AGP  PART I

Emergence of a Debate: AGPs and Public Health

Human Health and Antibiotic Growth Promoters (AGPs): Reassessing the Risk

A. Bezoen, W. van Haren, J. C. Hanekamp

3   Antibiotics: Use and Resistance Mechanisms

3.1 Summary

Below we shall summarise point by point the issues described in this chapter.

• Antibiotics are chemical compounds, produced by living organisms (such as fungi or bacteria), that are detrimental to other competing organisms. Usually these compounds kill or inhibit growth of bacteria or other microorganisms.
• Antibiotics are used both in human and animal medicine and as growth promoters (AGPs) in animal feed.
• AGPs discussed in this report are primarily active against Gram-positive bacteria (with a limited overlap towards Gram-negative bacteria) thus resistant Gram-positive bacteria are of our main concern.
• Bacteria can either have an intrinsic or an acquired resistance against antibiotics. Intrinsic resistance can only be passed on through cellular multiplication (bacterial offspring).
• Acquired resistance against antibiotics is in principle transferable to other organisms. This is the point of concern in the AGP discussion in relation to human health.
• Bacterial antibiotic resistance can be acquired in basically the following ways:
  • through chromosomal mutations (without selective antibiotic pressure)
  • through DNA transfer (with selective antibiotic pressure)
• In general transfer of resistance traits can be achieved by:
  • Transformation (DNA uptake from the environment)
  • Transduction (DNA transfer with the aid of a bacteriophage(a virus))
  • Conjugation (DNA transfer by direct cell to cell contact)
• A number of biochemical resistance mechanisms against antibiotics are:
  • Enzymatic breakdown or modification of the antibiotic(_-lactamases)
  • Overproduction of target
  • Two versions of antibiotic target; one sensitive, one resistant
  • Change of target site so that antibiotic does not bind
  • Eliminate entry ports of the cell (decreased uptake)
  • Produce pumps that export antibiotics out of the cell (decreased uptake)
  • Missing of target enzyme or metabolic pathway (intrinsic)
• The most important risk factor for the emergence of resistant bacteria is contact with antibiotics. Every use of antibiotics selects for bacteria that are less susceptible for that antibiotic (and related antibiotics).
• The continued use of small amounts of antibiotics as AGPs in animal feed will promote bacterial resistance to this antibiotic within livestock.
• The prolonged presence of antibiotics in animal feed increases the risk of resistance transfer within livestock.

In this chapter it will be shown that the use of antibiotics as AGPs results in the rise of resistant strains of Gram-positive bacteria within livestock. This is not a point of discussion. However, to what extent (if at all) the existence of resistant strains of bacteria in livestock is a human health threat is still an open question that needs answering. In the next chapter we shall look at this issue more closely.

3.2 Antibiotics: Categories

3.2.1 General

Antibiotics are chemicals produced by specific types of bacteria or fungi. They can be used to treat bacterial infections because they stop the growth of bacteria or are able to kill them (respectively bacteriostatic and bactericidal activity). In this way the infection can be stopped and the immune system of the animal or human infected is capable of dealing with the (remaining of) the bacteria (Bryan, 1982).

Some antibiotics are active towards many bacterial species, while others are more specific (broad/wide and narrow spectrum antibiotics respectively). Antibiotics with a broad spectrum are aminoglycosides, tetracycline and imipenem. Structural unrelated antibiotics are able to act on the same place in/at the bacterial cell. For instance the antibiotics D-cycloserin, fosfomycin, bacitracin, glycopeptides all act on cell wall synthesis.

3.2.2 Categories of antibiotics

Antibiotics can be divided in different groups, according to their structure or their target site in the cell. Some of these, like the _-lactam antibiotics and the tetracyclines have been used in human medicine since the 1940s (Levy, 1998). In the early days of twentieth century of medicine the antibiotic as formed by the producing organism was used. To increase the performance and specificity of antibiotics the 'basic' antibiotic can be chemically modified. For instance, ampicillin and methicillin are semi-synthetic penicillins derived of penicillin G (Schlegel, 1992).

Table 3.2.2.1. Structural subdivision of antibiotics (Bryan, 1982; Leclerq and Courvalin, 1991; Lambert et al., 1992; Schlegel, 1992; Allignet et al., 1996; SCAN 1998a; Murray, 1998).

Another way antibiotics can be subdivided is their mode of action. Antibiotics act specifically on bacterial cells or on processes in these cells. For the scope of this report it is not necessary to understand the mode of action of all antibiotics towards the cell or cellular processes. What will be described is the mode of action for the specific antibiotics important for this report. These are antibiotics that might cause a problem for human health. The classes of antibiotics that will be considered are the glycopeptides, macrolides and streptogramins. These antibiotics interfere with cell wall production (glycopeptides) and the synthesis of proteins (macrolides, streptogramins).

Table 3.2.2.2. Modes of action of antibiotics (Bryan, 1982; Russell and Chopra, 1990; Levy, 1998).

3.3 Antibiotic Usage

3.3.1 General
Humans mainly receive antibiotics to treat bacterial infections. Physicians or dentists prescribe antibiotics in hospitals or within the community. In hospitals antibiotics can also be provided prophylactically, preventing infections (for example previous to and during operations; Gopal Rao, 1998).

Antibiotics are given to farm animals for a number of purposes. The prophylactic use is more common in farm animals then it is in humans. When one animal in a herd or pig-house has an infectious disease, often the whole herd is treated. Besides the therapeutic or prophylactic use most of the animals reared on farms receive antibiotics in their daily feed. This is done because of the positive effect these antimicrobials have on animal growth and the amount of feed needed to reach slaughter weight.

Table 3.3.1.1. Indication of use of antibiotics in different fields (Harrison and Lederberg, 1998).

Amounts of antibiotics used cannot be compared easily between humans and animals. Below the most important factors that complicate matters are listed (Mudd, 1998; Van den Bogaard, 1997a; Levy, 1997).

• Dosages applied are different for each antibiotic and each application
• A difference in potency of antibiotics affects the total weight used
• The potency of the antibiotic preparations used in animal feed might vary
• The time-scale on which antibiotics are used influences the impact the antibiotic has on the animal/human and its environment

Usually rough figures are given about the yearly antibiotic use on animal farms. For comparison between different antibiotics and different applications the most accurate way is to compare the defined daily dosages (of the active compound) that are given to an animal or human. When a human or animal is treated for an infection, the dosage of antibiotics is usually recorded. Also the amounts of antibiotics present in the feed obtained from feed companies that mix antibiotics with the product is traceable.

Some attempts have been made to compare amounts used by humans and animals. According to Van den Bogaard (1997a) the total amount animals receive in one year is in the same order of magnitude as the amount humans receive (430 mg/kg body weight for poultry, 125 mg/kg for pigs, 55 mg/kg for cattle and 100 mg/kg for humans). In the UK, according to FRANA/Nefato, animals use less antibiotics than humans, 57 million people uses three times more antibiotics than the 198 million animals do.

However, by only comparing total amounts of antibiotics the situation is presented in an oversimplified manner. To be able to assess the risk of a certain use of antibiotics, not only amounts are important. The impact the antibiotic has on the flora in the intestines of the individual/animal treated is of paramount importance. This is not only related to the amount of an antibiotic administered, but also on the type of antibiotic and the time-scale of treatment. In general, the impact on the environment will be larger when treatment is prolonged and when more individuals/animals are treated per geographic area (Levy, 1997; Nord, 1993).

3.3.2 Antibiotics used in animal husbandry

Below is out-lined why antibiotics are used in feed, which antibiotics are used, in what amounts and on which scale.

The positive effect of antibiotics on growth was discovered incidentally. Stokstad and Jukes (1949) used the remaining of a fermenter culture of Streptomyces aureofaciens as a cheap source of vitamin B12. Cultures of this actinomycete were used to produce chlortetracycline. The chickens receiving the remaining material grew better than could be expected from the vitamin B12 alone and it seemed that the presence of chlortetracycline was responsible. Soon other antibiotics showed to have similar effects. Since the 1950s it became a routine to add low levels of antibiotics to animal feed (Van den Bogaard, 1997b). In many countries turkeys, chickens, pigs and calves receive feed that contains several antibiotics in low amounts. In this report principally the use of antimicrobials in Europe will be discussed.

According to studies in the Netherlands, the use of AGPs leads to an increase in growth of 1 to 8 % compared to animals that do not receive AGPs (Jongbloed, 1998; Westerhuis, 1998). The profit depends on the age of the animal and the animal species. Little pigs show a growth improvement of 3 - 8 %, while for broilers the effect is 2 - 4 %. The effect growth promoters have on older pigs, sows, laying hens and cows is less clear and lies between 0 - 3 %.

Growth promoters enhance digestion of the feed, the feed conversion (amount of feed (kg) needed to obtain 1 kg increase in weight) is improved. For pigs (22 up to 103 kg) it has been shown that the feed conversion decreased from 2.84 to 2.74 (Nefato, 1997).

In Sweden, where antimicrobials were forbidden in the feed in 1986, it was shown that it took 3 to 5 days more before pigs reached 25 kg of weight without growth promoters (Viane, 1997). So more feed is needed in the absence of growth promoters. This leads to an increase in the amount of faeces that is produced till the time the animals reach their final weight (Nefato, 1997).

A wide range of antimicrobial additives is used or has been used in animal husbandry to promote growth. Often the drug used as growth promoter is not used therapeutically for animals. The amount of an individual growth promoter animals receive lies in the range of 5 - 100 parts per million (ppm= mg/kg feed). The most common dosage is 20 - 50 ppm. The amount depends on the antimicrobial given, animal species and its age. The amounts that are applied usually are the maximum allowed dosages or amounts close to this maximum (see Feed Additive Directive 70/524 EC). AGPs in feed are as follows:

Table 3.3.2.1. Kinds of antimicrobial growth promoters added to the feed (provided by Op den Kamp, 1998; Gezondheidsraad, 1998).

The amount of feed animals consumes and the percentage of the feed that contains antibiotics in the Netherlands is given below.

Table 3.3.2.2. Amounts of feed consumed by different animal species and percentage of feed with added AGPs in the Netherlands in 1996-1997 (Gezondheidsraad, 1998; PDV, 1997; IKC, 1998).

In total, approximately 250 - 300 ton of antibiotics are mixed yearly with the feed in the Netherlands (Nefato, 1998; Piron (FEFANA-Alpharma), 1998).

3.3.3 Regulations for the use of antimicrobial agents

Already in the Swann report (1969) it was stated that antibiotics showing cross-resistance with antibiotics used in human health care should not be used as growth promoters. The ban of tetracycline and penicillins as growth promoters was recommended because these antibiotics are also used as a human medicine. In the 1970s the use of tetracycline and penicillin as growth promoter was banned in the European Community (Witte, 1997; Gezondheidsraad, 1998). In 1970, a European directive was published (70/524 EEC) that contained prerequisites for the use of antimicrobial growth promoters (Butaye et al., 1998c). Only growth promoters should be used that:

Scheme 3.3.3.1. Growth promoter regulations.

In 1997 more items have been added to directive 70/524 EEC (Nefato, 1997):

• Every product is checked on the basis of a dossier containing safety, quality and effectivity measurements
• For every product one company is responsible
• Every 10 year the component has to be re-evaluated, with the latest scientific knowledge as guideline

Before an antibiotic is accepted for usage in animal feed some important features needs to be analysed: chemical structure, mechanism responsible for drug resistance and cross-resistance with antibiotics used in human healthcare. Once approved, new registration-files are made every few years, containing the latest facts about the antibiotics involved (safety of use).

Not all the growth promoters currently used meet the prerequisites mentioned in directive 70/524 EEC. Especially the point that antibiotics used as growth promoter should not be related to antibiotics in medicine or should not be used in medicine is often not complied with. As can be seen in the table below many growth promoters have structurally related relatives that are used in human health care.

Table 3.3.3.1. Antimicrobials that are used or have been used in animal husbandry in Europe as AGPs and their analogues used in human health care (Bryan, 1982; Witte, 1997; Butaye et al., 1998c; Op den Kamp, 1998; Aarestrup et al., 1997, MAFF, 1998).

The antibiotics causing most (political) concern are avoparcin (not used anymore in Europe), virginiamycin, tylosin and Zn-bacitracin. The human health risk these antibiotics might cause will be discussed and evaluated in this report.

3.3.4 Antibiotics in animal feed

Avoparcin

Avoparcin is a glycopeptide antibiotic which has been widely used as feed additive since 1975 (Witte, 1997). This antibiotic is not metabolised when ingested by pigs and chickens, so it leaves the body in the active form (Bager, 1997). In humans, vancomycin and to a lesser extent teicoplanin are important tools, albeit limited, in treatment of bacterial infection caused by multiple resistant bacteria (mainly multiresistant staphylococci, enterococci and pneumococci). Bacteria resistant to avoparcin have been shown to be cross-resistant to vancomycin and teicoplanin (Cormican et al., 1997).

Avoparcin was banned in the EC in January 1997. Denmark decided to ban avoparcin already in 1995 as a result of a report of the Danish Veterinary Laboratory (1995). In January 1996 Germany was the second country to ban avoparcin. The decision of the German Federal Institute for Consumer Health Protection and Veterinary Medicine to ban avoparcin was partly based on the Danish study mentioned above. In enterococci the vanA gene cluster mediates the high level of resistance to vancomycin. The possibility that the vanA gene may be transferred to other bacteria like methicillin-resistant Staphylococcus aureus (MRSA) was important to choose for a preventive protection of human health.

In October 1995 a question (no. 82) concerning the continued use of avoparcin as a feed additive was addressed to the SCAN. At the European level, the SCAN is the advisory board about the use of antibiotics as feed additives. The SCAN evaluated the report of the Danish Veterinary Laboratory (1995), especially on the point of relevant scientific data that supported the need for banning avoparcin. According to the SCAN the Danish demonstrated the presence of glycopeptide-resistant enterococci in isolates from the majority of pig and poultry farms that used avoparcin. Also it was made clear that the transfer of resistance genes from E. faecium to E. faecalis could be achieved in the lab (Noble et al., 1992). A third point was that resistance to avoparcin leads to cross-resistance to vancomycin and teicoplanin. The DVL report, however, did not present evidence that the use of avoparcin as a growth-promoting agent caused disease in man or that existing diseases in animals or man increased or worsened notably. So, SCAN concluded that there was no direct evidence that the use of avoparcin in animal feed presented a risk for human health.

The European Community, however, decided to ban the use of avoparcin by January 1997 as a precautionary measure, partly based on the argument that the risk for human health could not be ruled out. SCAN namely also concluded that:

'... [it] cannot be ruled out with sufficient certainty that the use of avoparcin in feed may lead to the spread of glycopeptide-resistance beyond the sphere of livestock production.' (SCAN, 1996)

Such a conclusion is by definition derived from the fact that no amount of scientific experiments will be sufficient to exclude with absolute certainty a certain risk related to the use of AGPs.

Macrolides and streptogramins

The antibiotics tylosin, spiramycin and virginiamycin belong to the macrolide-lincosamide/streptogramin B (MLSB) group of antibiotics. Tylosin and spiramycin belong to the macrolides, while virginiamycin is a member of the streptogramin group. It is known that within the group of MLSB antibiotics cross-resistance can occur. Antibiotics of the MLSB class are used as medicines in humans and animals. Macrolides are used to treat respiratory tract infections (caused by Gram-positive bacteria) outside the hospital, but are also applied to treat infections with (Gram-negative) Campylobacter spp. (Aarestrup et al., 1998). Resistance mechanisms are known that lower the susceptibility of multiple compounds belonging to this antibiotic class (Allignet et al., 1996).

Tylosin and spiramycin were approved for use as feed additives in the EEC in 1970. Tylosin is allowed for use in pigs and piglets, while spiramycin can also be used for poultry, calves, lambs and fur animals (SCAN, 1998a).

In Denmark (1995) 52.3 tonnes of tylosin were used as additives in pig feed, while 0.5 tonnes of spiramycin was added to the feed of broilers. Also 9.5 tonnes of macrolides were used in therapy of animals. In Finland 0.74 tonnes of macrolides were solely used to treat diseased animals (mainly Serpulina infections).

Denmark banned the streptogramin virginiamycin as a feed additive in 1998 under a safeguard clause. SCAN was asked to review the scientific material on which the Danish government based its ban. SCAN is highly critical in its comments on the scientific evidence presented to them (SCAN, 1998b). SCAN concludes the following:

1. '...  no new evidence has been provided to substantiate the transfer of a streptogramins or vancomycin resistance from organisms of animal origin to those resident in the human digestive tract and so compromise the future use of therapeutics in human medicine
2. the development of vancomycin resistance amongst E. faecium and methicillin-resistant strains of Staphylococcus aureus, ..., are evidently a cause for concern. However, the data provided in the Danish report does not justify the immediate action taken by Denmark to preserve streptogramins as therapeutic agents of last resort in humans.
3. 3. as survey data ... failed to detect a single case of VRE, as Denmark has amongst the lowest incidence of MRSA in Europe and North America, and as coagulase-negative staphylococci remain sensitive to vancomycin, there are no clinical reasons to require the introduction of streptogramins as human therapeutics in Denmark now or in the immediate future. ...

In countries that permit the use of streptogramins in both animal production and human medicine, notably France and the USA, the use of pristinamycin (a human therapeutic antibiotic) has not been compromised by the use of virginiamycin as a growth promoter.

Zn-bacitracin

To date, as a human curative, bacitracin is only used topically to cure infections of the skin or mucous membranes. Lately also patients with VRE infections are treated (Chia et al., 1995). In the future bacitracin might be used to treat MRSA infections as well.

3.3.5 Antibiotics in human health care

As antibiotics kill or inhibit growth of bacteria, they can have a serious impact on the human intestinal flora. In the human intestine many bacteria are present. The predominant genera are the anaerobic Bacteroides, Fusobacterium, Bifidobacterium, Clostridium, Propionibacterium, Eubacterium and the facultatively anaerobic Enterobacteriacea (E. coli, Enterobacter), Lactobacillus, Enterococcus and Streptococcus (Drasar and Barrow, 1985). A precise composition of 'normal human intestinal flora' (quantitatively and qualitatively) can not be given, partly because not all bacteria can be cultured easily.

If a patient suffers from an infection caused by Gram-negative bacteria, this usually is treated with broad-spectrum antibiotics like cephalosporines and fluoroquinolones. The Gram-negative bacteria are killed and Gram-positive bacteria, like enterococci can then cause overgrowth (Murray, 1990). As a consequence, these bacteria can cause severe damaging health effects.

There are three groups of antibiotics that can cause severe effects on the intestinal flora:

• orally administered antibiotics; these are not well absorbed from the gastrointestinal tract
• antibiotics that are absorbed but subsequently excreted in the bile
• * parenterally given antibiotics that are subsequently excreted in the intestinal tract

A group of antibiotics that causes strong suppression of the intestinal flora are the MLSB antibiotics. This leads to overgrowth (or sometimes new colonisation) of streptococci, staphylococci, clostridia and enterococci (Nord, 1993). Also glycopeptides, like vancomycin and teicoplanin, lead to serious effects when administered orally. These antibiotics act against staphylococci, enterococci and pneumococci. It has been used clinically since the 1950s, but frequent use started in the late 1970s and the early 1980s (Murray, 1998). Teicoplanin is also used in human medicine, but to a lesser extent.

Kirst et al. (1998) have collected data about the vancomycin usage in the United States and several European countries. From the beginning of the 1980s the use in the Unites States increases rapidly until 1992. It now seems that vancomycin use is stabilising around 10,000 kg a year. In France the use is also more or less constant the last years, around 1,100 kg a year. The same holds true for the Netherlands, where around 60 kg is used. In Germany, Italy and the United Kingdom vancomycin consumption is still increasing (1996: 629 kg, 538 kg and 349 kg respectively). Not only the total use is important, but also the use per inhabitant. This is listed in the table below.

Table 3.3.5.1. Use of vancomycin in the USA and in Europe (Kirst et al., 1998).

In the United States the use of vancomycin is clearly higher than in Europe. For developing resistance (impact of the antibiotic) the amount used, the number of treated individuals as well as the population density (for AGPs: farm animal density) (Levy, 1997). In the USA, being a large country, the amount of antibiotic prescribed is high, as well as the number of individuals treated. This, combined with the effect vancomycin has on the intestinal flora, may cause resistant strains to emerge and spread easily.

Proper use of antibiotics can decrease the risk of selecting for resistant bacteria. Antibiotics should only be given when necessary, this means in the case of (serious) infections caused by bacteria. They should not be used to treat common colds and other infections caused by viruses (Levy, 1998; Gopal Rao, 1998; Huovinen, 1997). Often antibiotics are provided without knowing which organism is causing the infection. Tests to determine the microorganism causing the infection are not carried out routinely partly because most tests are time-consuming and thus costly. This also holds true for the testing of susceptibility of the infectious bacteria. Another point to be considered is the way antibiotics are administered. It is important to finish the whole treatment. Another point of concern is that in many countries antibiotics can easily be bought without a medical receipt. Moreover, hygienic measures taken in hospitals reduce the spread of resistant bacteria and will keep the rise of resistant bacteria more or less in check.

3.4 Cellular Processes and Antibiotics

3.4.1 Cell wall synthesis

An important difference between mammalian and bacterial cells is the presence of cell walls in the latter, positioned outside the cytoplasma membrane. The basic structure of the cell wall is a polymeric peptidoglycan, called murein. Gram-positive bacteria contain larger amounts of peptidoglycan in their cell wall compared to the Gram-negative bacteria (Schlegel, 1992). N-acetylglucosamine (N-Gluc) and N-acetylmuramic acid (N-Mur) building blocks form the backbone of murein. Muramic acid contains a peptide chain of four or five amino acids.

Polymer strands can be connected by peptide bonds formed between peptide chains of the muramic acid. A whole layer or even a net work (Gram-positive cell walls) of peptidoglycan can be composed in this way (Russell and Chopra, 1990; Schlegel, 1992).

In the cytoplasma the cell wall precursors are formed. In enterococci and in S. aureus a pentapeptide is linked to the muramic acid. First a tripeptide consisting of L-Ala, D-Glu and L-Lys is attached to the muramic acid, after which the dipeptide D-Ala-D-Ala is added. N-Gluc and N-Mur are coupled and together form disaccharides. Subsequently, the completed disaccharide N-Gluc-N-Mur is transported through the cytoplasma membrane by the aid of a lipid carrier (Arthur et al., 1996; Reynolds et al., 1998). Then these subunits are incorporated into a growing peptidoglycan chain, which after some modifications will form part of the cell wall.

Antibiotics have been developed that interfere with bacterial cell wall synthesis. Known antibiotics interfere with pentapeptide/disaccharide formation, transport of peptidoglycan precursors through the membrane (Zn-bacitracin) and cross-linking of peptidoglycan (vancomycin). The advantage of these antibiotics is that they act specifically on bacteria and are (in principal) not toxic to humans.

• Vancomycin (avoparcin, teicoplanin): mode of action
  Vancomycin binds to the D-Ala-D-Ala side of the pentapeptide in N-Gluc-N-Mur disaccharides, inhibiting the incorporation of these dimers into the growing peptidoglycan (Baptista et al., 1996). The two D-Ala molecules are present in pentapeptides of muramic acid in enterococci (Baptista et al., 1996), as well in S. aureus (Schlegel, 1992).

• Zn-bacitracin: mode of action
  Bacitracin indirectly inhibits the transport of peptidoglycan building blocks (N-acetyl glucosamide - N-acetylmuramic acid dimers) through the cytoplasma membrane. A monophosphate lipid carrier transports this disaccharide. After the dimer is released at the site of the cell wall the lipid molecule remains in the membrane in its pyrophosphate (PP) form. Bacitracin binds to the PP-lipid and inhibits its dephosphorylation. In this way the lipid carrier is not able to transport new disaccharides through the membrane (Russell and Chopra, 1990). Zn-bacitracin is active against Gram-positive bacteria. The antibiotic is very active towards Clostridium perfringens (Alpharma, 1998).

3.4.2 Bacterial protein synthesis

Proteins are constituted of amino acids coupled together. The mRNA (formed by transcription of DNA) possesses the code for the sequence of these amino acids. Before protein synthesis starts, amino acids that need to be incorporated in the protein are coupled to specific tRNAs, leading to aminocacyl-tRNA molecules.

In bacteria protein synthesis is mediated by 70 S ribosomes. These ribosomes exists of two subunits, of 50 S and 30 S. Both subunits contain rRNA and proteins (Watson et al., 1987). There are three stages in protein synthesis involving ribosomes: the initiation, the elongation and the termination phase (Cocito et al., 1997).

In the initiation phase the mRNA is bound to the 30S ribosome. After the first amino acid (bound to tRNA) in the protein code is found, the two ribosomal units are joined into the 70S ribosome (Russel and Chopra, 1990; Cocito et al., 1997). In the elongation phase amino acids are added to the growing protein chain. The 50S subunit of the ribosome consists of two important sites: the A (acceptor) site, which binds the next tRNA-amino acid molecule and the P site, which binds the growing peptide chain (peptidyl-tRNA). The addition of the next amino acid to the peptide chain is catalysed by the peptidyl transferase centre (PTC) (Watson et al., 1987; Russel and Chopra, 1990; Cocito et al., 1997). When termination sequences in the mRNA are reached the protein synthesis stops. The completed polypeptide is removed from the ribosome. The mRNA also leaves the ribosome, followed by separation of the two ribosomal subunits (Russel and Chopra, 1990; Cocito et al., 1997).

The following antibiotics discussed in this report interfere with bacterial protein synthesis. Antibiotics can interfere with different processes in protein synthesis. They can bind to 30 S or 50 S ribosomal subunits or to the mRNA. When they bind to the 30 S ribosomal subunit before the 70 S ribosome is formed, initiation of protein synthesis is prevented. Some antibiotics interfere with the linking of the mRNA codon to the tRNA anticodon, preventing elongation of protein synthesis (Cocito et al., 1997). Antibiotics that bind to the 50 S ribosomal subunit or to elongation factors, that are connected to the ribosome for short periods, inhibit elongation of protein synthesis.

The macrolides, streptogramines B and lincosamines together form the MLS group of antibiotics. These antibiotics (mainly) disturb functioning of the ribosome during protein elongation. The AGPs tylosin, spiramycin and virginiamycin belong to this class of antibiotics.

• Macrolides: mode of action
  Macrolides contain a ring constituted of C en O-atoms (lactone ring), which is substituted with one or two (amino) sugar moieties (Russel and Chopra, 1990). The lactone ring can be 14-, 15- or 16-membered. This group of antibiotics binds to the 50 S ribosomal subunit. Probably they act on the release of peptidyl-tRNA from ribosomes when translocation from the P to the A site takes place.

• Lincosamides: mode of action
  Lincosamides consist of 14, 15 of 16-membered lactone rings. These antibiotics are inhibiting the peptidyl transferase function of the 50 S ribosomal subunit. This means that the growing peptide chain can not be transferred from the peptidyl to the acceptor site.

• Streptogramins: mode of action
  Streptogramins can be divided into two groups, group A and B. Both types are macrocyclic lactone rings. The A-group streptogramins contain a large unsaturated non-peptide ring. The B-group consists of cyclic hexadepsipeptides which contain unusual amino acids (Russel and Chopra, 1990; Cocito et al., 1997).

Streptogramins of the A group can bind to 50S subunits or 70S ribosomes when they are not in the elongation phase. Most likely streptogramins A bind to the free peptidyl transferase catalytic centre (Chinali et al., 1987; Russell and Chopra, 1990). In this way protein synthesis cannot enter the elongation phase. To the streptogramin A group belong virginiamycin M and virginiamycin S (Cocito et al., 1997).

The B group streptogramins belong to the MLSB group of antibiotics. They are acting on the elongation step of protein synthesis. Binding of aa-tRNA to the A site and peptidyl transfer from the P site is prevented. Translocation of the growing polypeptide chain is not inhibited (Cocito et al., 1974; Ennis and Duffy, 1972).

The two groups of streptogramins are acting synergistic towards Gram-positive bacteria. When an antibiotic of the A type binds to the ribosome, conformational changes in the 50 S ribosome occur. This leads to an increase affinity for the B group streptogramins towards this ribosome subunit (Moureau et al., 1983). When streptogramin A and B are acting individually, they are only bacteriostatic which means that growth is stopped, but can be resumed when cells are transferred to antibiotic free medium. When administered together the effect is bactericidal (Chinali et al., 1987).

3.5 Bacterial Resistance and its Transfer: Basics

3.5.1 Location of resistance genes

Bacteria can obtain antibiotic resistance in a few distinct ways (Van Egeraat, 1991a; Bryan, 1984; Russel and Chopra, 1990). Resistance traits can be present on different parts or pieces of DNA: the chromosome, plasmids and/or transposons. To be able to discuss transfer of resistance, it is necessary to gather insight in how resistance against antibiotics in bacteria is accomplished and where resistance genes are located. The location is highly influential on the possibilities of transfer.

• Plasmids
  Besides the large chromosome, bacteria often possess small circular pieces of DNA called plasmids. In general, plasmids contain genes that are not necessarily needed for the host bacterium. Multiple copies are usually present in the bacterial cell. A plasmid can replicate (multiply) independent of the DNA of the chromosome. Plasmids can be transferred to bacteria of the same species or bacteria that are less related. Resistance genes are often present on plasmids. The presence of more than one resistance gene on one plasmid is not uncommon.
  There are conjugative plasmids and nonconjugative plasmids. The conjugative plasmids are capable of moving to another cell. These plasmids are usually larger than the nonconjugative plasmids (Bryan, 1982).
• Transposons: insertion sequences and complex transposons
  Transposons are pieces of DNA that can migrate through the genome of an organism (Saedler and Gierl (eds.), 1996). They can be part of plasmids and bacteriophages but also occur on the bacterial chromosome.
  Insertion sequences (simple transposons) are mobile DNA elements present in bacteria. They usually contain only the transposase gene. They can transpose themselves, this means they are cut out of their location in the DNA and are residing somewhere else. In doing this, the IS cause genome rearrangements, such as deletions, inversions, duplications and replicon fusions (Ohtsubo and Sekine, in Saedler and Gierl (eds.), 1996). Insertion sequences usually consist of 800 - 2500 base pairs and have a few to a few hundred copies per genome. The sequence codes for a transposase enzyme and often resistance genes are also present. The left and right ends of an IS contain inverted repeats of 10 - 40 bp. These repeats play a role in the transposition of the sequence. This transposition is different from the homology dependent recombinations that can occur in cells.
  Complex tranposons can be part of plasmids but also occur on the bacterial genome. A transposon is a piece of DNA of 750 up to 40.000 base pairs. The transposon consists of genes coding for enzymes that cut themselves out of a larger piece of DNA and incorporate the transposon somewhere else. Complex transposons contain one or more genes with different functions. These can be genes for antibiotic resistance.
  When a transposon containing resistance genes inserts itself in a plasmid it can be transferred to another cell. When the plasmid is able to replicate itself in the new host, or if the transposon moves to another replicable plasmid or inserts in the chromosome, this cell becomes resistant to the antibiotic (Summers, 1996).

3.5.2 Intrinsic and acquired resistance

Bacteria acquiring resistance against an antibiotic is a form of adaptation under biochemical stress. The information thus generated is stored and passed on to other bacterial organisms in several ways.

First, the two types causing resistance will be discussed namely:

• Intrinsic resistance
• Acquired resistance

Subsequently, the possible mechanisms of resistance in a bacterial cell are described. Then the resistance mechanisms towards the growth promoters and antibiotics of importance for this report will be discussed. The last step is to explain the possibilities of transfer of genes from one bacterium to another.

• Intrinsic
  Already before antibiotics were used throughout the world, bacteria resistant to some antibiotics existed. This resistance called intrinsic resistance, is due to properties of the cell and mediated by chromosomal genes (Russell and Chopra, 1990). The antibiotic is prevented from entering the cell or reaching its target, or the target is not sensitive to the antibiotic. The intrinsic type of resistance cannot be transferred to other bacteria, only to the offspring of the cell.
  The predominant form of intrinsic resistance is resistance mediated by the shape and constituents of the cell wall. This barrier prevents some antibiotics from entering the cell. In Gram-negative bacteria this type of resistant often has been noticed. The outer membrane of Gram-negative bacteria can prevent the entrance of some _-lactams into the cell. Also large antibiotics like bacitracin, vancomycin and teicoplanin cannot pass the porins of the Gram-negative outer membrane.
  Enterococci can be intrinsically resistant to penicillins, cephalosporins, aminoglycosides, clindamycin and Zn-bacitracin (Murray, 1998; Baquero, 1997; De Neeling et al., 1997; Alpharma, 1998). E. faecium can also be intrinsic resistant to sulphonamides