-06 466–475. doi:10.1016/j.mib.2006.08.011
(30) European Food Safety Authority and European Centre for (51) Smet, A., Martel, A., Persoons, D., Dewulf, J., Heyndrickx, M.,
Disease Prevention and Control ( 2012 ) EFSA J. 10, 2598. Catry, B., Herman, L., Haesebrouck, F., & Butaye, P. (2008)
doi:10.2903/j.efsa.2012.2598. Antimicrob. Agents Chemother. 52, 1238–1243. doi:10.1128/
(31) Van den Bogaard, A.E., & Stobberingh, E.E. (2000) AAC.01285-07
Int. J. Antimicrob. Agents 14, 327–335. doi:10.1016/S0924- (52) Dahmen, S., Haenni, M., & Madec, J.Y. (2012) J. Antimicrob.
8579(00)00145-X Chemother. 67, 3011–3012. doi:10.1093/jac/dks308
(32) Johnson, J.R., & Russo, T.A. (2002) J. Lab. Clin. Med. 139, (53) Huang, X., Yu, L., Chen, X., Zhi, C., Yao, X., Liu, Y., Wu, S.,
155–162. doi:10.1067/mlc.2002.121550 Guo, Z., Yi, L.,Zeng, Z., & Liu, J.H. (2017) Front. Microbiol. 8,
(33) Karlowsky, J.A., Hoban, D.J., Decorby, M.R., Laing, N.M., & 562. doi:10.3389/fmicb.2017.00562
Zhanel, G.G. (2006) Antimicrob. Agents Chemother. 50, (54) Liu, Y.Y., Wang, Y., Walsh, T.R., Yi, L.X., Zhang, R.,
2251–2254. doi:10.1128/AAC.00123-06 Spencer, J., Doi, Y., Tian, G., Dong, B., Huang, X., Yu, L.F.,
(34) Pitout, J.D., & Laupland, K.B. (2008) Lancet Infect. Dis. 8, Gu, D., Ren, H., Chen, X., Lu, L., He, D., Zhou, H., Liang, Z.,
159–166. doi:10.1016/S1473-3099(08)70041-0 Liu, J.H., & Shen, J. (2016) Lancet Infect. Dis. 16, 161–168.
(35) Tadesse, D.A., Zhao, S., Tong, E., Ayers, S., Singh, A., doi:10.1016/S1473-3099(15)00424-7
Bartholomew, M.J., & McDermott, P.F. (2012) Emerg. Infect. (55) Xavier, B.B., Lammens, C., Ruhal, R., Kumar-Singh, S.,
Dis. 18, 741–749. doi:10.3201/eid1805.111153 Butaye, P., Goossens, H., & Malhotra-Kumar, S. (2016) Euro
(36) USDA National Antimicrobial Resistance Monitoring Surveill. 21. doi:10.2807/1560-7917.ES.2016.21.27.30280
System ( 2014 ) 2011 NARMS Animal Arm Annual (56) Yin, W., Li, H., Shen, Y., Liu, Z., Wang, S., Shen, Z.,
Report, U.S. Department of Agriculture, Agricultural Zhang, R., Walsh, T.R., Shen, J., & Wang, Y. (2017) mBio 8,
Research Service Athens, GA, https://www.ars.usda.gov/ e00543–17. doi:10.1128/mBio.00543-17
922 Caruso: Journal of AOAC International Vol. 101, No. 4, 2018

(57) Campos, J., Mourão, J., Pestana, N., Peixe, L., Novais, C., & (73) Al-Bakri, A.G., & Afifi, F.U. (2007) J. Microbiol. Methods 68,
Antunes, P. (2013) Int. J. Food Microbiol. 166, 464–470. 19–25. doi:10.1016/j.mimet.2006.05.013
doi:10.1016/j.ijfoodmicro.2013.08.005 (74) CLSI ( 2010 ) Methods for Antimicrobial Dilution and
(58) Jakobsen, L., Spangholm, D.J., Pedersen, K., Jensen, L.B., Disk Susceptibility of Infrequently Isolated or Fastidious
Emborg, H.D., Agersø, Y., Aarestrup, F.M., Hammerum, A.M., & Bacteria; Approved Guideline—Second Edition, CLSI document
Frimodt-Møller, N. (2010) Int. J. Food Microbiol. 142, M45-A2, Clinical and Laboratory Standards Institute (CLSI),
264–272. doi:10.1016/j.ijfoodmicro.2010.06.025 Wayne, PA
(59) Miles, T.D., McLaughlin, W., & Brown, P.D. (2006) BMC Vet. (75) Valgas, C., DeSouza, S.M., Smânia, E.F.A., & Smania, A.
Res. 2, 7. doi:10.1186/1746-6148-2-7 (2007) Braz. J. Microbiol. 38, 369–380. doi:10.1590/S1517-
(60) Founou, L.L., Founou, R.C., & Essack, S.Y. (2016) Front. 83822007000200034
Microbiol 7, 1881. doi:10.3389/fmicb.2016.01881 (76) Pfaller, M.A., Sheehan, D.J., & Rex, J.H. (2004) Clin.
(61) Vincent, C., Boerlin, P., Daignault, D., Dozois, C.M., Microbiol. Rev. 17, 268–280. doi:10.1128/CMR.17.2.
Dutil, L., & Galanakis, C. (2010) Emerg. Infect. Dis. 16, 88–95. 268-280.2004
doi:10.3201/eid1601.091118 (77) Paparella, A., Taccogna, L., Aguzzi, I., Chaves-Lòpez, C.,
(62) Graham, J.P., Leibler, J.H., Price, L.B., Otte, J.M., Serio, A., Marsilio, F., & Suzzi, G. (2008) Food Control 19,
Pfeiffer, D.U., Tiensin, T., & Silbergeld, E.K. (2008) Public 1174–1182. doi:10.1016/j.foodcont.2008.01.002

Downloaded from https://academic.oup.com/jaoac/article/101/4/916/5654005 by guest on 16 November 2023

Health Rep. 123, 282–299. doi:10.1177/003335490812300309 (78) Robinson, A.M., Medlicott, N.J., & Ussher, J.E. (2016) Future
(63) Hamelin, K., Bruant, G., El-Shaarawi, A., Hill, S., Edge, T. A., Sci. OA 2, FSO142. doi:10.4155/fsoa-2016-0042
Fairbrother, J., Harel, J., Maynard, C., Masson, L., & (79) Card, R., Zhang, J., Das, P., Cook, C., Woodford, N., &
Brousseau, R. (2007) Appl. Environ. Microb. 73, 477–484. Anjum, M.F. (2013) Antimicrob. Agents Chemother. 57,
doi:10.1128/AEM.01445-06 458–465. doi:10.1128/AAC.01223-12
(64) Guenther, S., Grobbel, M., Lubke-Becker, A., Goedecke, A., (80) Kirchner, M. Abuoun, M., Mafura, M., Bagnall, M.,
Friedrich, N.D., Wieler, L.H., & Ewers, C. (2010) Vet. Hunt, T., Thomas, C., Weile, J., & Anjum, M.F. (2013) PLoS
Microbiol. 144, 219–225. doi:10.1016/j.vetmic.2009.12.016 ONE 8, e84142. doi:10.1371/journal.pone.0084142
(65) Marshall, B.M., & Levy, S.B. (2011) Clin. Microbiol. Rev. 24, (81) Köser, C.U., Ellington, M.J., & Peacock, S.J. (2014) Trends
718–733. doi:10.1128/CMR.00002-11 Genet. 30, 401–407. doi:10.1016/j.tig.2014.07.003
(66) Manges, A.R., & Johnson, J.R. (2012) Clin. Infect. Dis. 55, (82) Gupta, S.K., Padmanabhan, B.R., Diene, S.M., Lopez-Rojas, R.,
712–719. doi:10.1093/cid/cis502 Kempf, M., Landraud, L., & Rolain, J.M. (2014)
(67) Depoorter, P., Persoons, D., Uyttendaele, M., Butaye, P., Antimicrob. Agents Chemother. 58, 212–220.
De Zutter, L., Dierick, K., Herman, L., Imberechts, H., doi:10.1128/AAC.01310-13
Van Huffel, X., & Dewulf, J. (2012) Int. J. Food Microbiol. 159, (83) Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M.,
30–38. doi:10.1016/j.ijfoodmicro.2012.07.026 Rasmussen, S., Lund, O., Aarestrup, F.M., & Larsen, M.V.
(68) Holmes, A.H., Moore, L.S., Sundsfjord, A., Steinbakk, M., (2012) J. Antimicrob. Chemother. 67, 2640–2644.
Regmi, S., Karkey, A., Guerin, P.J., & Piddock, L.J. (2016) doi:10.1093/jac/dks261
Lancet 387, 176–187. doi:10.1016/S0140-6736(15)00473-0 (84) Gibson, M.K., Forsberg, K.J., & Dantas, G. (2015) ISME J. 9,
(69) CLSI ( 2012 ) Performance Standards for Antimicrobial Disk 207–216. doi:10.1038/ismej.2014.106
Susceptibility Tests; Approved Standard—Eleventh Edition, (85) Fouhy, F., Stanton, C., Cotter, P.D., Hill, C., & Walsh, F. (2015)
CLSI document M02-A11, Clinical and Laboratory Standards Front. Microbiol. 6, 172. doi:10.3389/fmicb.2015.00172
Institute (CLSI), Wayne, PA (86) Perry, J.A., & Wright, G.D. (2014) BioEssays 36, 1179–1184.
(70) White, R.L., Burgess, D.S., Manduru, M., & Bosso, J.A. (1996) doi:10.1002/bies.201400128
Antimicrob. Agents Chemother. 40, 1914–1918 (87) Dobrindt, U., & Hacker, J. (2008) Curr. Opin. Microbiol. 11,
(71) Baker, C.N., Stocker, S.A., Culver, D.M., & Thornsberry, C. 409–413. doi:10.1016/j.mib.2008.09.005
(1991) J. Clin. Microbiol. 29, 533–538 (88) Wieser, A., Romann, E., Magistro, G., Hoffmann, C.,
(72) Prakash, V., Lewis, J.S.II, & Jorgensen, J.H. (2008) Antimicrob. Norenberg, D., Weinert, K., & Schubert, S. (2010) Infect.
Agents Chemother. 52, 4528. doi:10.1128/AAC.00904-08 Immun. 78, 3432–3442. doi:10.1128/IAI.00174-10