www.elsevier.com/locate/addr

Advanced Drug Delivery Reviews 56 (2004) 1497–1521

Antimicrobial drug delivery in food animals and microbial food

safety concerns: an overview of in vitro and in vivo factors

potentially affecting the animal gut microflora

S. Steve Yan, Jeffrey M. Gilbert*

Division of Human Food Safety, Center for Veterinary Medicine, Food and Drug Administration, 7500 Standish Place,

HFV-150, Rockville, MD 20850, USA

Received 22 September 2003; accepted 18 February 2004

Available online 2 April 2004

Abstract

This review provides an overview of considerations particular to the delivery of antimicrobial agents to food animals.

Antimicrobial drugs are used in food animals for a variety of purposes. These drugs may have therapeutic effects against disease

agents, or may cause changes in the structure and/or function of systems within the target animal. Routes of administration,

quantity, duration, and potency of an antimicrobial drug are all important factors affecting their action(s) and success. Not only

might targeted pathogens be affected, but also bacteria residing in (or on) the treated food animals, especially in the intestines

(gastrointestinal tract microflora). Resistance to antimicrobial agents can occur through a number of mechanisms. The extent to

which resistance develops is greatly affected by the amount of drug [or its metabolite(s)] a bacterium is exposed to, the duration

of exposure, and the interaction between an individual antimicrobial agent and a particular bacterium. The impact of

antimicrobial agents on the emergence of resistance in vitro and in vivo may not readily correlate.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Antimicrobial agent; Activity; In vitro and in vivo; Microflora; Gastrointestinal tract; Food animals; Antimicrobial resistance

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1498

2. Microflora of the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

2.1. An ecological system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

2.1.1. Diverse nature of microflora in food animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

2.1.2. Harmonious co-existence of microflora in the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . 1500

2.2. Host–microflora equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501

2.3. Foodborne pathogens among microflora of food animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502

2.4. Primary location of zoonotic foodborne pathogens in the gastrointestinal tract . . . . . . . . . . . . . . . . . . . 1502

3. Characteristics of antimicrobial drug activities in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503

0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2004.02.010

* Correspondence author. Tel.: +1-301-827-0233.

E-mail address: jgilbert@cvm.fda.gov (J.M. Gilbert).

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211498

3.1. Antimicrobial activity is measurable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503

3.2. Antimicrobial activity is specific. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503

3.3. Antimicrobial activities exist at low concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505

3.4. Antimicrobial activities can be persistent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1507

3.5. Other factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1507

4. Antimicrobial drug delivery in food animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1508

4.1. Drug delivery routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1508

4.1.1. Parenteral administrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1508

4.1.2. Oral administrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1508

4.1.3. Other drug delivery methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509

4.2. Drug absorption and transport through the intestinal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . 1509

4.3. Role of biliary excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1510

5. Antimicrobial drug delivery and antimicrobial resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1510

5.1. Mutant prevention concentration for fluoroquinolones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511

5.2. Stepwise changes in susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511

5.3. Horizontal transmissibility of resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512

5.4. Concerns of antimicrobial resistance among the gut microflora . . . . . . . . . . . . . . . . . . . . . . . . . . 1512

6. Correlation of data from in vitro and in vivo studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.1. Discrepancy between in vitro and in vivo data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.1.1. Bacterial generation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.1.2. Protein binding of antimicrobial agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.1.3. Metabolites with antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.2. A collective application of post-antibiotic and sub-MIC effects in vivo . . . . . . . . . . . . . . . . . . . . . . 1513

6.3. Retaining resistance property in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513

6.4. Drug–bacterium specifics for microbial food safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1514

7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1514

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515

1. Introduction

The use of antimicrobial drugs in veterinary

medicine generally parallels their uses in human

medicine. Many of the antimicrobial agents ap-

proved for use in humans are approved for thera-

peutic and other purposes in food (or food-

producing) animals. Under the current requirements

for approval of new antimicrobial drugs for food

animals in the United States, a rigorous evaluation

process exists to ensure that the uses of antimicro-

bial drugs are safe and effective for the targeted food

animals, and that they are safe to the environment

and to humans that consume food products derived

from these animals [1]. In addition, approved anti-

microbial agents must have demonstrated a consis-

tency of quality, including strength, purity, and

potency.

In recent years, antimicrobial resistance associated

with the use of antimicrobial drugs in food animals

has received an increasing amount of public attention

[1–3], and related information can be found at many

organization’s sites (e.g., http://www.fda.gov/cvm/

antimicrobial/antimicrobial.html; http://www.cdc.

gov/drugresistance/; http://www.who.int/infectious-

disease-report/2000/, accessed as of August 29,

2003). Antimicrobial agents administered to animals

will result in residual levels in the gastrointestinal

(gastrointestinal) tract of the treated animals. This

exposure to antimicrobial agents may result in the

disruption of the host’s intestinal microflora. This

exposure may also select for resistant bacteria which

may ultimately cause food safety concerns for humans.

Human food safety is an important and indispensable

component of any new animal antimicrobial drug

application process for food animals. [3].

Evaluation of the human food safety of antimicro-

bial drugs for use in food animals takes into consid-

eration: (1) the evaluation of the quantitative drug

residues in edible tissues and their possible toxicolog-

ical effects in humans, including the potential disrup-

tion of human intestinal microflora [3]; and (2) the

effect of the subject antimicrobial drug on the gener-

ation or selection of resistant, zoonotic foodborne

pathogens. This latter aspect is termed microbial food

safety [1]. The goal of microbial food safety evalua-

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1499

tion is to determine whether the use of a specific

antimicrobial drug in food animals, under labeled

conditions of use, will create (or make worse) a state

of antimicrobial resistance among foodborne or other

enteric bacteria in the gastrointestinal tract of the

treated animals [1–3].

This review provides an overview of what is

currently known about the interactions among antimi-

crobial agents and bacteria in vitro. In addition,

similarities and differences between in vitro and in

vivo antibacterial activities of various antimicrobial

drugs will be discussed. The intent of this discussion

is to help further the understanding of the impact of

exposure to antimicrobial agents on the gastrointesti-

nal tract microflora in food animals.

2. Microflora of the gastrointestinal tract

2.1. An ecological system

The digestive system of animals hosts several

hundred species of bacteria. These bacteria (microbial

flora or microflora) in the gastrointestinal tract form a

complex ecosystem [4,5] that plays an important role

in maintaining the integrity of the host’s enterocyte,

providing enzymes for metabolism of ingested of

foods, modulation of metabolic processes for host

needs (such as converting steroids and unconjugating

bilirubin into more water soluble forms of urobilino-

gen), and protecting the host epithelial cells from

colonization by toxin-producing or invasive intestinal

pathogens [6]. The intestinal microflora also provide

certain nutrients and vitamins that are beneficial to the

host [4,7]. The relationship between the non-harmful

microbiota and the host is often viewed as symbiotic;

therefore, the organisms in the gastrointestinal tract

are commensals under normal conditions. A complete

understanding of the balance and composition of the

gut microflora in the host animal species remains

incomplete.

Food animals include mammalian and non-mam-

malian species that inhabit terrestrial and aquatic

environments. Major food animals include cattle,

swine, and poultry (chickens and turkeys). Addition-

ally, there are many other farmed food animals such

as sheep, goats, ducks and numerous species of fish.

Animals raised for human consumption vary widely

in size from large species (cattle) to relatively small

species (chicken or fish). They have considerable

variations in their gastrointestinal anatomies and

physiologic functions. Among mammals, there are

distinct differences between species with respect to

anatomy and physiology, especially relating to the

alimentary canal. For example, cattle, sheep, and

goats are ruminants with the dietary habits of herbi-

vores, while swine and poultry are monogastric

omnivores. It is beyond the scope of this review to

identify detailed differences in the gastrointestinal

tract microflora among food animals. Instead, the

focus here is to identify general features of micro-

flora in the gastrointestinal tracts of food animals as

well as factors that may affect the intrinsic nature of

the microflora.

2.1.1. Diverse nature of microflora in food animals

In order to understand differences among the

microflora of food animals, it is useful to compare

the anatomy and physiology of the digestive systems

of three representative food animals: cattle, swine, and

poultry. Cattle have a large, complex, compartmented

pre-gastric system, while swine have a true monogas-

tric system. Poultry have a simple, modified gastric

system that is different from that of either cattle or

swine. In cattle, the three pre-gastric compartments

(the rumen, reticulum, and omasum) physically pro-

cess feedstuffs and subject them to fermentation with

resident symbiotic microorganisms, prior to entry into

the abomasum (the fourth compartment), where more

typical acidic digestive conditions prevail. Of these

four compartments, the rumen is the largest, where

billions of microorganisms break down grasses and

other coarse vegetation that monogastric animals

cannot digest [8]. The rumen of cattle is colonized

by several hundred species of microorganisms, in-

cluding bacteria, fungi, and protozoa, and the compo-

sition of the microorganisms in the rumen differs

considerably from the microorganisms found in the

lower gastrointestinal tract of cattle [8].

The gastrointestinal tract of swine is a monogastric

system similar to that of humans. The acidic environ-

ment in the stomach serves as a natural barrier

between the upper alimentary canal, e.g., mouth and

esophagus, and the lower alimentary canal, e.g.,

duodenum, ileum, cecum, and colon. This barrier

blocks the entry of bacteria, including potential patho-

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211500

gens, from entering the lower alimentary canal. The

components and relative proportion for some seg-

ments of the swine gastrointestinal tract is different

from that of cattle.

The gastrointestinal anatomy of poultry is different

from either the ruminant, or monogastric system of

swine. Poultry do not have a sharp distinction between

the pharynx and mouth (oropharynx). Salivary glands

are well developed in poultry, and their esophagus is

divided into the cervical and thoracic regions. The

crop, an expanded region of the esophagus, primarily

serves the function of food storage. The feed is then

passed through the proventriculus (stomach) to the

gizzard, an organ where grinding of feed occurs [9].

The lower gastrointestinal tract of poultry is different

from either cattle or swine in many aspects.

The gastrointestinal anatomic variations found in

the different food animal species are suited to the

diverse physiologic needs of each species, which may

explain differences in the resident microflora among

animal species. Microorganisms that colonize the gut

of a given animal species may be determined by the

gastrointestinal anatomy, its associated patho-physio-

logical characteristics, as well as the diet. In fact,

understanding the differences in how bacterial species

colonize various animal species is not completely

understood. Moreover, there are differences in micro-

flora across locations within the alimentary canal as

well as between the different animal species. For

example, Campylobacter are more frequently found

in chickens than in swine, and are rare in adult cattle.

When analyzed at the bacterial species level, Cam-

pylobacter jejuni is the predominate species isolated

from chickens, while Campylobacter coli is the pre-

dominant species isolated from swine [10–12].

2.1.2. Harmonious co-existence of microflora in the

gastrointestinal tract

Microflora distributions within the gastrointestinal

tract differ enormously from segment to segment. In

humans, such differences are clearly observed be-

tween the stomach, upper small intestine, lower small

intestine, colon, and rectum [13]. Similarly, the nature

and quantities of microorganisms vary greatly from

segment to segment within the gastrointestinal tract

for each food animal species. A gradual but distinct

change in the distribution of microflora along various

segments of the gastrointestinal tract has long been

recognized [14]. This transition of microflora ranges

from the sparsely populated flora in the stomach to the

diverse, densely populated bacterial flora in the colon

[15–17].

The gastrointestinal tract provides a chamber-like

environment with oxygen tension transitionally de-

creasing from the upper to the lower gastrointestinal

tract. This environment of low oxygen tension may

favor the co-existence of aerobes, facultative anae-

robes, and obligate anaerobes that could mutually

benefit each other. The aerobes utilize oxygen that

helps maintain a reduced oxygen level suitable for

facultative and obligate anaerobes. The gastrointesti-

nal tract also provides an ecological niche for those

facultative anaerobes (such as Escherichia coli) that

are capable of switching their metabolic enzymes in

response to variations in oxygen tension within the

local gastrointestinal environment. The intrinsic and

adaptive nature of the gut microflora population in

each animal species may have evolved as a result of

the co-existence of various microorganisms adaptable

to the host gastrointestinal environment [18].

The lower gastrointestinal tract contains a wide

variety of both Gram-positive and Gram-negative

aerobes, facultative anaerobes, and obligate anae-

robes, the majority being commensals [19,20]. Com-

mon intestinal commensals include aerobes or

facultative anaerobes such as Enterobacteriaceae

[21], Streptococcus spp., Enterococcus spp., Staphy-

lococcus spp., Lactobacillus spp., fungi, and obligate

anaerobes including Bacteroides spp., Provetella,

Porphyromonas, Bifidobacterium spp., Peptostrep-

toccus spp., Clostridium spp., and Eubacterium

spp. [22]. Many bacterial species from the gastroin-

testinal tract may not be routinely recoverable by

standard in vitro culturing methods because their

growth conditions are undefined, or there is a lack

of suitable symbiotic ingredients that normally exist

in the lumen; therefore, the exact contents of micro-

flora have not yet been determined in most animal

species. Recent advances in molecular methodology

have dramatically expanded the list of the known gut

microbiota [23–25]. Techniques such as polymerase

chain reaction based amplification targeting the 16S

rDNA have been extensively used in recent years for

detection and identification of the various micro-

organisms that compose the microflora from fecal

materials [26,27].

g Delivery Reviews 56 (2004) 1497–1521 1501

2.2. Host–microflora equilibrium

The digestive system of unborn animals is consid-

ered to be sterile in utero. During the birthing and

nursing processes, the alimentary canal of the neonate

becomes exposed to the microbial flora of the dam.

These organisms colonize the various sites of the

alimentary canal and, for the most part, remain there

throughout life. The majority of microflora live in the

gastrointestinal tract in a harmonious manner so that

symbiosis and commensalism are established.

Colonization processes vary among bacterial spe-

cies [28–30]. Random and non-specific interactions

between the microflora and the host may play a major

role during the initial colonization process. Coloniza-

tion is also an equilibrium process between the host

and the microorganisms, determined by specific fea-

tures of microbes and host factors [31–33].

On the microbial side, specific determinants on the

bacterial cell surface can mediate cell-to-cell adher-

ence between bacteria and the host gastrointestinal

epithelial cells. Cellular structures of bacteria, such as

fimbriae [34,35], fibrillae [36,37], glycocalyx and

capsules [38], may enhance the stability of the adhe-

sion process. For example, C. jejuni colonization of

the gastrointestinal tract of chickens is facilitated by

bacterial expression of a 37-kDa outer membrane

protein, CadF, which binds to the extracellular protein,

fibronectin, on the surface of host cecum epithelial

cells. A mutant strain lacking CadF and a parental

strain with CadF were used to study their ability to

colonize the cecum of newly hatched chicks. The

parental wild strain of C. jejuni readily colonized

the cecum. However, when chicks were experimen-

tally challenged with the CadF mutant, none of the

microorganism could be recovered from any of the

chicks, indicating that disruption of the CadF gene

rendered the C. jejuni incapable of colonizing the

cecum [39,40].

Several host factors play a role in microbial colo-

nization of the gastrointestinal tract, including pH,

intestinal motility, characteristics of the gut epitheli-

um, diet, and physical components of the host im-

mune system. The acidic pH in the stomach eliminates

most bacteria ingested and prevents these bacteria

from colonizing either the stomach or in the proximal

small intestine. Consequently, the bacterial load in the

stomach of non-ruminant mammalian animals is low-

S.S. Yan, J.M. Gilbert / Advanced Dru

er than the colonization observed in other portions of

the gastrointestinal tract [18,41]. Intestinal peristaltic

motility prevents overgrowth and antigrade migration

of the gut microorganisms by mixing and balling

bacteria within intestinal mucus and propelling them

in a caudal direction [42–45]. However, bacteria are

able to take advantage of the existing environment by

utilizing the gut epithelial cell surface proteins, struc-

tures, and extracellular matrix proteins that are

expressed and secreted by host cells. These attributes

of the gastrointestinal tract provide a bridging effect

between microorganisms and host epithelial cells in

the lumen [46–51].

Complex carbohydrate moieties linked to proteins

on the cell surface most likely play an important role

in cell–cell adhesion in the brush border epithelial

region. Phase-contrast and electron microscopy were

used to study the adhesion of C. jejuni and C. coli in

isolated porcine intestinal brush border membranes.

Approximately 45% of the bacterial cell population

adhered to the brush borders. Pretreatment of the

brush borders with trypsin or pronase, and competi-

tive inhibition with L-rhamnose caused a reduction in

adhesion. These results suggest that specific cellular

structures on the host tissues/cells contribute to the

adhesion by microorganisms [52].

Homeostasis of the microbial population within the

gut is greatly affected by host diet [46]. The diet

provides the substrate and affects the composition of

microflora in animals. Fermentable fibers have an

especially profound effect on gut microflora [4]. Intes-

tines of conventional and germ-free rats fed fiber-free

or fiber-supplemented diets were quantified by mor-

phological indexes to demonstrate that fiber has several

direct and indirect effects on the gut [53]. These effects

include muscle hypertrophy in the colon, an increase in

the number of crypts per circumference, an increase in

the number of branched crypts in the proximal colon,

and an increase in goblet cell count in the gastrointes-

tinal glands [53]. Additionally, since the mucous mem-

branes of the gastrointestinal tract are exposed to an

enormous load of microflora, the host depends on its

immunity to prevent potential systemic invasion by

indigenous flora. The gastrointestinal tract has special-

ized components of the immune system, including

mucous secretions, Peyer’s patches, scattered mucosal

lymphoid nodules and nodes, and secretory immuno-

globulins [49,54,55] that are important in regulating the

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211502

balance of beneficial and harmful properties of bacte-

rial colonization.

Differing pathogeneses of Salmonella among sero-

types in host animal species may provide an example

that the host–parasite equilibrium is in a continuous

process of adaptation [56,57]. A serotype pathogenic

to one animal species may be a non-harmful colonizer

in another, and some serotypes are invasive to multi-

ple host animal species depending upon their viru-

lence factors [58–60]. For example, S. Typhimurium

and S. Newport may affect multiple animal species,

while S. Typhi and S. Paratyphi host’s range is

restricted to humans [61]. A potential key factor

defining a particular Salmonella serotype as either a

successful pathogen or simply a colonizer in a partic-

ular animal species may rely on its capability to

invade the host gut epithelial cells, acting at the

molecular level to induce bacterial uptake by the

epithelial cells, and its ability to adapt to the intracel-

lular environment of that particular host species. A

successful pathogenic process also requires a series of

events involving interactions between the invading

microorganism and that host’s local defense systems

[58,59,62].

2.3. Foodborne pathogens among microflora of food

animals

Zoonotic foodborne pathogens can be commensal

colonizers of the gastrointestinal tract of various food

animal species. The most commonly recognized ani-

mal-derived bacterial foodborne infections are those

caused by Campylobacter, Salmonella, and E. coli

O157:H7 [63–66]. Other potential bacterial food-

borne pathogens that may or may not be associated

with food animals include Yersinia enterocolitica,

Listeria monocytogenes, Bacillus cereus, Staphylo-

coccus aureus, Shigella spp., and Clostridium spp.

[63,65,67–72].

C. jejuni is the most commonly identified bacterial

cause of diarrheal illness in the world. Human cam-

pylobacteriosis may include fever, diarrhea, and ab-

dominal cramps [73], and poultry have been identified

as the most common source for human infections

[40,74]. Salmonella is also a group of enteric bacteria

that is widespread in the intestines of birds, reptiles,

and mammals, including cattle and swine, and may be

spread to humans via consumption of contaminated

foods of animal origin [21,75]. There are over 2000

serotypes under the species of Salmonella enterica,

but not all have been identified as pathogenic [76].

E. coli O157:H7 is a bacterial pathogen that has a

reservoir primarily in cattle. It also can be found in

other food animals (such as sheep, pigs, and goats) and

among wild animals (such as deer) [77–80]. E. coli

O157:H7 and a few other serotypes produce a shiga or

shiga-like toxin which is responsible for human illness

characterized by severe and bloody diarrhea, often

painful abdominal cramps, with or without fever

[81–83]. E. coli O157:H7 does not cause disease in

ruminants (e.g., cattle) partly because of the lack of

vascular receptors on the gut epithelial cells for shiga-

like toxin. This may explain how cattle serve as an

asymptomatic reservoir for E. coli O157:H7 [84].

2.4. Primary location of zoonotic foodborne patho-

gens in the gastrointestinal tract

In food animals, understanding the location of

certain microflora species that are pathogenic to

humans is important to public health. To elucidate

which sections within the ruminant gastrointestinal

tract harbor most of the shiga toxin producing E. coli,

E. coli O157:H7 was orally inoculated into sheep and

cattle via a gastric tube [81]. Animals were then

euthanized at weekly intervals during post-inoculation

period, and tissue and digesta cultures were taken

from the rumen, abomasum, duodenum, lower ileum,

cecum, ascending colon, descending colon, and rec-

tum. It was found that E. coli O157:H7 was most

prevalent in the lower gastrointestinal tract, specifi-

cally the cecum, colon, and feces, which suggest the

colon as the primary site for E. coli O157:H7 persis-

tence and proliferation in mature ruminant animals.

Campylobacter typically colonizes the intestine of

poultry [10,45,52,65]. Studies in chickens demonstrat-

ed that the cecum is the primary site for Campylo-

bacter adhesion and colonization [39,40,85].

However, Campylobacter can also be found in the

crop of newly hatched chicks [11]. Initial data suggest

that the mechanism for adhesion in the crop by

Campylobacter may be different from that in the

cecum. Therefore, colonization of Campylobacter

may involve multiple mechanisms, allowing them to

colonize various sections of the gastrointestinal tract

in poultry [11].

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1503

Various Salmonella serotypes can be found in

swine, poultry, cattle, and other food animals [56,57,

83,86,87] with the small intestine and cecum as the

primary sites of their colonization [62,88]. In a germ-

free mouse model, the ileal and cecal Peyer’s patches

and the mesenteric lymph nodes contained 103–104

viable Salmonella bacterial cells within 24 h of intro-

duction of the inoculum into the stomach [89], suggest-

ing that Salmonella may target selected specialized

tissues/cells within the gastrointestinal tract.

Clinically important zoonotic foodborne pathogens

mostly reside in the lower gastrointestinal tract. Anti-

microbial action upon the host’s gut microflora, includ-

ing commensals and pathogens, varies with individual

antimicrobial agent. An understanding of general char-

acteristics of antimicrobial drug activities may be

helpful. Accordingly, microbial food safety mainly

focuses on the microflora in the lower gastrointestinal

tract exerted by antimicrobial drug uses. This under-

standing is an important consideration to product for-

mulators since minimizing intestinal drug exposure can

markedly affect microbial human food safety concerns.

3. Characteristics of antimicrobial drug activities

in vitro

Substantial information has been accumulated on

how antimicrobial drugs work against bacteria, both at

the cellular and molecular levels. For in-depth reviews

in this area, readers are encouraged to refer to spe-

cialized journal articles [90–94]. The brief discussion

below is intended to provide an overview of the in

vitro activity of antimicrobial drugs for the purpose of

understanding the potential effect of antimicrobial

drug delivery in food animals on the microflora of

their gastrointestinal tract.

3.1. Antimicrobial activity is measurable

The antibacterial activity of an antimicrobial agent

may bemeasured in vitro by two bacterial growth-based

methods. These include determination of the inhibitory

and killing capabilities of the antimicrobial agent.

Results generated from measuring the inhibitory activ-

ity are normally referred to as the minimum inhibitory

concentration (MIC). Thismethod is commonly used in

in vitro antimicrobial susceptibility testing. The MIC is

defined as the lowest concentration of an antimicrobial

agent that inhibits visible growth of a bacterium. [95].

Interpretation of results generated from this testing

method may be semi-quantitative (expressed with the

actual MIC value), or qualitative (e.g., susceptible,

intermediate or resistant). Susceptibility testing can also

be performed by the disk diffusionmethod, and activity

is measured relative to the size of the zone of inhibition

surrounding a disk embedded with an agent. In vitro

susceptibility testing is normally performed either in

clinical laboratories or inveterinary laboratories.Within

the United States, interpretation of these in vitro results

is based upon standards recommended by the National

Committee for Clinical Laboratory Standards (NCCLS,

http://www.nccls.org/, accessed as ofAugust 29, 2003).

Some antimicrobials are lethal to bacteria at the

concentration equal to or higher than the MIC, while

other agents have primarily bacteriostatic activity. The

terms bacteriostatic or bactericidal describe an agent’s

antibacterial mode of action. While the bactericidal

effect of a particular drug can be quantitatively deter-

mined in vitro, it is not routinely tested due to technical

and interpretive difficulties. The minimum bactericidal

concentration (MBC) is defined as the minimum con-

centration of an antimicrobial agent that kills 99.9% or

more of the inoculated organism under specified test

conditions [94,96,97].

Antibacterial killing effect may be measured by

exposure of bacteria to a given concentration of drug,

and the resultant reduction of the bacterial population

is quantified over time. The killing of such an in vitro

exposure–response relationship is referred as the

‘‘killing curve’’. The bactericidal effect may also be

measured in the presence of serum, which may better

reflect in vivo antimicrobial activity due to the impact

of serum components. For this reason, it may provide

a better prediction of the host response to an antimi-

crobial treatment [98].

In vitro antibacterial effects can also be demon-

strated by bacterial morphological changes imposed

by an antimicrobial agent. Fig. 1 shows the morpho-

logical changes of an isolate of E. coli following

exposure to a fluoroquinolone.

3.2. Antimicrobial activity is specific

Antimicrobial agents act upon bacteria by targeting

one or more specific components of the bacterial cells

Fig. 1. Morphological changes of E. coli following exposure to a fluoroquinolone drug. An overnight culture of an E. coli strain (ATCC 25922)

was used to inoculate a Mueller-Hinton agar plate. The plate was incubated in a non-CO2 incubator under 35 jC for 12 h to allow a light lawn to

grow before putting on an Etest strip containing levofloxacin. The plate was incubated for another 4 h. Bacterial cells were collected in a small

area from the lawn above the concentration of MIC (approximately between two and six times the MIC) along the edge of the strip. Cells were

fixed, stained, and subjected to transmission electronic microscopy. (A) Cells grown without exposure to levofloxacin (control). (B, C) Cells

exposed to levofloxacin. Notice that the size and basic shape of the cells remained unchanged. However, compared to controls, cellular materials

became unevenly distributed, enlargement of the inter-space between the outer membrane and the cytoplasmic membrane occurred, disruption

of intact of cell wall and membranes was visible, and most apparently, vacuoles of variable sizes formed in the cytoplasma, suggesting a

bactericidal effect from the drug. The insert bar represents 0.4 Am in length. Courtesy of Drs. Xiaotian Zheng and Yi Tang.

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211504

that are essential for their physiological function and

replication. Many antimicrobials are divided into

classes based on their mechanisms of action. For

example, h-lactam drugs achieve their effect by inhib-

iting a group of bacterial membrane proteins called

penicillin-binding proteins (PBPs). PBPs are respon-

sible for cell wall synthesis [99,100], and those with

high molecular weights usually have multiple enzy-

matic functions. Thus, these proteins provide essential

targets for h-lactam drug activity. Using radiolabeled

penicillin as a tool, it has been demonstrated that the

binding of h-lactam drug(s) to each PBP is specific,

concentration-dependent, and saturable (Fig. 2). The

interaction between h-lactams and their target proteins

mimic a ligand–receptor relationship, with a thresh-

old at which the physiological function of the target

proteins is hampered to a degree that it affects

bacterial cell growth, ultimately leading to cell death.

Other examples of specific drug–target interac-

tions are: (1) fluoroquinolones that target bacterial

DNA gyrase and topoisomerase IV, two enzymes

essential during cell replication; (2) macrolides that

target the 50S subunit of ribosome and block bacterial

protein synthesis; (3) aminoglycosides that inhibit the

Fig. 2. Binding of the penicillin to the penicillin-binding proteins in a group G Streptococcus. Membrane proteins (200 Ag) from a strain of the

Lancefield Group G Streptococcus were incubated with a series of concentrations of 125I-labeled penicillin V for 10 min, followed by SDS-

polyacrylamide gel electrophoresis and fluorography. The concentrations of radiolabeled penicillin V from lanes a to i were as follows: 2.0, 1.0,

0.5, 0.125, 0.06, 0.03, 0.015, 0.004, and 0.001 Ag/ml. The penicillin-binding proteins are revealed because they covalently bound to

radiolabeled penicillin, which are named numerically according to their molecular weights. Binding of radiolabeled penicillin by each PBP is

uneven and demonstrates a concentration-dependent manner. This work was performed in the laboratory of Dr. Dennis L. Stevens.

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1505

function of 30S subunit of the ribosome; and (4)

glycopeptides that inhibit the synthesis of the cell

wall by binding to the D-alanyl-D-alanine terminus of

the cell wall precursor units [101]. Table 1 illustrates

examples of mechanisms of action and other proper-

ties of selected antimicrobial classes and agents.

Inhibition of bacterial cells by antimicrobial agents

is target-specific and dependent upon the mode of

action of a given drug. However, the bactericidal

effect exerted by antimicrobial drugs can be either a

direct event subsequent to the inhibition of the same

cellular targets or a cascade of reactions resulting from

a particular drug–bacterium interaction. For example,

it is recognized that h-lactam drugs are characterized

by time-dependent killing [102]. For streptococci,

bacterial cell death following exposure to penicillin

requires an activation of a process of autolysis, which

is not the direct drug target. Normally, penicillin may

inhibit the essential PBPs at or slightly above the MIC

[103,104]. An increase in concentration does not

necessarily enhance antibacterial activity once the

target proteins are saturated. The associated but com-

pletely independent cellular events of autolysis trig-

gered by the interaction of penicillin with the drug

targets (PBPs) in streptococci may explain in part why

h-lactam antimicrobials mostly demonstrate a time-

dependent exposure–response relationship.

3.3. Antimicrobial activities exist at low

concentrations

Some antimicrobial agents demonstrate activities at

concentrations below the MIC (i.e., sub-MIC). How-

ever, the sub-MIC effects cannot be demonstrated by

conventional susceptibility testing. These effects are

observed both under electron microscopy and by

studying bacterial growth rate [105,106]. The term

minimum antibiotic concentration (MAC) has been

used to indicate the lowest concentration that affects

bacterial structure, growth rate, or both [107]; there-

fore, the MAC could be equal to or lower than the

MIC for a particular ‘‘drug–bacterium’’ interaction.

However, MAC is usually performed for research

interests and is rarely used in clinical laboratories

because of its time-consuming and labor-intensive

procedures [102,106–109].

Among antimicrobial drugs, h-lactams produce the

most dramatic alterations in bacterial morphology at

sub-inhibitory concentrations. Inhibition of the bacte-

rial PBPs by these drugs provides a biochemical basis

for the morphological alterations [106]. At sub-MICs,

penicillin is able to bind to one or more of the

essential PBPs, while at concentrations at or above

the MIC, it binds to most or all PBPs. Morphological

changes exerted by penicillin at a lower concentration

Table 1

Properties of major classes of antimicrobial agents [90,94,195,196]

Antimicrobial class

(example agents)

Mechanism of action Mode of action Mechanisms of

resistance

PK–PD relationshipa

[197,198]

h-Lactams (penicillins,

cephalosporins, and

carbapenems)

Inhibition of the penicillin-

binding proteins (PBPs)

located on the cytoplasmic

membrane

Bactericidal h-lactamase production,

target (PBPs) modifications,

reduced permeability,

and efflux

Time-dependent

Aminoglycosides

(streptomycin and

neomycin)

Protein synthesis inhibition

through binding to the 30S

subunit of ribosome

Bactericidal Decreased permeability,

efflux, modification

enzymes, and target

(ribosome) modification

Concentration-

dependent

Macrolides (erythromycin,

tylosin and spiramycin)

Protein synthesis inhibition

through binding to the 50S

subunit of ribosome

Bacteriostatic Target (ribosome modification

enzymes, decreased

permeability, and efflux

Time-dependent

Fluoroquinolones

(enrofloxacin and

ciprofloxacin)

Inhibition of DNA gyrase

and topoisomerase

Bactericidal Target point mutations

decreased permeability,

efflux, and plasmid-mediated

mechanism

Concentration-/time-

dependent (AUC)

Glycopeptides (avoparcin

and vancomycin)

Inhibition of cell wall

synthesis by binding to the

terminal amino-acyl-

D-alanyl-D-alanine

Bactericidal Terminal peptide modification

of the cell wall ingredients;

cell wall thickness as a result

of enhanced glutamine

synthetase and aminotransferase

Time-dependent

Tetracyclines

(oxytetracycline and

chlortetracycline)

Protein synthesis inhibition

at the ribosomal level

(interfere with peptide

elongation)

Bacteriostatic Efflux, drug detoxification,

and ribosome modification

Time-dependent

Lincosamides (lincomycin

and clindamycin)

Protein synthesis inhibition

by binding to the 50S

subunit of ribosome

Bacteriostatic Decreased ribosomal

binding

Time-dependent

Folate synthesis

inhibitors (sulfonamides

and ormetoprim)

Inhibition of

dihydropteroate synthase,

and inhibition

of dihydrofolate reductase

Single—

bacteriostatic,

combination—

bactericidal

Decreased permeability,

formation of enzymes

with reduced sensitivity

to the drugs

Concentration-

dependent

Phenicols (florfenicol and

chloramphenicol)

Inhibit the

petidyltransferase

reaction at the 50S

subunit of ribosome

Bacteriostatic Decreased target

binding, reduced

permeability, efflux,

and modifying enzymes

Time-dependent

Pleuromutilin (tiamulin

and valnemulin)

Inhibit protein synthesis by

binding to the 50S subunit

of ribosome

Bacteriostatic Decreased membrane

permeability, and

ribosome change

Concentration-

dependent

Polypeptides (bacitracin

and polymyxin)

Acts as cationic detergent

and disrupt bacterial cell

membrane

Bacteriostatic Lipopolysacchride

modification

n/a

a Represents a generalization only, the actual relationship can be variable when an individual drug is involved.

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211506

can be demonstrated using electronic microscopy. For

example, in a streptococcal strain with an MIC of 0.06

Ag/ml to penicillin, apparent morphological alterations

are visible at one-half the MIC (Fig. 3).

In addition to morphological changes, sub-inhibi-

tory concentrations of antimicrobial agents have been

shown to have other effects on bacteria. For example,

sub-MICs of azithromycin significantly reduced the

percentage of gonococci that expressed assembled pili

(which facilitates bacterial adhesion to the host cells)

on their surfaces by decreasing pili subunit synthesis

[110]. Exposure of E. coli to one-half the MIC of

Fig. 3. Morphological alterations imposed by a sub-MIC concen-

tration of penicillin. Following an exposure to 1/2 the MIC of

penicillin for 2 h, group A streptococcal cells became very elongated,

enlarged, and distorted compared to control cells. Observation was

scanning electron microscopy. Insert represents control cells that

were processed in the same manner without of exposure to the

drug.

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1507

ciprofloxacin caused a decrease in the amount the of

bacterial capsular materials that contribute to E. coli

pathogenicity [111]. Similarly, treatment with a sub-

MIC of various antimicrobial agents resulted in thin-

ning of the capsule of Klebsiella pneumoniae and an

increase in the capsular hydrophobicity. As a result,

there was a decrease in the negative charge of the cell

surface, causing a reduction in the physical repulsion

between K. pneumoniae and phagocytes, which

enhances sensitivity of the bacteria to phagocytic

killing activity [112].

3.4. Antimicrobial activities can be persistent

Persistent activity of antimicrobial agents is often

referred to as the post-antibiotic effect (PAE). A PAE

is the consequence of a prior exposure to concentra-

tions of antibiotics rather than to persistent sub-inhib-

itory concentrations of antimicrobial agents [113]. In

therapeutics, PAE may prevent bacterial re-growth

after serum and tissue concentrations fall below in-

hibitory levels, which may enhance the therapeutic

activity of certain antimicrobial drugs [113]. Early in

the antibiotic era, Bigger [114] demonstrated that

staphylococci exposed to inhibitory concentrations

of penicillin G exhibited a delayed re-growth after

the drug was removed from culture. Later, PAE was

described for many other antibiotics and various

bacteria [113,115,116].

The PAE is an intrinsic property of a specific

‘‘drug–bacterium’’ combination [109,113], and like-

wise, the duration of a PAE is variable and depends

upon the drug–bacterium combination. In a study of

various antimicrobials for Pseudomonas aeruginosa,

the mean in vitro PAE was 1.35 h for ceftazidime,

2.38 h for ciprofloxacin, 2.39 h for imipenem, 2.16

h for piperacillin, and 1.77 h for tobramycin [117].

The glycopeptide teicoplanin showed a maximum in

vitro PAE of 5 h against methicillin-resistant S.

aureus, while only demonstrating a minimum of 0.6

h against Enterococcus faecalis [118]. In veterinary

medicine, the in vitro PAEs of tylosin and apramycin

were described for swine and bovine respiratory tract

pathogens [119], and that of marbofloxacin, enroflox-

acin, difloxacin, and ciprofloxacin against Bordetella

bronchiseptica isolates from non-food animals [120].

Previous studies on penicillin offered a good exam-

ple of explorations into the mechanisms of PAE. Ini-

tially, the PAE of penicillin was considered to be the

result of selection of a slowly growing subpopulation of

bacteria in the culture because penicillin becomes

bacteriostatic [121]. Other investigators suggested that

penicillin readily binds to crucial sites (i.e., the PBPs) of

the organism in sufficient amounts to maintain the

antibacterial effect, even after drug was removed

[122]. Subsequently, it was demonstrated that re-

growth in Streptococcus spp. and E. coli after removal

of penicillin was dependent on the re-synthesis of PBPs

[123]. Further investigation concluded that the irrevers-

ible binding of penicillin to PBPs 1–3 (i.e., the essential

targets of h-lactams for this organism) causes the PAE

of penicillin in S. pyogenes, and the PAE represents the

time necessary for the bacterial cells to synthesize new

PBPs in order to resume normal growth [124].

3.5. Other factors

The antimicrobial exposure–response relationship

reflects the dynamics of the drug (e.g., bacteriostatic

or bactericidal), dosing (concentration used, duration

of exposure, etc.), and characteristics of the organisms

(susceptibility level, growth stage, cell density, growth

medium, etc.). There are several other factors that may

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211508

affect the in vitro evaluation of this relationship: (1)

The in vitro testing system used for measuring anti-

microbial activities does not contain host-specific

proteins that may bind to an antimicrobial drug and

modulate its activity. Therefore, the effect observed in

vitro may not precisely correlate with in vivo results.

(2) Bacterial growth rates can affect the outcome of

antimicrobial effect. For example, h-lactams are more

active when bacterial cells are in the active growth

phase compared with those in the steady or stationary

phases [108,121,125]. The growth-dependent effect is

less apparent for non-h-lactam antimicrobials [126].

(3) When measuring inhibitory and killing effects, the

quantity of bacterial cells used (or the inoculum size)

in the testing systems may influence the results as well.

Henry Eagle first described this finding of inoculum

effect for penicillin, which was consequently called

Eagle’s effect [127]. (4) Eagle also described a para-

doxical phenomenon of penicillin in the action against

Gram-positive cocci in which penicillin, at a range of

concentrations above their MICs, demonstrated a less-

er inhibitory effect than that at the immediate lower

concentrations [128]. This paradoxical phenomenon

was also observed in Gram-negative E. coli with

ampicillin [129].

4. Antimicrobial drug delivery in food animals

The method of drug delivery in food animals varies

with animal species, the infectious disease target, the

drug formulation or purposes for which the antimicro-

bials are employed. Treatment can be short- or long-

term, and can be applied to an individual animal or an

entire herd or flock. A key microbial food safety aspect

of antimicrobial drug delivery in food animals is the

amount of antimicrobial that ends up in the gastroin-

testinal tracts of treated animals. This may potentially

have an effect on their microflora [75] and cause

adverse disturbances of the normal gut microflora.

This disturbance may include the proliferation of those

intestinal bacteria exhibiting a decreased susceptibility

or frank resistance to antimicrobial agents of interest

and that can become human pathogens.

In recent years, greater emphasis has been placed

on the emergence or selection of resistant bacteria in

the gut of food animals as a result of antimicrobial

drug uses [2,6,75,82,130]. The problem may be great-

er with specific formulations (e.g., immediate release

versus sustained release), routes of drug delivery, and

conditions of use. These factors are discussed below.

4.1. Drug delivery routes

4.1.1. Parenteral administrations

Parenteral routes of administration are often em-

ployed to minimize the challenges to drug bioavail-

ability that can occur with oral drug administration,

including the failure of a sick animal to consume an

adequate dose of the product when presented in feed or

delivered in water. Intramuscular and subcutaneous

administrations are two of the most common parenteral

routes. However, parenteral administration of antimi-

crobial drugs does not necessarily diminish concerns

about drug distribution to the gastrointestinal tract as

some parenterally administered compounds can enter

the gut via an enterohepatic cycling pathway. In

addition, antimicrobial agents in the plasma may be

secreted through the epithelial cells (by both active and

passive mechanisms) and diffuse into the gut lumen.

4.1.2. Oral administrations

The oral route is a convenient method to administer

drugs in feed or water to food animals, especially for

swine and poultry. For systemic infections, an ideal

oral formulation is readily absorbed and provides high

concentrations of the free drug to the site of the

infection. Conversely, if a desirable therapeutic goal

is for control of infections within the digestive system,

a drug that is poorly absorbed from the intestinal track

could theoretically be advantageous.

Oral administration of tetracyclines is widely used

in swine; however, the systemic bioavailability of

tetracyclines is reportedly poor in this target animal

species. For example, the bioavailability for oxytetra-

cycline hydrochloride was only 23% when adminis-

tered orally to fasted gilts [131]. For chlortetracycline,

oral bioavailability ranges between 11–27% in fasted

pigs and 12–22% in the fed pigs [132]. Besides

absorption of the drug itself, unfavorable conditions

in the gastrointestinal luminal environment (e.g., pH,

ionic tension, food material interference, etc.) may

affect bioavailability of a particular compound [133,

134]. Strategies for improving the bioavailability of a

drug include the use of protective particulate carriers

or coating drugs with target-specific ligands that

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1509

mediate site-specific delivery of the drug–carrier

complex [135].

Oral administration through drinking water is a

popular route of drug delivery for poultry and swine,

where large groups of animals are to be treated.

Drinking water medication offers the advantage of

low cost, minimal labor intensity, and ease of admin-

istration. Diseased animals generally continue to con-

sume fluids even after they have stopped eating [9].

The major disadvantage is the variation in drug intake

among individual animals, as well as drug solubility

and stability concerns. Administration of antimicro-

bials in feeds is another convenient oral regimen for

antimicrobial drug delivery in food animals. Similar to

oral regimens and drinking water medications, antimi-

crobial agents in feeds are subject to the variable

intake, which will impact systemic drug exposure.

One of the fundamental concerns associated with

oral drug administration is that as compared to paren-

teral administration, it may result in higher levels of

antimicrobial exposure of the gut microflora. This

exposure can be affected by the extent of drug absorp-

tion after oral administration, the intestinal site of drug

absorption, and the proportion of drug in the gastro-

intestinal tract remaining active (unmetabolized and

unbound). This information may not be well defined

for many veterinary antimicrobial agents. In part, this

is due to the many complex variables that can impact

gut microflora exposure to the active compound and/or

active metabolites. For example, within any segment

of the large bowel, those bacteria embedded in the

mucosal secretions may experience a level of drug

exposure that differs from those microbes that reside in

the luminal region and are mixed with food and other

organic materials. Without precise information on the

drug concentrations within the various horizontal and

vertical regions of the intestine, it is difficult to

accurately predict the impact of antimicrobial drugs

on the gastrointestinal tract microflora.

4.1.3. Other drug delivery methods

Other unique antimicrobial drug delivery methods

are used in food animals. Intrauterine infusion of

antimicrobial agents has been used in the treatment

of bacterial endometritis and management of peripar-

tum conditions in food animals [136–138]. Bacterial

mastitis is also a common infection in lactating dairy

cows and can be treated parenterally or via the intra-

mammary route with antimicrobial agents. Intramam-

mary treatment delivers drug into the udder through

the teat canal, where the drug passively diffuses into

the aqueous and lipid fractions of the milk [139]. Such

an organ-specific drug delivery methods allow anti-

microbial drug(s) to reach relatively high concentra-

tions at the infected site while minimizing distribution

to other tissues dependent upon pharmacokinetic

profiles of particular drugs. In general, antimicrobial

drugs delivered via these routes have less impact on

the gut microflora because systemic absorption can be

low and, consequently, the amount of drug entering

into the gastrointestinal tract can be relatively low.

4.2. Drug absorption and transport through the

intestinal epithelium

For an orally administered antimicrobial to reach the

site of an extra-intestinal infection, it must be absorbed

and enter into the systemic circulation. Identifying

compounds and formulations that optimize oral drug

absorption is an important aspect of product develop-

ment. Numerous reviews are available on drug absorp-

tion in the gastrointestinal tract and associated drug

transport systems [133,135,140–142]. Within the

small intestine, drug flow between the gut wall lined

with epithelial cells and the adjacent components of the

blood circulatory system contained in the connective

tissue is divided into two opposite directions: forward

uptake from the mucosa to serosa, and back flux from

the serosa to the mucosa. Several studies on antimicro-

bial drug uptake in the gastrointestinal tract suggest that

the rate of drug uptake in the gut lumen is dependent

upon factors including drug concentration, pH in the

lumen, net charge of drugmolecules, protein binding of

the drug, etc. Depending upon the particular com-

pound, passive diffusion, carrier–drug complex, and

active transport mechanisms may participate in both

the uptake of drug from the gut into the blood and the

efflux of drug from the blood back into the gastroin-

testinal tract [135,140,143–147].

Antimicrobial drugs are compounds with diverse

chemical structures. Each class and individual agent

within a class may have its own unique absorption

properties in the gastrointestinal tract. For example,

Mizen et al. [147] studied the uptake from the

gastrointestinal tract of the alkyloxyimino penicillin,

ampicillin, amoxicillin, and cyclacillin, in mouse

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211510

jejunal sac and small intestine perfusion models. They

demonstrated that although all of these are members

of the penicillin family, the mucosal to serosal uptake

for alkyloxyimino penicillin was approximately half

that of amoxicillin and four times less than that of

cyclacillin.

Drug penetration into the gastrointestinal tract can

be affected by the patho-physiological status of stud-

ied subjects. Pharmacokinetic parameters may be

different in infected hosts when compared to healthy

ones, potentially changing parameters such as tissue

distribution, body clearance, and mean elimination

half-life. Lindecrona et al. studied the differences of

pharmacokinetics (PK) and penetration of danoflox-

acin into the gastrointestinal tract between healthy and

Salmonella infected pigs [148]. They found that mean

elimination half-life of danofloxacin from the blood of

Salmonella infected pigs increased from the 6.7 h ob-

served in healthy pigs to 9.4 h in diseased animals.

The rate of body clearance decreased from 0.57 l h� 1

kg� 1 in healthy pigs to 0.29 l h� 1 kg� 1 in infected

pigs. This resulted in higher areas under the curve

(AUCs) in the infected pigs compared with the

healthy ones. High drug concentrations were achieved

in all segments of the intestinal tract. However, the

AUCcontent-to-AUCplasma ratios were 52.4 –99.4

among healthy pigs as compared to 6.5–8.8 in

infected pigs ( p < 0.05). These findings indicate that

antimicrobial drug concentration in the gastrointesti-

nal tract of food animals may be different in healthy

versus diseased animals.

4.3. Role of biliary excretion

Many drugs, and their metabolites, are excreted

into the gastrointestinal tract through biliary secretion,

and some may be re-absorbed into the blood (enter-

ohepatic cycling) [101,149]. Substances such as glu-

curonide may conjugate with a drug or its

metabolite(s), and then be secreted into the bile from

liver and enter into the gastrointestinal tract [147]

through a non-selective carrier mediated transport

system [101]. In some cases, drug metabolites may

have more potent antimicrobial activity than the

parent drug, as in the case of enrofloxacin which is

partially converted to the human drug ciprofloxacin,

[150–153]. Enrofloxacin is found in the liver, bile,

and kidneys [154] while ciprofloxacin is highly con-

centrated in the bile. In a piglet model, the bile-to-

serum ratio of ciprofloxacin was 586.4F 140.3%

[155]. Thus, both enrofloxacin and ciprofloxacin can

enter the gastrointestinal tract. In contrast, penicillin G

has a minimal clearance through the biliary system,

and only about 5% of penicillin was recovered from

bile in a rabbit model [156]. Some drugs may enter

bile in a more time-dependent fashion. For example,

only 2.5% of oxytetracycline in turkeys is secreted via

the biliary route at 6 h after a dose of 1 mg/kg

intravenously [157]. However, at 2 h after adminis-

tration, a significantly higher concentration of oxytet-

racycline was found in the bile, and the peak bile-to-

plasma ratio was as high as 60:1 [157].

In summary, the pharmacokinetic properties of

antimicrobial agents intended for use in food animals

may result in effects on the animal gut microflora that

are difficult to predict, due to variable pharmacoki-

netic parameters between drugs. Variable conditions

of use and differences in formulation may further

confound this uncertainty.

5. Antimicrobial drug delivery and antimicrobial

resistance

Based upon the definition set forth by the NCCLS,

resistant bacterial isolates are not inhibited by usual

systemic concentrations of the agent following normal

dosage schedules, and/or fall in the range where

specific microbial resistance mechanisms are likely

and clinical effectiveness has not been reliable in

treatment studies [158]. A bacterium can become

resistant to a particular drug through one or more

mechanisms. The term ‘reduced susceptibility’ is used

to describe those organisms that are less susceptible

than previously observed, but have not become frankly

resistant. It is probable that bacteria exhibiting reduced

susceptibility to a particular antimicrobial drug have

attained that state through the same mechanisms that

will lead them to becoming fully resistant. The subject

of antimicrobial resistance and its mechanisms are

beyond the scope of this review. However, there is a

wealth of literature available on the subject.

When an antimicrobial agent is used therapeutical-

ly, the drug does not distinguish between target

pathogens (those that cause the infection) and normal

gut microflora. The exposure–response considera-

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1511

tions previously described apply equally well to target

pathogens and commensals when the antimicrobial

drug is delivered to food animals. Accordingly, resis-

tance can develop in gut microflora through mecha-

nisms similar to those described for target pathogens.

Common mechanisms of resistance employed by

bacteria against representative antimicrobial agents

of various classes are shown in Table 1. In the

following sections, we address a few aspects that

may be associated with resistance development.

5.1. Mutant prevention concentration for

fluoroquinolones

As previously stated, fluoroquinolones inhibit both

Gram-positive and Gram-negative bacteria by binding

to DNA gyrase and topoisomerase IV which interferes

with the DNA synthesis [159,160]. Mechanisms of

decreased susceptibility or resistance to fluoroquino-

lones include point mutations at key positions in the

target genes [161], poor membrane permeability, acti-

vated efflux pumps [160], and newly reported plasmid-

mediated resistance [159]. Point mutations in DNA

gyrase and/or topoisomerase are one of the primary

mechanisms of resistance to fluoroquinolones found

both in Gram-positive and Gram-negative bacteria.

The relationship between fluoroquinolone concen-

tration and induction/selection of point mutations in

clinical isolates was studied in vitro by plating bacte-

rial cell suspensions onto agar containing increasing

concentrations of fluoroquinolone drugs [162–165].

As the drug concentration in the agar increased, the

number of colonies on the agar plates decreases. Along

with the series of fluoroquinolone concentrations on

the plates, two apparent declines in colony count were

observed as the drug concentration increases. The first

decline occurred at the concentration equivalent to the

MIC, at which majority of the inoculated bacterial cells

are not recovered. At concentrations above the MIC,

recovery of colonies declined slowly (plateau region)

until reaching a higher concentration, where a second

noticeable decline of colonies was observed. This high

concentration represents an inhibited growth of the

entire population. The plateau region represents a

concentration range where mutants with decreased

susceptibility or resistance to the tested drug may be

encouraged to emerge. The concentration that caused

the second drop is defined as the mutant prevention

concentration (MPC). The MPC values are more

closely related to the bactericidal potential of the

specific fluoroquinolone.

In theory, a mutant that developed at concentrations

below the MPC presumably carries a single point

mutation in the primary target (e.g., DNA gyrase in

Gram-negative rods). To a specific bacterium, the

MPC varies among fluoroquinolones, and the MPC

of a particular fluoroquinolone is usually higher than

the MIC. Therefore, an MPC/MIC ratio can be estab-

lished [161–165]. It has been suggested that the

concentration window between the MIC and the

MPC (also called mutant selection window) for sus-

ceptible bacterial populations may favor induction of

point mutations [161]. However, this needs to be

specifically characterized for each fluoroquinolone

versus a particular bacterium of interest and needs to

be revisited in vivo.

When fluoroquinolone is administered in food

animals, there can be a wide range of drug concen-

trations within the gut lumen or in different segments

of the gastrointestinal tract. Exposure of the gut

microflora is therefore potentially subject to an un-

certainty of mutant selection pressure, and the promo-

tion of resistance may become a concern.

5.2. Stepwise changes in susceptibility

A stepwise decrease in antimicrobial susceptibility

has been widely reported, and resistance occurs in a

stepwise fashion. This makes it important to identify

organisms with low level resistance that may consti-

tute the genetic platform for development of higher

resistance levels [166]. In the laboratory, this has been

demonstrated by repeated exposure to media contain-

ing progressively higher concentrations of penicillin

or other h-lactams [167–170]. Stepwise induction of

resistant mutants has also been demonstrated in other

classes of antimicrobials [92,166].

Penicillin resistance can be used to illustrate how a

stepwise event could contribute to resistance devel-

opment. Resistance to penicillin and other h-lactamantibiotics can occur through three basic mechanisms:

(1) enzymatic inactivation through hydrolysis of drug

by h-lactamases, (2) reduction of bacterial drug per-

meability through cell membrane, and (3) drug target

modifications (e.g., change in PBPs). As discussed

earlier, the PBPs are a group of bacterial membrane

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211512

proteins or enzymes (transpeptidases and carboxypep-

tidases) that are responsible for cross-linking process-

es in the final steps of cell wall synthesis. h-Lactamantibiotics mimic the normal substrate of the enzymes

and covalently inhibit the PBPs, especially those of

higher molecular weights (or essential PBPs). With

repeated exposure to penicillin, both streptococci and

enterococci can modify the amino acid composition of

their PBPs, leading to a conformational change in an

essential PBP that decreases its affinity to penicillin

[168,171]. Stepwise reduction in susceptibility

through other mechanisms has also been described.

In a Salmonella strain, stepwise resistance to ampicil-

lin was observed through decreased outer membrane

permeability associated with diminished expression of

a porin protein [167]. However, the stepwise changes

in the susceptibility of the gut microflora in vivo are

largely unclear and require more research in the area.

5.3. Horizontal transmissibility of resistance

Some antimicrobial resistance traits are transfer-

able. Acquired resistance can evolve via genetic

alterations in the microbes’ own genome (vertical

transmission), or by horizontal transfer of resistance

genes through various types of mobile DNA elements.

The vertical transmission of self-replicating plasmids,

prophages, transposons, integrons, and resistance

islands all represent DNA elements that frequently

carry resistance genes into susceptible organisms

[166]. In a typical form of vertical transmission, a

plasmid carries a resistant gene that is easily replicated

and passed on to daughter cells. In so doing, this

resistance becomes prevalent in the bacterial popula-

tion linked to that particular clone.

Resistance may also transfer horizontally within

and between species, and across genera [172]. Resis-

tant genetic elements can be introduced to the microbe

through site-specific recombinases/integrases. Their

integration into the genome and resistance may also

be created by homologous recombination events cre-

ating mosaic genes where each piece of the gene may

originate from a different microbe [166]. It is impor-

tant to note that horizontal transfer of resistant genetic

materials has also been observed in vivo.

Using molecular biology methodologies, conjuga-

tive transposons carrying erythromycin and tetracy-

cline resistant genes can be freely transferred among

human colonic bacteria, suggesting that extensive gene

transfer occurs among bacteria in the human colon,

both within the genus Bacteroides and between other

genera including Gram-positive bacteria.[173]. The in

vivo transfer of the resistant elements can occur in the

absence of antibiotics [173,174] and has also been

shown to occur in animals [175]. In food animals, a

plasmid encoding streptomycin resistance was identi-

fied in 13 of 32 Pasteurella multocida isolates from

cattle and swine studies, and was found to be able to be

transformed into an E. coli strain that subsequently

expressed streptomycin resistance [176]. Tetracycline

resistance in strains of C. jejuni and C. coli was found

to be mediated by plasmids, and it was demonstrated

that intra- and inter-species transfer of the plasmid

within the genus Campylobacter could occur [177].

5.4. Concerns of antimicrobial resistance among the

gut microflora

Data accumulated over the past decades suggests

that antimicrobial agents are potent factors in the

disruption of the host microflora [75,178–185], and

that a disruption of the established balance of the

microflora can lead to adverse consequences in the

host animal [186]. Antimicrobial drug delivery in

food animals may also cause resistance among the

gut microflora, either through selection of resistant

clones or by the induction of a resistant subpopula-

tion. In some cases, resistance has little association

with the use of antimicrobial drugs because of its

intrinsic nature. For example, vancomycin is virtually

ineffective against Gram-negative bacilli because the

molecule is too large to penetrate their outer mem-

brane. Likewise, h-lactams which affect the structural

integrity of the bacterial cell wall have no effect on

mycoplasma because they lack a cell wall. From the

microbial food safety perspective, antimicrobial resis-

tance development in the intestinal microflora focuses

on those bacteria that may potentially cause infections

in humans through foodborne routes. Zoonotic food-

borne pathogens are of the greatest concern due to

their potential impact on public health, particularly

when they become resistant to the drugs of choice [1–

3]. Therefore, it is important to understand what in

vitro and in vivo factors are relevant in the contribu-

tion to the development of resistance as a result of the

delivery of antimicrobial to food animals.

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1513

6. Correlation of data from in vitro and in vivo

studies

In general, in vitro results cannot be simply ex-

trapolated to in vivo conditions for antimicrobial

drugs [187]. Exactly which factors determine the

correlation between in vitro and in vivo data remain

largely unknown. Therefore, data generated from both

systems are necessary and irreplaceable. A few ap-

parent differences between in vitro and in vivo are

noticeable and worthy of mention.

6.1. Discrepancy between in vitro and in vivo data

6.1.1. Bacterial generation time

A typical in vitro bacterial growth curve indicates

an initial phase of slow growth followed by a rapid

exponential replication phase, a relative steady station-

ary phase, and a decline phase in a typical enriched

medium. Bacteria numbers may double in as short as

20 min during the exponential growing stage in a rich

medium under optimal in vitro conditions. However,

the generation time in vivo is dependent upon a

number of host and site specific factors, and can be

as long as several hours in the gastrointestinal tract

[188,189]. The difference in bacterial growth can have

a profound effect on the activities of certain classes of

antimicrobials, especially h-lactams [125].

6.1.2. Protein binding of antimicrobial agents

It is important to remember that the antibacterial

activity of antimicrobials is only exerted by the

available free drug, and in vitro antimicrobial activity

reflects the activity of unbound (free) drug. When

used in vivo, antimicrobial drugs may bind to host

proteins, which facilitates their tissue distribution to

different compartments within the body [190]. Protein

binding of antimicrobial drugs is highly variable

among antimicrobial drug classes as well as between

members within a single class. In addition, the per-

centage of protein binding of a particular compound

can vary across animal species [149]. Therefore, the

antibacterial activity of antimicrobials in vivo needs to

be assessed on a case-by-case basis.

6.1.3. Metabolites with antibacterial activity

While antimicrobial drugs can be metabolized in

vivo, they undergo degradation reactions in vitro.

Some drugs, such as fluoroquinolones and amino-

glycosides, are relatively stable in vitro. Others, such

as h-lactams, are less stable. As discussed earlier, some

drug metabolite(s) may have antimicrobial activities

that can augment the effect of the parent compound.

For example, enrofloxacin is converted to a more

potent intermediate product, ciprofloxacin [152,191].

6.2. A collective application of post-antibiotic and

sub-MIC effects in vivo

The PAE has been suggested to contribute to the in

vivo exposure–response [119,120]. However, the in

vivo PAE of particular drugs may be either enhanced

or reduced as compared to that in vitro. For example,

the in vivo PAEs for selected antibiotics against S.

aureus originating from mastitic milk was compared to

the PAE for the same S. aureus strain cultured in vitro.

It was observed that the PAE of penicillin, pirlimycin,

and tilmicosin were lower for the S. aureus strain

originating in vivo as compared to the same strain

grown in vitro. In contrast, the PAE of cephapirin was

actually increased in vivo as compared to in vitro

[192].

In reality, it is difficult to separate the PAE in vivo

from the activities exerted by the sub-MICs of anti-

microbial drugs. As drug clearance occurs in vivo, the

serum or tissue concentration of the drug may fall into

a range of sub-MICs for a particular pathogen or

commensal following an exposure to prior high con-

centrations [106]. The term of post-antibiotic sub-

MIC effect (PA SME) has been proposed to describe

these two integrated effects, since a period with sub-

inhibitory concentrations often exists between doses

[109,193]. Therefore, antimicrobial drug effectiveness

may be enhanced for target pathogens from the mixed

effects of both the PAE and sub-MICs. This PA SME

may similarly affect the gut microflora.

6.3. Retaining resistance properties in vitro and in vivo

What has been learned about resistant bacteria

from in vitro systems needs to be revisited in vivo

when potential antimicrobial resistance development

is a consideration. In vitro models are very useful in

elucidating mechanisms of antimicrobial resistance,

determining what factors may promote the resistant

property and explaining how they are spread both

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–15211514

within and between bacterial species. However, in

vitro models cannot replace the need for an in vivo

model to understand what may actually occur under

a particular setting or conditions of drug use, partic-

ularly since the complexity of in vivo systems far

exceeds that of in vitro systems. For example, when

tetracycline resistance in E. coli was evaluated in an

in vitro chemostatic model, the isogenic R-plasmid-

free E. coli strain outgrew the plasmid-bearing strain

in the absence of the drug, suggesting that tetracy-

cline resistance in this laboratory strain demonstrated

a disadvantage in the maintenance of the resistance

trait in vitro [194]. The same isolates were then

tested in an in vivo mouse model by inoculating

into the stomach [187]. In the absence of tetracycline

selection pressure, the R-plasmid-bearing E. coli

strain became dominant in the feces. When the R-

plasmid-bearing strain was given to mice four days

after the inoculation with the R-plasmid-free strain, a

rapid spread of the R-plasmid was observed and the

tetracycline resistant bacteria became dominant with-

in one day, replacing the tetracycline susceptible E.

coli. The tetracycline resistance plasmid did not

disadvantage itself in the expression of the resistance

trait in the gut, even in the absence of antibiotic

pressure [187]. Possible factors affecting the discrep-

ancies between the in vitro and in vivo findings

include differences such as bacterial generation

times, oxygen availability, nutrient variation, host

characteristics, and co-expression of other gene prod-

ucts on the same plasmid.

6.4. Drug–bacterium specifics for microbial food

safety

As a result of the numerous in vitro and in vivo

factors already discussed, microbial food safety data

for a particular antimicrobial drug under specified

conditions of use can be helpful in determining wheth-

er in vivo antimicrobial activities are as predictable

from the in vitro perspectives, and whether the poten-

tial for emergence of resistance predicted from the in

vitro findings would actually occur in vivo. Although a

tremendous amount of knowledge has been accumu-

lated for most antimicrobial agents in vitro and in vivo,

pertinent correlation of data on the emergence of

resistance associated with the drug delivery in food

animals are still needed to make an informed evalua-

tion of the microbial food safety of antimicrobial drugs

used in food animals.

The information base for the human microbial food

safety evaluation done as part of the pre-approval

process for new antimicrobial animal drugs for food

animals should consider all relevant available infor-

mation about the drug. This should encompass a

broad scientific base, including such factors as phar-

macokinetic and pharmacodynamic parameters, in

vitro susceptibility profiles, and in vivo exposure–

response data of gut microflora in the target food

animal species, especially those posing public health

concerns. Focusing on drug–bacterium specific mi-

crobial food safety information may help eliminate

ambiguity and uncertainty, ensuring that a compre-

hensive and timely evaluation can be achieved.

7. Summary

Strategies for delivering antimicrobial drugs in

food animals will need to continue to evolve as both

disease conditions and animal management practices

change. Veterinary pharmaceutical companies will

continue to tune their antimicrobial product formula-

tions and drug delivery regimens to offer the best

protection and treatment schemes with delivery sys-

tems responsive to emerging needs. For some food

animal species, delivery of antimicrobial drugs

through feed and water is most efficient, allowing

for drug administration to targeted groups of animals

on the farm. Parenteral delivery of antimicrobial drugs

is often the route of choice for cattle and occasionally

swine, but is only practical when animals are pre-

sented in manageable numbers. Hundreds of cattle or

swine are not beyond consideration for an injectable

antimicrobial drug; however, 100,000 or more chick-

ens would be. While the parenteral route requires

some skill and can be labor-intensive, it ensures that

the individual animal receives the appropriate pre-

scribed label dose.

As we gain a better understanding of the antimi-

crobial exposure– response relationship, we can

move towards developing new delivery strategies

and methods that focus on minimizing cost and

labor, while ensuring delivery of the prescribed dose

to the targeted animal to optimize effectiveness. By

better understanding the mechanisms through which

S.S. Yan, J.M. Gilbert / Advanced Drug Delivery Reviews 56 (2004) 1497–1521 1515

exposure of the gut flora to antimicrobial drugs can

be reduced, novel delivery approaches may be

employed to minimize the impact on the gut flora

in the target animal species. This should also help

minimize concerns regarding the spread of resistant

bacteria and promote confidence in the human food

safety evaluation during the new antimicrobial ani-

mal drug process.

Acknowledgements

The authors would like to thank Drs. Julia W.

Punderson, Bernadette Abela-Ridder, Karen Lampe,

Ana Haydee Fernandez, Richard Ellis, Mark M.

Robinson, and Robert D. Walker for their comments

on the manuscript.

Note

The opinions and information in this review article

are those of the authors, and do not represent the

views and/or policies of the U.S. Food and Drug

Administration.

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