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Antibiotic Resistance

Introduction

The generic purpose of an antibiotic is to prevent the growth and/or survival of invading organisms whilst causing minimal damage and toxicity to the host. The typical mechanism of antibiotic action involves targeting specific enzymes or substrates of the invading bacterial species. Antibiotics may be either bacteriocidal (i.e. kill bacteria, e.g. β-lactam antibiotics) or bacteriostatic (i.e. slow bacteria growth and reproduction e.g. tetracyclines). However, the majority of bacteria are actually killed by the host immune system, the administration of antibiotics is typically only to aid the immune system and thus speed up recovery.

Some antibiotics may be synergistic, that is, when used with other antibiotics the overall therapeutic effect is greater than the sum of their individual effects, this allows for reduced doses to be administered.

Antibiotics can have either target a narrow spectrum of bacteria (such as the penicillins and macrolides), or a broad spectrum of bacteria (such as the aminoglycosides, cephalosporins, quinolones and some synthetic penicillins).

The use of antibiotics in food animals is subject to EU legislation which governs maximum residue limits (MRLs), the maximum concentration of antibiotic allowed to be administered to the animal for it still to be fit for human consumption. Antibiotics must be prevented from entering the food chain in this manner, so MRLs have been categorised into 4 annexes:

  • Annex 1 – MRL has been fixed
  • Annex 2 – MRL not required
  • Annex 3 – Provisional MRL
  • Annex 4 – No MRL can be set

The use of annex 4 antibiotics (e.g. chloramphenicol) is prohibited in animals destined for human consumption, with use of annex 1 or 2 antibiotics being preferred.

Choice of Antibiotics

There are many factors to consider when selecting an appropriate antibiotic for use. The first question to ask should be, ‘Is the infection bacterial?’ Antibiotics should only be administered for bacterial infections as they are of no use against viral infections. It is also important to remember that antibiotics only treat the infection and do not act as anti-inflammatories or anti-pyretics.

The site of infection is another consideration, as whilst some antibiotics will be of great use for skin infections, they may not be as useful for respiratory tract infections. The species of bacteria must also be considered, this will firstly require the culture and identification of the invasive organism to determine the species. However, within a species there may be variations of antibiotic susceptibility so it is important to generate a resistance profile i.e. determine which antibiotics the bacteria is resistant to and thus avoid prescribing these antibiotics. It must then be decided whether to use a broad or wide spectrum of antibiotic and wether this antibiotic should be bacteriocidal or bacteriostatic.

Bacteriostatic antibiotics will require a duration of therapy which gives the host cellular and humoral immune responses enough time to eradicate the bacteria. Alternatively bacteriocidal antibiotics are used when the host immune system is considered to be ineffective against the invasive organism.

Other factors to consider include:

  • The distribution of the drug throughout the body
  • The cost for a course of the antibiotic
  • The toxicity posed to the host
  • Any underlying disease which may be affected by the use of the antibiotic
  • Whether the host is pregnant or juvenile
  • The practically of the drug i.e. the route of administration, dosage, frequency of dose and the duration of action etc.

The Cascade

One final consideration to be made when administering antibiotics for animals is ‘The Cascade,’ which is a regulation governing restrictions on the administering of veterinary medical products.

In animals, an antibiotic must be licensed for use with a particular species and disease. However if no such antibiotic exists, to avoid unacceptable suffering, the veterinary surgeon may prescribe another antibiotic in order of the cascade:

  1. A veterinary medicine licensed for use in another species or in the same species but for a different use. This is known as ‘off-label’ use.
  2. A medicine licensed for use in humans.
  3. An unlicensed medicine, created and prescribed as a ‘one-off’ by the veterinary surgeon.

Determination of Resistance

Two important factors to consider when attempting to determine whether a bacteria is resistant to a particular antibiotic are:

  • The Minimal Inhibitory Concentration (MIC) – The minimum concentration of a drug which prevents the growth of bacteria
  • The Minimum Bacteriocidal Concentration (MBC) – The concentration of a drug which causes a 99% reduction in a bacterial innoculum over a given period of time.

Also of importance are:

  • Pharmacodynamics – The effect of a drug on a pathogen i.e. the MIC and MBC
  • Pharmacokinetics – The effect of the body on a drug i.e. the absorption, distribution, metabolism and excretion of the drug

Breakpoints are predetermined concentrations of an antibiotic, which determine whether a bacteria is deemed susceptible or resistant to that antibiotic. If this concentration of antibiotic causes a reduction in bacterial numbers or prevents further growth then that strain of bacteria is defined as susceptible to the antibiotic in question. If the bacteria does not at least show signs of prevented growth, the bacteria is defined as resistant to the antibiotic.

Disc Diffusion

One method of determining antibody resistance is disc diffusion. A species of bacteria is isolated and cultured on growth medium. After an incubation period that allows for visible colonies to be observed on the growth medium, diffusion discs are placed around the growth plate. These discs are impregnated with different antibiotics. After a short incubation period, susceptible bacteria around the discs will have been killed, indicated by a clearing in the colonies. The diameter of the clearing can be used to determine how susceptible the species is to an antibiotic. If there is no clearing (or a very small clearing) this indicates resistance to the antibiotic which impregnated the disc.

Mechanisms of Antibiotic Resistance

Intrinsic Resistance

Intrinsic resistance is that which is not acquired, inherent resistance, such as a natural low permeability to antibiotics due to a bacterial envelope. This type of resistance is characteristic to almost all representatives of a species. An example of this type of resistance is observed in Pseudomonas species. Their resistance to antibiotics is owed to the low permeability of their bacterial envelope to certain antibiotics and the presence of a multi-drug efflux pump.

This efflux pump occurs naturally in bacteria and usually removes waste products from the cell, such as bile, fatty acids and organic solvents. However a mutation in some species also permits the efflux of antibiotics from the cell thus preventing them from exerting their action upon the bacteria. This trait is also acquirable however, such as the efflux pump gene responsible for tetracycline resistance.

Acquired resistance

Antibiotic resistance genes found on plasmids, can be transferred between individual bacteria, hence non-resistant bacteria can easily acquire resistance. Acquired resistance can also occur due to mutations. Beneficial mutations can then be transmitted around the bacterial population. The biggest resistance problems occur in Gram-negative organisms due to the large numbers of plasmids found within the population, another resistance problem occurs in Staphylococcus species i.e. MRSA and MRSP.

Targets of Antibiotics

Antibiotics can work in a number of ways:

  • Inhibit cell wall synthesis e.g. penicillins, cephalosporins, carbapenems, glycopeptides
  • Inhibit DNA synthesis e.g. fluoroquinolones
  • Inhibit RNA synthesis e.g. rifampicin
  • Inhibit folic acid synthesis e.g. sulfonamides, trimethoprim
  • Inhibit protein synthesis e.g. macrolides, chloramphenicol, tetracycline, aminoglycosides

However, bacteria have a number of mechanisms to resist the effects of antibiotics:

  • Decrease permeability to the antibiotic
  • Inactive the antibody by chemically altering it with enzymes
  • Efflux of the antibody via efflux pumps in the membrane
  • Alter the target of the antibiotic, so it no longer has an effect
  • Bypass steps in metabolism which the antibiotic targets

How do Antibiotics Work?

β-Lactams

β-lactams can be both natural and semi-synthetic. They work by inhibiting enzymes associated with the synthesis of peptidoglycans, so essentially inhibit cell-wall synthesis. Such antibiotics include; penicillin and its derivatives, cephalosporins, monobactams and carbapenems.

However over use of these antibiotics has selected for bacteria which have developed resistance, in the form of β-lactamase – an enzyme which hydrolyses β-lactams. The hydrolysation of β-lactam antibiotics will alter their conformation and thus they will no longer have an antimicrobial effect.

To combat this, β-lactamase inhibitors are now used alongside β-lactam antibiotics. Although β-lactamase inhibitors do not act as antibiotics alone, coupled with β-lactams, they can effectively target bacteria. β-lactamase inhibitors, inhibit the enzyme responsible for hydrolyzing β-lactam antibiotics, thus when coupled with β-lactams, the antibiotics are free to act upon their targets again – enzymes associated with cell wall synthesis. An example of this is Synulox, which contains the β-lactam amoxicillin and the β-lactamase inhibitor clavularic acid.

Bacterial β-lactamase enzymes work by hydrolyzing the ring-bond of the β -lactam which denatures their structure thus preventing them from exerting their antibiotic action.

β-lactamases can be either chromosomally derived (typically in Gram-negative bacteria) or  their genes can be found on plasmids and thus spread throughout a population. Extended spectrum β-lactamases are encoded for on plasmids (plasmids; TEM, SHU and CTX-M). These extended spectrum β-lactamases can catalyse a broad spectrum of β-lactam antibiotics.

Other mechanisms for penicillin (and derived antibiotics) resistance include; alterations in the penicillin binding protein found in bacterial cell membranes. Alterations in this protein reduces the bacterial affinity for penicillin. Similarly, the acquisition of a novel penicillin binding protein (mecA) also reduces affinity for penicillin. This gene is found in MRSA and is what makes it so resistant to penicillin its derivatives.

Aminoglycosides

Aminoglycosides irreversibly bind to the 30S ribosome and freeze the initiation complex. This effectively inhibits protein synthesis within the bacterium. Aminoglycosides have a broad spectrum but do not target anaerobes. They can also have a synergistic effect when used with β-lactams. They do however pose a toxicity risk; nephrotoxicity (toxic to the kidneys) and ototoxicity (toxic to the ear, specifically the cochlea or auditory nerve).

The mechanisms for resistance against aminoglycosides include; alteration of the bacterial ribosomes which prevent aminoglycosides from binding, decreased permeability to the antibiotics or the inactivation of aminoglycosides by mechanisms such as acetylation, phosphorylation and adenylation. These mechanisms are facilitated by aminoglycoside enzymes found in the bacteria.

Tetracyclines

Tetracyclines are bacteriostatic and similarly to aminoglycosides, they bind to the 30S ribosome (however, they bind irreversibly). They also inhibit RNA from binding to the 70S ribosome and thus inhibit protein synthesis. They have a broad spectrum of targets, but there are also many tetracycline resistant strains of bacteria.

Fluoroquinolones

Fluoroquinolones inhibit DNA synthesis, they do this by targeting the DNA enzymes gyrase and topoisomerase. They too have a broad spectrum, but bacterial plasma-mediated QNR genes provide resistance against the antibiotic. QNR genes offer low level protection for gyrase and topoisomerase from fluoroquinolones, however, the presence of QNR genes increases the mutation rate of gyrase and topoisomerase genes. Mutation of these genes can further increase resistance to fluoroquinolones.

Trimethoprim

Trimethoprim inhibits the bacterial dihydrofolate reductase (DHFR) enzyme which therefore inhibits folic acid synthesis. Again, trimethoprim has a broad spectrum, it is often used in combination with other antibiotics.

Resistance to trimethoprim occurs if the DHFR enzyme becomes less susceptible to the antibiotic e.g. through mutation, or if the metabolic step in folic acid production which requires DHFR is skipped altogether thus removing the target for trimethoprim completely.

Antibiotic Resistance

Resistance has always existed, even before antibiotic use. However the use of medical antibiotics have increased the prevalence of resistance. It is important to note though, that antibiotics do not cause resistance. They do however select for resistance already prevalent in a population.

Staphylococci Virulence

Introduction

Staphylococcus is a gram positive, cocci shaped, genus of bacteria. Observed under a microscope will reveal they exist in microscopic ‘grape-like’ clusters. One species of staphylococci, Staphylococcus aureus, can grow at temperature ranges of 15-45ºC and at  a relatively high NaCl concentration of 15%.

Catalase Test

One of the main tests used to differentiate between bacterial species is the catalase test. This test determines whether or not the enzyme catalase is present. Catalase, is responsible for the breakdown of hydrogen peroxide. The test consists of adding a bacterial colony to a drop of hydrogen peroxide, if the bacteria is catalase-positive (i.e. has the catalase enzyme) bubbles of oxygen will be produced. This will not occur in catalase-negative bacteria. Staphylococci species are catalase-positive.

Oxidase Test

The oxidase test, is another major test used to differentiate between bacteria. It tests to see if certain cytochrome c oxidase enzymes are present. These enzymes are involved with the electron transport chain. There are a number of ways to perform this procedure, but they all typically involve adding a reagent to the sample bacteria and observing for a colour change. Typically the development of a blue colour indicates a positive result (oxidase enzymes are present), with no colour change indicating a negative result. Staphylococci species are oxidase-negative.

Coagulase Test

The final major test used for differentiation is the coagulase test. This test determines whether the enzyme coagulase is present, an enzyme responsible for the formation of blood clots and primarily associated with staphylococci species. However, within the Staphylococcus genus, there are both coagulase-positive and coagulase-negative species. S. aureus is an example of a coagulase-positive species, and S. epidermidis is an   example of a coagulase-negative species. The procedure involves adding blood plasma to the test sample, the development of agglutination after a short period of time indicates a positive result, with no agglutination indicating a negative result.

Staphylococci & Animals

Currently, around 34 species of staphylococci have been identified, with many of these being found in multiple species of animals. Different species of Staphylococcus have different preferred hosts, Staphylococcus tend to co-evolve with their hosts, but they are still able to cross species barriers. The major Staphylococcus found in dogs and cats are:

  • S. intermedius
  • S. felis

S. aureus is not commonly found in dogs and cats, it is found more often in other domesticated species however.

Staphylococci can be found in certain locations all over the body where they result in no disease. These are known as resident populations and include; mucosal surfaces, mucocutaneous junctions (such as the lips, nostrils, vagina, etc.) and the ear canal.

Staphylococci of Veterinary Importance

Coagulase positive:

  • S. aureus
  • S. intermedius
  • S. hyicus

Coagulase negative:

  • S. sciuri
  • S. equorum
  • S. epidermidis

Staphylococcus pseudintermedius

S. pseudintermedius is most commonly found on the skin of domestic dogs and cats. This species is zoonotic and thus has the potential to infect humans, although human cases of infection with this species are rare. Of increasing concern is the rise in human S. pseudintermedius infectious, particularly because of the resistance to antibiotics shown by this species.

Some clinical signs associated with infection of this bacteria are; otitis externa (inflammation of the outer ear), mastitis, infective endocarditis (inflammation of the inner layer of the heart), abscess formation, infection of wounds and primarily, chronic pyoderma (an inflammatory skin disease).

Alterations of the skin’s micro-environment can promote the growth of bacteria such as S. pseudintermedius. For example, inflammation provides humidity and warmth, both of which promote bacterial multiplication. Any trauma caused to the skin will further reduce epidermal defenses. Continuing with the inflammation example, irritation may cause the the animal to scratch intensely which can lead to damage of the upper layers of the epidermis and make infection for S. pseudintermedius easier. Any allergic skin diseases, or underlying immunological disorders will also contribute to the degree of infection.

Pyoderma

Pyoderma is a bacterial skin disease, caused by bacteria normally found on healthy skin (commensal) such as Staphylococcus. Because these bacteria are opportunistic, if skin becomes diseased or damaged, they may proliferate which can cause problems. Whilst pyoderma is primarily caused by underlying skin problems, it is also possible for pyoderma to occur on healthy skin, however this often indicates underlying immune system problems.

Pyoderma becomes a problem when commensal bacteria breach the epidermis and begin to proliferate and adhere to keratinocytes – the predominant cell type of the epidermis. Areas prone to infection are those where the skin creates folds which reduces air circulation and provides a warm, humid environment, perfect for bacterial growth.

Treatment of pyoderma typically involves the use of antimicrobials either topically or systemically for around 8 weeks. Whilst this may remove the initial pyoderma, it is also important to treat the underlying conditions responsible for the outbreak as reinfection may occur. Reinfection will require more treatment, which is of concern as this can lead to antimicrobial resistance. Methicillin-resistant Staphylococcus pseudintermedius (MRSP, i.e. similar to MRSA) has recently been identified.

Staphylococcus hyicus

S. hyicus is responsible for causing exudative epidermitis, an oozing inflammation of the skin, also known as ‘Greasy Pig Disease’. It occurs when abraded skin is invaded by S. hyicus bacteria which then cause infection. Lesions begin to develop after infection, which then spread to the hair follicles (known as folliculitis). Inflammation soon follows this causing erosion and ulceration and the lesions continue to growth, engulfing large amounts of the skin surface. Alongside this, the sebaceous glands will produce a black, greasy, exudate.

Whilst this disease can be treated with antibiotics, death often occurs as a result of starvation of dehydration, so it is important to offer the infected pigs electrolytes by mouth to ensure a steady recovery.

Virulent strains of S. hyicus produce an ‘exfoliative toxin’. A toxin responsible for the epidermal necrolysis. Isolation of this toxin and reintroduction into a healthy pig will interestingly, reproduce the observed disease.

Exfoliative Toxin

The histopathology of S. hyicus induced exudative epidermitis, is very similar to that of S. aureus scalded skin syndrome. A disease which causes fluid filled blisters to appear on the skin of humans. Their similarities are owed to the exfoliative toxins, A & B which they secrete. These toxins cause a detachment of the epidermal layer and result in the observed lesions and deformations of the skin. This is beneficial to the bacteria as it allows them to further penetrate and proliferate beneath the skin.

Staphylococcus aureus Diseases

Staphylococcus aureus is responsible for a wide array of diseases, these include:

  • Superficial lesions
    • Skin and soft tissue infections
    • Mastitis
  • Invasive infections
    • Osteomyelitis – inflammation of the bone marrow
    • Endocarditis – inflammation of the inner layer of the heart
    • Pneumonia – inflammatory condition of the lungs
    • Septicaemia – The presence of pathogenic organisms in the blood which can lead to a body-wide inflammatory state
  • Toxinoses
    • Food poisoning
    • Scalded skin syndrome – Formation of fluid filled blisters on the skin
    • Toxic shock syndrome – A potentially fatal disease caused by a S. aureus toxin

Virulence & Virulence Factors

Staphylococcus species can express virulence in a number of ways, these include:

  • Adherence and surface associated proteins
  • Bacterial capsules which prevent phagocytosis
  • Exoenzymes, extracellular enzymes secreted by bacteria
  • Proteases, enzymes which break down proteins e.g. nuclease
  • Exotoxins, toxins secreted by the bacteria

Adhesion

Bacterial adhesion to host cells is the first step in colonization. Adhesion typically requires the presence of bacterial adhesins. These include pili, fimbrae, the flagella or the cell surface itself. These surface proteins allow for the attachment to host cells, in particular the host proteins laminin and fibronectin. Adhesins which promote attachment to collagen fibres can lead to osteomyelitis (infection of bone marrow) and arthritis.

Clumping Factor

Clumping factor is a protein which binds to fibrinogen, it is responsible for the formation of blood clots and it what causes the agglutination in the coagulase test. Clumping factor also helps to evade the immune system, by coating the itself in fibrinogen the bacterium avoid opsonisation and thus phagocytosis.

Protein A

A surface associated protein found in S. aureus, it binds to the Fc regions (tail regions of antibodies, which bind to Fc receptors on cell surfaces) of immunoglobulins, particularly IgG. This causes incorrect orientation of the bacteria in relation to the IgG, this is beneficial  because it disrupts opsonisation and thus phagocytosis. Protein A can also bind to IgM associated with B-cells (the lymphocytes associated with the humoral immune response), this induces apoptosis in these cells and thus causes depletion.

The gene which encodes for this protein is the spa gene, this gene is often used for molecular typing of Staphylococcus.

Capsular Polysaccharides

Capsules are bacterial structures which encase the entire bacterium, they are useful for protecting the bacterium against phagocytosis but they can also be a virulence factor. The presence of certain capsules can act as a potent abscess potentiator, isolation and injection of such a capsule can produce sterile intra-abdominal abscesses.

Super Antigens

Normal antigens are able to generate an immune response which induces a reaction from only the appropriate cells of the immune system. Typically much less than 1% of body T-cells (the lymphocytes associated with cell-mediated immunity) are activated. A super antigen on the other hand, generates a much more potent immune response. It is believed super antigens can activate up to 20% of the T-cells in the body.

Such a large immune response actually works in favour of Staphylococcus species. It is thought that these bacteria can produce more than 20 types of super antigens, in the form of enterotoxins and exotoxins. Enterotoxins specifically target the intestines and can cause diarrhoea and vomiting. In severe responses to super antigens (such as the exotoxins) the host can develop Toxic Shock Syndrome (TSS). TSS is where the body cannot cope with the large amounts of inflammatory cytokines released by the T-cells due to the binding of the super antigen, this can lead to shock and multiple organ failure.

The vast majority of TSS cases are caused by S. aureus and S. pyogenes. The most potent inducer of TSS however is Toxic shock syndrome toxin 1, a toxin produced by S. aureus which is responsible for around 75% of all TSS cases. Again, this leads to over stimulation of T-cells and a drastic systemic release of inflammatory cytokines which leads to lowered blood pressure, fever and in severe cases shock and organ failure.

Exfoliative Toxins

Another virulence factor of staphylococci is their ability to produce exfoliative toxins, exfoliative toxin A and B (ETA & ETB). These toxins result in blister formation at the epidermis surface which allows the bacteria to further penetrate and spread beneath the skin.

Membrane Damaging Toxins

There are three main forms of membrane damaging toxins (also known as type-II exotoxins) excreted by Staphylococcus, these are:

  • α-toxin – A potent membrane damaging toxin which is important for tissue invasion. In humans, platelets and monocytes are particularly sensitive to it. Cells susceptible to the α-toxin have specific receptors which it binds to, allowing it to cause damage.
  • β-toxin – This membrane damaging toxin targets membranes rich in lipids. A classic test to determine whether β-toxin is present, is to plate a sample on sheep erythrocyte-enriched growth medium. If β-toxin is present, it will lyse the erythrocytes which will be apparent on the growth medium as a clear area (as opposed to red).
  • Panton-Valentine Leukocidin (PVL) – This membrane damaging toxin, targets the cell membrane of leukocytes for which it has a high affinity. It can cause skin and soft tissue infections as well as pneumonia. Its structure allows for the formation of a pore through membranes, causing leukocyte cell contents to leak out.

MRSA – Methicillin resistant Staphylococcus aureus

Methicillin resistant S. aureus are bacteria which have developed resistance to beta-lactam antibiotics – those which target cell wall synthesis of bacteria. Such antibiotics includes the penicillin family of drugs. Their resistance is conferred by the gene meAc, which encodes for the protein penicillin-binding protein 2a (PBP2)

To determine whether a species of S. aureus is resistant:

  • The sample is first cultured
  • It must then be identified as S. aureus
  • The next step is to determine wether or not the strain is methicillin resistant
  • Once resistance is confirmed, the strain is then typed

Samples should be obtained from typical sites of carriage which include; the nasal passageway, skin, perineum and faeces. To culture MRSA, the growth medium must be selective i.e. contains a beta-lactam antibiotic, so only MRSA will grow.

At-risk patients include:

  • Dogs/Cats – Recent surgery, presence of non-healing wounds, recent or current treatment with multiple courses of broad spectrum antibiotics.
  • Horses – As above, also any length in-patient hospitalization