Anti-infective drugs – introduction.

Antibiotics – their classification,

mode of action, and combination.

Microbial resistance and the risk of

antibiotic residues in food.

Infection is the invasion of a host organism's bodily tissues by disease-causing organisms,

their multiplication, and the reaction of host tissues to these organisms and the toxins they

produce. Infections are caused by microorganisms such as viruses, bacteria and fungi.

When infection attacks the body, anti-infective drugs can suppress the infection. Three types of

anti-infective drugs exist:

1. antibacterials (antibiotics and chemotherapeutic agents),

2. antivirals

3. antifungals

ANTIBACTERIAL drug is an agent that interferes with the growth and reproduction of bacteria.

An antibiotic is a chemical substance produced by a microorganism that inhibits the growth or

kills other microorganisms. Antimicrobial chemotherapeutic is a substance produced by

chemical synthesis that kills or inhibits the growth of microorganisms.

ANTIVIRAL drugs are a class of medication used specifically for treating viral infection. Like

antibiotics for bacteria, specific antivirals are used for specific viruses.

ANTIFUNGAL drug is a medication used to treat fungal infections such as ringworm

(dermatophytes), candidiasis (thrush), or serious systemic infections (cryptococcal meningitis).

ANTIBACTERIAL DRUGS (ANTIBIOTICS AND CHEMOTHERAPEUTICS)

The noun “antibiotic” was first used in 1942 by Dr. Selman A. Waksman, soil microbiologist.

Dr. Waksman and his colleagues discovered several actinomycetes derived antibiotics. The word

“antibiotic” will be used to describe: a chemical substance derivable from a microorganism that

kills or inhibits microorganisms and cures infections.

Each class and individual antibiotic acts in a different way and may be effective against either

a broad spectrum or a specific type of disease-causing agent.

Chemotherapy is the treatment of a disease by the use of pure chemicals (synthetic

antimicrobials) which have specific antagonistic effects on the organism causing the disease.

The action is achieved by interference with the metabolic processes of the organism (either

directly or indirectly), by antagonizing enzymes or preventing coenzyme formation, thus

starving the organism of a substance essential for normal life (sulphonamides).

SOURCES OF ANTIBACTERIAL AGENTS

A constant search is required to ensure that disease treatment can continue to be effective in the

face of antibacterial resistance. The sources of antibacterial agents are:

1. Mould and fungal metabolites (Penicillium spp.); bacteria (e.g. Streptomyces spp.,

Micromonospora spp., Bacillus spp.);

2. Semi synthetic variants of natural products (amoxicillin from benzyl penicillin).

3. Synthetic chemistry (sulfonamides, chinolones)

REQUIREMENTS FOR AN IDEAL ANTIBIOTIC AGENT

1. Selective target – target unique

2. Bactericidal – kills

3. Narrow spectrum – does not kill normal flora

4. High therapeutic index – ratio of toxic level to therapeutic level

5. Few adverse reactions – toxicity, allergy

6. Various routes of administration – IV, IM, PO

7. Good absorption

8. Good distribution to site of infection

9. Emergence of resistance is slow

DIVISION OF ANTIBIOTICS

1. ACCORDING TO CHEMICAL STRUCTURE:

a. Beta-lactams (penicillins, cephalosporins, carbapenems and monobactams)

b. Tetracyclines

c. Amphenicols

d. Polypeptides and glycopeptides

e. Aminoglycosides

f. Macrolides

g. Lincosamides

h. Ansamycins (rifamycins)

i. Pleuromutilins

j. ATB with various structures (aminocoumarin and steroid).

2. ACCORDIG TO EFFECT ON MICROORGANISMS

a. Bacteriostatic antibiotic – suppresses the growth of bacteria (tetracycline, tylosin)

b. Bactericidal antibiotic – kills bacteria (penicillin, streptomycin, bacitracin)

3. ACCORDING TO MECHANISM OF THEIR ACTION

a. Cell envelope antibiotics – cell wall synthesis inhibitors: β-lactams, glycopeptides,

polymyxins

b. Protein synthesis inhibitors: tetracyclines, amphenicols, aminoglycosides, macrolides,

pleuromutilins, lincosamides, steroid substances (fusidic acid)

c. Nucleic acid synthesis inhibitors: rifamycins

4. ACCORDING TO THE SPECTRUM OF ACTIVITY

a. Broad-spectrum antibiotic – one that is effective against a wide range of bacteria. It

also means that it acts against both G+ and G- bacteria (tetracyclines).

b. Slightly-broad spectrum (middle/mean broad) antibiotic (ampicillin, neomycin)

c. Narrow-spectrum antibiotic – one, which is effective against only specific families of

bacteria (penicillin G, streptomycin).

ANTIBACTERIAL SPECTRUM

Antibiotics differ in the range of bacteria against which they are active. The most common

distinction is between those active against Gram-positive and Gram-negative microorganisms.

Typically a 'broad-spectrum' antibacterial will affect a range of micro-organisms, including

some Gram-positive and Gram-negative bacteria.

Thus benzylpenicillin affects only certain Gram-positive organisms (narrow spectrum),

mecillinam affects only certain Gram-negative organisms (narrow spectrum), while

oxytetracycline, amoxycillin and chloramphenicol affect a range of Gram-positive and Gram-

negative organisms (broad spectrum).

The spectrum of certain antibacterials may also extend to include mycoplasmas (e.g. the

fluoroquinolones) or protozoa (e.g. dimetridazole).

ANTIBACTERIAL ACTION

Antibacterial action can be bacteriostatic or bactericidal.

The following antimicrobials at usual concentrations are generally bacteriostatic:

tetracyclines, amphenicols, macrolides, lincosamides, pleuromutilins, sulphonamides. These

produce stasis of bacterial growth in vitro. This means that, in vivo, the bacteria are made

susceptible to the body's defense mechanisms. Their successful use does, of course, depend on

these defenses being competent to achieve this effect, and this may be questionable in animals

that are very old, very young or greatly weakened and debilitated by the disease that is being

treated.

In general, the following common antimicrobials at usual concentrations are bactericidal:

penicillins (including semisynthetic), cephalosporins, aminoglycosides, polypeptides,

glycopeptides, potenciated sulfonamides, quinolones and nitrofurans. These produce actual

death of the cell in vitro, so when used clinically they should produce their therapeutic effect

without the aid of the body's defense mechanisms. Thus they are indicated in the

immunocompromised animals referred to above or where the immune system may be

depressed, e.g. in severe virus disease.

Classification of antimicrobials as bactericidal or bacteriostatic can also be misleading

because "bactericidal" drugs can be rendered bacteriostatic if sufficient drug concentrations are

not achieved at the site of infection.

MECHANISM OF ACTION OF ANTIBACTERIALS

CELL WALL SYNTHESIS INHIBITION

Some of the antibacterial compounds interfere with the cell wall synthesis by weakening the

peptidoglycan structures in bacterial cell wall, by this integrity of bacterial cell wall structure

weakens and eventually disrupts. Mammalian cells only have plasma membrane so these

antibiotics specifically target only bacterial cells. That is these antibiotics do not induce any

negative effect on the host mammalian cells. Several agents affect cell-wall synthesis, the most

important being penicillins and cephalosporins.

The major constituent of the cell wall of Gram-positive organisms is a mucopeptide and

that of a Gram-negative organism is a combination of a mucopeptide and a lipoprotein. The cell

wall is built up of layers of N-acetylmuramic acid and poly-N-acetylglucosamine molecules,

which unite in long strands with trailing polypeptides. In the final step cross linkages are

formed.

Gram-positive cell structure

The Gram-positive cell wall is thick and consists of 90% peptidoglycan. Teichoic acids link various

layers of peptidoglycan together. Teichoic acids also regulate the autolysin activity in this complex

equilibrium.

The cytoplasmic membrane (which defines the intracellular space) consists of a lipid bilayer;

intrinsic proteins which are hydrophobic (mostly enzymes involved in respiration and transmembrane

transport); extrinsic proteins which are hydrophilic; Penicillin-Binding Proteins (PBPs): periplasmic

space proteins involved in peptidoglycan synthesis (glycosyltransferase, transpeptidase and

carboxypeptidase activities)

Gram-negative cell structure

The outer membrane is made up of phospholipids, endotoxin or lipopolysaccharide (LPS) - plays

an important role in the antibiotic entry into the cell; proteins including the porins (complexes of three

proteins) form aqueous channels that provide a route across the outer membrane for all the water-soluble

compounds needed by the bacterium. The periplasmic space contains peptidoglycan – 5-20% of cell

wall; various enzymes (in particular, ß-lactamases). The cytoplasmic membrane (which defines the

intracellular space) consists of: a lipid bilayer; intrinsic proteins which are hydrophobic (mostly

enzymes involved in respiration and transmembrane transport); extrinsic proteins which are

hydrophilic; Penicillin-Binding Proteins (PBPs) - periplasmic space proteins involved in peptidoglycan

synthesis (glycosyltransferase, transpeptidase and carboxypeptidase activities)

PROTEIN SYNTHESIS INHIBITION

Some of the antibiotic compounds inhibit bacterial cell multiplication by inhibiting protein

synthesis in them. Protein synthesis is a multi-step process. Majority of antibiotics inhibit the

process that occurs in the 30S or 50S subunit of 70S bacterial ribosome, this in turn inhibits the

protein biosynthesis.

Most of the antibiotics inhibits the formation of 30S initiation complex or altogether inhibits

the formation of 70S ribosome by the 30S and 50S ribosome subunits or they inhibit assembling

of amino acids into a polypeptide chain. Tetracyclines block protein synthesis by preventing the binding of aminoacyl-tRNA in 30S ribosome

subunit. They interrupt the cycle by which amino acid is carried and attached to the ribosome. These

compounds block protein synthesis in both prokaryotic and eukaryotic system.

Aminoglycosides interfere with the formation of 30S initiation complex hence inhibits the protein

biosynthesis.

Amphenicols interrupt the transfer of a growing peptide chain to newly attached amino acids. Macrolides interfere with the assembly of 50S subunit of ribosome hence inhibit the protein synthesis.

Lincosamides inhibit enzyme peptidyl transferase, hence prevent the protein synthesis. Macrolides and

lincosamides inhibit the movement of an amino acid to a donor site on the ribosome (translocation).

NUCLEIC ACID INHIBITION

Replication of the nucleic acids of the bacterial cell is prevented directly by nalidixic acid and

rifamycins and indirectly by the sulphonamides.

1. Effect on DNA. Sulphonamides ultimately deprive the cell of nucleic acid and the

presence of nalidixic acid prevents its replication.

2. Effect on RNA. Rifamycin specifically inhibits the bacterial enzyme concerned with the

replication of RNA. This is achieved by the binding of one molecule of the antibiotic to one

molecule of the enzyme.

CLINICAL USE OF ANTIMICROBIAL AGENTS - PRINCIPLES

Successful antimicrobial therapy is based on 4 principles:

1. Identification and characterization of the pathogen, including its antimicrobial

sensitivity, and selection of a drug based on the sites of infection and the lesions.

2. Effective concentrations of the indicated antimicrobial agent for a sufficient period at

the site of infection.

3. A dose rate, frequency, and route of administration of the antimicrobial agent, as

well as a duration of therapy, that maximizes the likelihood of a cure, prevents

relapse, and minimizes the risk of resistance without causing any harmful drug-

induced effects in the animal.

4. Specific and appropriate supportive therapy to enhance the animal's ability to

overcome the infection and associated disease conditions.

The immunocompetence of the animal should always be assessed, recognizing that

extraneous factors, stress, disease, malnutrition, and the effects of concurrently administered

drugs may influence the animal's ability to resist infection. Host defense systems are seriously

compromised in bone marrow depression, hypogammaglobulinemia, conditions in which

alveolar macrophages are incapacitated, tracheobronchitis, necrotic enteritis, starvation, and

other conditions. In addition, immunosuppressants (e.g., corticosteroids and antineoplastics)

compromise immune response. Any depression of immune capabilities can be expected to

modify the effectiveness of many antimicrobial agents, especially those with only bacteriostatic

activity.

REQUIREMENTS FOR SUCCESSFUL ANTIMICROBIAL THERAPY

CLINICAL DIAGNOSIS

Successful antimicrobial chemotherapy usually requires a specific diagnosis, even though a

reasonable preliminary diagnosis is often all that is possible, at least initially.

MICROBIOLOGICAL DIAGNOSIS

Treatment should be aimed at a specific pathogen whenever feasible. However,

polymicrobial infections are common. The ideal is a conclusive microbiologic diagnosis, but

frequently this must be presumptive (at least initially), and treatment must be based on

experience. Rational deduction may be necessary under field conditions. The examination of a

direct smear stained with Wright's or Gram's stain may help to establish what types of

pathogens are involved (gram-positive or gram-negative rods or cocci).

CULTURE AND SUSCEPTIBILITY TESTING

Isolation and characterization of the causative pathogen, susceptibility testing, and

determination of the MIC provide a sound foundation from which to select the antimicrobial

drug, as well as the dosage regimen. However, under field conditions, it is often difficult to

attain laboratory support for antimicrobial therapy.

MIC (Minimum Inhibitory Concentration) is the lowest concentration, which prevents

visible growth of bacteria; quantitative measure of the in vitro sensitivity of a particular

bacterium to a particular antibiotic.

APPROPRIATE SELECTION OF ANTIMICROBIAL AGENTS

Among the factors to be considered are the causative microorganisms, results of sensitivity

tests, pathogenicity of organisms, pathologic lesions, acuteness of infection, pharmacokinetics of

the drug indicated, expense, potential drug toxicity, organic dysfunctions (especially kidney and

liver function), and possible interactions with drugs administered concurrently.

Broad-spectrum antibiotics are properly used in the following medical situations:

1. Empirically prior to identifying the causative bacteria when there is a wide

differential and potentially serious illness would result in delay of treatment. This

occurs, for example, in meningitis, where the patient can become so ill that he/she

could die within hours if broad-spectrum antibiotics are not initiated.

2. For drug resistant bacteria that do not respond to other, more narrow-spectrum

antibiotics.

3. In super-infections where there are multiple types of bacteria causing illness, thus

warranting either a broad-spectrum antibiotic or combination antibiotic therapy.

CORRECT DOSAGE AND ROUTE OF ADMINISTRATION

The dosage selected should result in adequate therapeutic concentrations at the site of

infection for sufficient time without causing side effects or toxicity. For dose-dependent drugs,

higher dosages are more likely to enhance therapeutic success than shorter intervals. For β-

lactam and other time-dependent drugs, therapeutic success appears to be greater if the

concentration remains above the MIC for about one-half to two-thirds of the dosage interval,

and efficacy is likely to be improved more by decreasing the interval than by increasing the

dose. The advocated dosage schedules should be carefully followed for at least 7 days (although

response should be apparent in 3 – 4 days for most infections), or longer if needed, to ensure

elimination of the pathogen and to prevent relapse, reinfection, or development of antimicrobial

resistance.

ANCILLARY TREATMENT, NUTRITIONAL SUPPORT AND NURSING CARE

Supportive treatment, optimal nutrition, and general nursing care are often critical for

successful management of infectious disease. Ancillary treatment might include the use of anti-

inflammatory agents, antidiarrheal preparations, expectorants, bronchodilators, inotropic

agents, urinary acidifiers and alkalinizers, immunopotentiators, and fluid and electrolyte

replacement. Attention should be given to caloric and nutrient intake, especially of protein and

vitamins. These nutrients play a cardinal role in immune responsiveness.

FACTORS INFLUENCING CLINICAL USE OF ANTIBIOTICS

A number of important details concerning absorption, circulation and metabolism of

antibiotics within the animal must be considered if the full benefit is to be obtained and the

antibiotics used correctly.

1. THE USE OF COMBINED ANTIBIOTICS AND JOINT USE OF ANTIBIOTIC AND OTHER

CHEMOTHERAPEUTICS.

2. THE BLOOD AND TISSUE LEVELS OF AVAILABLE ANTIBIOTICS ARE OF PARAMOUNT IMPORTANCE

WHEN ANTI INFECTIVE ACTIVITY IN VIVO IS BEING CONSIDERED.

The attainment of early high levels and their subsequent maintenance will depend on the

route of application and the nature of the formulation of the antibiotic. Thus the choice of

intravenous, intramuscular, subcutaneous, intraperitoneal or other parenteral route will

influence the rate of attainment and duration of activity. Further oral therapy may be used to

continue treatment or it may be used in some cases of less severity as the sole means of

treatment, particularly when an infection appears to be mainly in the gut.

3. REACTION WITH BLOOD AND TISSUE FLUIDS.

Some antibiotics such as penicillin are not decreased in potency by contact with blood,

serum, pus, cerebrospinal fluid, etc., whereas others such as streptomycin and sulphonamides

are influenced unfavorably by these fluids.

The absorption of some antibiotics from the intestine may be inhibited by the presence of

other chemical substances in the intestinal contents. Thus a high calcium level in the ingesta

(e.g. milk) will reduce the systemic availability of tetracyclines given orally. To overcome this

specially reduced calcium diet may be given for a short period.

4. PHYSIOLOGICAL BARRIERS.

There are a number of physiological barriers which some antibiotics will cross quite easily

but others only with difficulty. These barriers consist of:

a. The blood-brain barrier, which is a physiological division between the blood and

cerebrospinal fluid. Some antibiotics such as penicillin and streptomycin do not pass

the blood-brain barrier; in fact penicillin is actively pumped away from the

cerebrospinal fluid in a manner very similar to that in which it is eliminated in the

kidney. When the meninges are inflamed the pumping action is impaired and so

penicillins can enter the cerebrospinal fluid, although even then therapeutic levels may

not be achieved. Intrathecal injections should therefore be given at least to initiate

treatment. Other antibiotics such as oxytetracycline will also normally achieve

therapeutic concentrations in the cerebrospinal fluid only when the meninges are

inflamed. A further group, of which chloramphenicol is outstanding, passes the blood-

brain barrier fairly easily and achieves concentrations in the cerebrospinal fluid as

high as 50% of the blood levels.

b. The placental barrier does not normally resist the passage of antibiotics.

c. The intestinal barrier. When given orally many antibiotics are not absorbed into the

system in therapeutic concentrations. Streptomycin and neomycin are examples. The

reverse may also hold good. For example, when streptomycin is injected, very little is

excreted into the intestinal lumen except through the biliary system. This is important,

for in such cases only parenteral injection is suitable for treating systemic infections

and only the oral route can be used for treating intestinal infections. The failure of

neomycin to be absorbed is important in limiting its toxicity, which is quite

considerable by the parenteral route, but low by the oral route.

d. Serous membranes. Some antibiotics such as penicillin will not readily pass either

way across the normal pleural or peritoneal membrane, whereas others, for example

chlortetracycline hydrochloride, diffuse quite readily into the pleural and peritoneal

cavities.

e. Milk. The concentration of antibiotic in milk after parenteral administration depends

on the antibiotic involved. Benzylpenicillin and the semisynthetic penicillins are found

in the milk in considerably lower concentrations than in the serum, although the

concentration is raised in mastitis. Other antibiotics, such as spiramycin, tylosin and

the penethamate derivative of penicillin, are found in the milk in concentrations higher

than those in the plasma. Thus the latter are the most appropriate for systemic

treatment of mastitis. However, the intramammary route is generally preferred as it

gives direct access to the site of infection.

5. EFFECT ON INTESTINAL FLORA IN HERBIVORES.

Ruminants are highly dependent on the integrity of the rumenal microflora. Thus oral

administration of broad-spectrum antibiotics such as tetracyclines results in acute diarrhoea.

While this has been well known for some years, it has recently been recognized that

tetracyclines (by whatever route) can occasionally cause acute and even fatal intestinal

disturbance in horses. Although this is not yet fully understood, it probably results from a

change in the caecal flora, perhaps similar to that described below for hamsters and guinea-pigs.

Penicillin G, outstandingly safe in most species, has long been known to be toxic to guinea-

pigs. This has been ascribed either to an allergy or to a disturbance in the intestinal flora. The

latter explanation now seems the most likely. It has been shown not only that penicillin is toxic

to the guinea-pig, but also that most broad-spectrum antibiotics produce the same effect, and

that similar toxicity is seen in the hamster.

It has been shown that the condition in hamsters is the result of overgrowth of Clostridium

difficile and that this organism is also the cause of the severe diarrhea in man. While it is not

clear if the same organism occurs in other species suffering from antibiotic-induced diarrhea,

this seems quite possible.

However, it is clear that extreme care should be taken in the use of antibiotics in herbivores.

In particular, tetracyclines, ampicillin, other penicillins, lincomycin and chloramphenicol are to

be avoided in hamsters and guinea-pigs. Ampicillin should also be avoided in rabbits, although

in this species tetracyclines seem relatively safe. Problems in herbivores are not avoided by

using the parenteral route, since toxicity can and does ensue in sensitive species.

6. ANTIBIOTICS AND IMMUNITY.

Some diseases do not recur because natural recovery from an attack results in the creation

and maintenance of adequate levels of immunoglobulins in the blood and tissues. If, however,

large doses of antibiotics or indeed of any other anti-infective agents are given, and the infection

is rapidly eliminated, the immunological reactions are not always properly stimulated and the

body is susceptible to further infection as soon as the antibiotic is withdrawn. Such situations

have arisen in the control of coccidosis by chemotherapeutic food medication.

COMBINATION OF ANTIBIOTICS

The administration of 2 or more agents may be beneficial in the following situations:

1. to treat mixed bacterial infections in which the organisms are not susceptible to a

common agent - to broaden the spectrum of activity,

2. to achieve synergistic antimicrobial activity against particularly resistant strains

(e.g., Pseudomonas aeruginosa),

3. to overcome bacterial tolerance,

4. to prevent the emergence of drug resistance,

5. to minimize toxicity, or

6. to prevent inactivation of an antibiotic by enzymes produced by other bacteria that

are present.

Additive or synergistic effects are seen when antibacterial agents are used in

combination, but antagonism may also emerge, sometimes with serious consequences.

Generally, bacteriostatic agents act in an additive fashion, whereas bactericidal agents are often

synergistic. However, the effects of several bactericidal antibiotics are substantially impaired by

simultaneous use of drugs that impair microbial growth or "bacteriostatic" drugs (eg, most

ribosomal inhibitors), so bacteriostatic and bactericidal combination may be antagonistic.

This antagonism results from the fact that many bactericidal antibiotics act only on dividing

cells. Thus bacteriostatic drugs, whose effect is to produce stasis of growth, will clearly interfere

with the bactericidal drugs. This is a general guideline only; many exceptions are known, and

confounding factors also play a role.

The most widely used combination of bactericidal drugs in Europe is penicillin G and streptomycin;

synergy can be demonstrated for this combination in vitro. A combination of bacteriostatic drugs which

produces synergy is that of sulphonamides with trimethoprim. This combination can even be bactericidal

under some conditions. The danger of combining bacteriostatic and bactericidal antibiotics was

dramatically demonstrated some years ago when it was shown that there was a higher mortality in

human patients with pneumococcal meningitis when they were treated with a combination of penicillin

and chlortetracycline rather than with penicillin alone.

Ideally, antimicrobial selection should be based on mechanisms of action that are different

and on spectra of activity that are complementary. β-lactams are often selected because their

action is unique and not only complements other drugs but also facilitates movement of other

drugs through the damaged cell wall into the microbe. Examples of combination therapy for

mixed infections include the use of clindamycin, metronidazole, or the semisynthetic penicillins

for their anaerobic coverage in combination with aminoglycosides for their gram-negative

efficacy.

Preventing the development of resistance with combination antimicrobial therapy is best

exemplified by the use of carbenicillin or amikacin together with gentamicin or tobramycin for

the treatment of Pseudomonas infections.

Bacterial enzymatic inactivation of β-lactam antibiotics, such as the penicillins and

cephalosporins, can be decreased by concurrent administration of a β-lactamase inhibitor, such

as clavulanic acid or sulbactam.

RESISTANCE OF MICROORGANISMS TO ANTIBACTERIAL AGENTS

The emergence of bacteria resistant to antimicrobial agents within an animal population or

during therapy is of great concern. When resistance develops, a previously used therapeutic

approach may no longer be successful, and a suitable alternative antimicrobial drug must be

sought. Additionally, there frequently is concern from an epidemiologic and public health point

of view.

ANTIBIOTIC RESISTANCE

1. Natural (intrinsic) – implies an intrinsic property in an organism that confers resistance;

found where an antibiotic is ineffective against a particular species of bacteria, such as

where benzylpenicillin is by nature ineffective against E. coli (this is a reflection of the

inability of benzylpenicillin to penetrate the bacterial cell wall, and so reach the target

enzymes situated there).

2. Acquired - suggests that an organism has obtained, by one mechanism or another, the

means to survive exposure to an antimicrobial agent; occurs when a previously

susceptible population becomes resistant following exposure to antibiotic. Most simply,

this occurs where small numbers of bacteria genetically able to resist the antibiotic were

contained within the original population. The removal of the susceptible bacteria by the

antibiotic gives the resistant bacteria a population advantage, they multiply and soon the

previously susceptible population becomes replaced by a resistant population. This so-

called one-step process can be seen with streptomycin, where resistance development is

rapid.

MECHANISMS BY WHICH BACTERIA MANIFEST RESISTANCE

1. Organisms may produce enzymes, constitutive or inducible, which inactivate the drug

(e.g., penicillins, cephalosporins, aminoglycosides, chloramphenicol). Or defective

production of autolytic enzymes ("tolerance"), e.g., penicillins and cephalosporins. Or

decreased enzyme affinity, e.g., trimethoprim.

2. The permeability to or uptake of the drug by organisms may be decreased (inhibition or

changes in membrane transport systems to prevent entry of the antibacterial, e.g.,

aminoglycosides) or transport out of the cell may be increased (induction of membrane

transport systems to remove the antibacterial, e.g., tetracyclines).

3. Alteration of the drug receptor or binding site (specific configuration of target sites) may

result in reduced drug affinity at target loci (e.g., oxacillin, cloxacillin, macrolides,

lincomycin, streptomycin).

4. The organism may develop alternate metabolic or synthetic pathways to bypass or

repair the effects of the antimicrobial (e.g., sulfonamides, trimethoprim).

5. Development of impermeable cell walls with extremely narrow porins, e.g.,

Pseudomonas aeruginosa in response to many antibiotics.

In each of these cases, a modification of protein synthesis and enzyme activity is necessary to

confer resistance; thus, this adaptation is genetically determined.

Bacteria have 2 types of genetic structures that may confer resistance-chromosomes and

plasmids. Both consist of double-stranded DNA, and both are associated with the bacterial inner

cell membrane at some time. Plasmids are not essential for survival but do carry genetic

determinants that confer both antibiotic resistance and virulence on bacteria. Plasmid-mediated

resistance (R-factor or acquired resistance) is far more complex. Plasmids may contain 20-500

genes that can carry resistance to a number of different antibacterial agents (3-6 is common; up

to 9 have been recorded) and specific virulence factors. The 3 possible mechanisms by which

plasmids may migrate from one bacterium to another are transformation, transduction, and

conjugation.

MECHANISMS BY WHICH BACTERIA DEVELOP RESISTANCE

1. Mutation. Within a large population of bacteria, chromosomal mutations may occur,

which confer resistance either slowly, in a step-wise fashion with each succeeding

generation of the mutant more resistant or rapidly, in a single step in which the

bacterium is resistant after the initial mutation. Mutation is a random event.

Antimicrobials do not induce mutations but may exert a selecting out of resistant strains

by suppression of susceptible bacteria. Mutated bacteria are often metabolically

deranged and are at a selective growth disadvantage; they usually disappear with time

in the absence of the antimicrobial agent.

2. Conjugation. Certain Gram(-) bacteria undergo conjugation, a type of reproduction in

which genetic material is transferred from cell to cell via a pilus that is encoded by a

resistance transfer factor (RTF) on a plasmid (the DNA passes from the donor cell to the

recipient via a bridge formed during direct cell-to-cell contact). Resistance factors (R--

factors) from plasmid DNA and/or chromosomal DNA may encode for resistance to

multiple drugs and may be rapidly transferred to the bacterial population. This is

termed infectious drug resistance or transferable drug resistance and has been

observed clinically in enteric infections with Salmonella spp., Shigella spp., and

Escherichia coli. General facets of conjugation make it an important process for gene

transfer under natural conditions. Many types of bacteria can act as recipients, and

resistance can pass freely from organisms normally saprophytic in the gut of animals to

pathogenic bacteria. Conjugation allows the passage of a number of distinct genes at one

time. Thus, resistance to several antibiotics, all mediated by different biochemical

means, may be acquired in a single step.

3. Transduction. The process of transference of drug resistant genes by bacteriophage

(that makes use of its specialized molecular equipment adapted for inserting DNA into

recipient bacteria) is termed transduction. Normally, it is phage DNA that is transferred;

in certain cases, however, some DNA from the episome in the bacterial cell replaces the

proper phage nucleic acid sequence. It may be important in the development of resistant

strains of Staphylococcus aureus.

4. Transformation. Bacteria may incorporate DNA encoding for drug resistance from their

environment after its secretion or release by resistant organisms (naked DNA seems to

pass from the donor to the recipient through the growth medium.). Acquisition of

resistance by this mechanism is relatively infrequent.

Genetic sequences capable of coding for resistance can migrate from a plasmid to a

chromosome and, then back to the plasmid. These sequences are then transpositional and are

known as transposons. A number of transposons responsible for the transfer of R-factor

resistance also have been isolated, characterized, and identified.

The clinical relevance of plasmid-mediated resistance principally concerns the following:

1. intestinal infections, in which the reservoir of R-factors may be carried by saprophytic

flora in the gut;

2. the use of low levels of antibiotics (as in animal feeds) or improper dosing regimens,

which may lead to a high incidence of R-factors in a given population (low -

subtherapeutic concentrations of antibiotics used as growth promoters in farm animals

may assist in the induction of resistance, too); and

3. the indiscriminate use of antibiotics, which may eliminate the effectiveness of many

antimicrobial agents in the future

When antibiotics are removed from an environment containing a resistant population the

selection pressure is withdrawn and the previously susceptible bacteria (or at least those which

have survived) can now compete on equal terms with the resistant population. In some cases,

this may allow a gradual return to a susceptible population. Such reversion to susceptibility has

been demonstrated, for instance with respect to ampicillin resistance. On the other hand,

tetracycline resistance, once established, has been shown to persist even after the tetracycline

has been removed from the environment.

The use of antibiotics in food animals, including use as growth promotants, may contribute to

the transfer of resistance genes among bacteria and ultimately from food animals to humans,

where the organisms become pathogenic. The bacterial resistance created in the animal

following veterinary use of a drug or drug class may result in resistance to human drugs of the

same class. Whereas the organism developing resistance might be nonpathogenic, transfer of the

resistance gene to other bacteria in the human intestinal tract may result in a pathogenic

organism becoming resistant and ultimately in therapeutic failure in the human patient.

The following guidelines will help to minimize the emergence of bacterial resistance:

1. A broad-spectrum antibacterial agent should not be used if a narrow-spectrum agent is

also active against the causative organism.

2. Information regarding endemic infections and sensitivity patterns should be obtained

and considered when choosing an antibiotic.

3. Appropriate dose rates should always be followed.

4. When a combination regimen is used to prevent the development of resistant strains,

individual agents should be used at full dosage.

5. Antibacterials for topical application should be selected from those against which

development of resistance is uncommon.

6. To the extent that it is consistent with reasonable practice, every effort should be made to

use antibiotics only when the medical indications are clear and to avoid overuse of newer

agents when already available agents are effective.

RISK OF ANTIBIOTIC RESIDUES IN FOOD

The use of antibiotics in food-producing animals can lead to residue occurrence in food of animal

origin contributing to resistance or adverse effects/toxicity. Generally we recognize:

• Therapeutic use of ATB - the risk of antibiotic resistance is relatively small where the

drugs are correctly used under veterinary supervision,

• Prophylactic use – healthy animals are being treated (antibiotics as feed additives) -

much resistance among bacteria – banned in EU!

• Metaphylaxis – where disease is already present in a group of animals (e.g., diarrhoea in a

litter of pigs) – an inevitable and reasonable use of the drugs.

ANTIMICROBIAL FEED ADDITIVES

Maintenance of healthy animals requires prevention of infection by pathogenic

organisms. In addition, specific alteration of a host's microflora may have beneficial effects on

animal production by alteration of ruminal flora, resulting in changes in the proportions of

volatile fatty acids produced during ruminal digestion. Thus, antimicrobial compounds may

improve production efficiency or growth of healthy animals fed optimal nutritional regimens.

Production-enhancing antimicrobial compounds can be classified as ionophore (e.g., monensin,

lasalocid) or nonionophore (e.g., virginiamycin, zinc bacitracin) antibiotics. Antimicrobial

compounds are administered in the feed at low dose rates relative to high doses required for

therapeutic effects.

There is circumstantial evidence that use of subtherapeutic doses of antimicrobials

creates selective pressure for the emergence of antimicrobial resistance, which may be

transmitted to the consumer from food or through contact with treated animals or animal

manure.

BAN ON ANTIBIOTICS AS GROWTH PROMOTERS IN ANIMAL FEED IN EU

Antibiotics have been widely used in animal production for decades worldwide. Added in low

doses to the feed of farm animals, they improve their growth performance. However, due to the

emergence of microbes resistant to antibiotics which are used to treat human and animal

infections ("anti-microbial resistance"), the Commission decided to phase out, and ultimately

ban, the marketing and use of antibiotics as growth promoters in feed. Antibiotics are now only

allowed to be added to animal feed for veterinary purposes. This decision was based on opinions

from the Scientific Steering Committee, which recommended the progressive phasing out of

antibiotics used for growth stimulation, while still preserving animal health.

Regulation (EC) No 1831/2003 of the European parliament and of the Council on additives

for use in animall nutrition:

Feed additives means substances, micro-organisms or preparations, other than feed material

and premixtures, which are intentionally added to feed or water in order to: favourably affect

the characteristics of feed, animal products, the colour of ornamental fish and birds; satisfy the

nutritional needs of animals; favourably affect the environmental consequences of animal

production, favourably affect animal production, performance or welfare, particularly by

affecting the GI flora or digestibility of feedingstuffs, or have coccidiostatic or histomonostatic

effect.

Antibiotics, other than coccidiostats or histomonstats, shall not be authorised as feed

additives.

Phasing out: Antibiotics, other than coccidiostats and histomonostats, could be marketed and

used as feed additives only until 31 December 2005; coccidiostats and histomonostats as feed

additives are phasing out of the use by 31 December 2012.

An EU-wide ban on the use of antibiotics as growth promoters in animal feed entered into

effect on January 1, 2006. The last 4 antibiotics which were permitted as feed additives to help

fatten livestock (monensin, salinomycin, avilamycin, flavofosfolipol) are no longer allowed to be

marketed or used from this date. The ban is the final step in the phasing out of antibiotics used

for non-medicinal purposes. It is part of the Commission's overall strategy and EU's food safety

strategy to tackle the emergence of bacteria and other microbes resistant to antibiotics, due to

their overexploitation or misuse.

PROBIOTICS

Probiotics promote the establishment and development of a desirable intestinal microbial

balance in the animal. There is a delicate balance between normal and pathogenic

microorganisms. This balance can be upset by poor husbandry conditions, disease, or stressors

(e.g. transport). Bacteria that produce lactic acid can, in general, be beneficial to the animal;

certain yeasts may also be beneficial.

Their ability to increase growth and promote health are claimed to be due to one or more of

the following factors: preventing colonization of the gut by pathogenic coliforms, altering GI

absorption rate, and inhibiting bacterial growth and influencing the balance of bacteria in the

gut.

The probiotic feed additives consist of selected strains of lactobacilli and streptococci that

alter the microbial species present in the GI system to the benefit of the treated animal.

Unicellular yeasts are also used. The production benefits are variable, and positive responses are

more likely when a stressful management change may result in a change in balance of gut

microflora.

Probiotics can help overcome the negative effects of certain conditions that detrimentally

modify the gut flora. Thus, they are useful in some cases to minimize GI upsets or to help

overcome stress due to weaning or transport. The unicellular yeast fungus may also have

beneficial effects on rumen fermentation and thereby improve digestion and feed efficiency.

The effect of probiotics in older animals may be reduced due to the well-established, balanced

population of micro flora that is less sensitive to minor detrimental husbandry challenges.