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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.

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Evolution of Bacterial Virulence

Point Mutations

Much like humans, bacteria are subject to evolution. Evolution occurs through beneficial mutations in the DNA. Because bacteria have much shorter generation times than humans, the process of evolution occurs much more rapidly. An example of how bacteria evolve through mutation is point mutation.

Point mutation is the random mutation of DNA nucleotides which occurs due to incorrect replication of DNA. The types of mutation that can occur are substitution of a nucleotide, deletion of a nucleotide or the addition of a nucleotide. For example, in a sequence of DNA a fragment of which codes for something (such as a protein) consists of multiple codons (sections of 3 nucleotides) if one of these nucleotides is altered in a point mutation, the codon (which codes for a specific amino acid) will be altered. Some alterations of codons will mean they encode for a different amino acid:

Initial DNA:                  … … TTA … …

After point mutation: … … TAA … …

In this case the selected codon in the initial DNA encodes for the amino acid leucine, however after the mutation, it no longer encodes for an amino acid at all as TAA is a stop codon (this closes the reading frame, meaning the rest of the gene is not read).

Point mutations may sometimes have no effect however, as multiple codons can encode for the same amino acid, for example:

Initial DNA:                  … … GCT … …

After point mutation: … … GCA … …

Both GCT and GCA codons encode for the amino acid alanine, so there are no alterations to the final protein which is encoded for by this gene.

Horizontal Gene Transfer

Bacteria are also able to ‘evolve’ by means other than mutation. Horizontal gene transfer is the sharing of genetic information between individuals in the population. Typical gene transfer occurs vertically i.e. parent to offspring, however with horizontal gene transfer genetic information can be spread to completely unrelated individuals.

There are three primary forms of horizontal gene transfer, these are:

  • Transformation – DNA is taken up from the environment
  • Conjugation – DNA is transferred directly between individual bacteria
  • Transduction – DNA transfer is mediated by bacteriophages (a virus which infects bacteria)

Transformation

Many bacteria are naturally transformable and have various means of DNA uptake from their environment. Bacteria able to do this have genes which encode for protein machinery which transports the DNA across the cell membrane from the environment. It is then possible for this DNA to be incorporated into the bacterial chromosome.

This ability has important implications in bacterial pathogenicity for example; Neisseria gonorrhea are able to uptake DNA from the environment which enables them to undergo possible conformational changes that make them more pathogenic. Another example would be Streptococcus pneumoniae and its possibility to acquire penicillin resistance.

Conjugation

Conjugation is the direct transfer of DNA between bacteria, this is of major advantage as advantageous genes (such as those which encode for antibiotic resistance) can be easily spread throughout the population.

DNA is transferred in the form of a plasmid (a circular DNA structure) in a unidirectional manner, that is only the donor requires the plasmid transfer-associated genes (i.e. those which encode for plasmid transfer mediating pili) for horizontal transfer via conjugation to occur.

Plasmids

Plasmids are typically circular in structure and consist of double stranded DNA. They replicate independently of the bacterial chromosome and are located in the cytoplasm, they are usually much smaller than the bacterial chromosome ranging from 1-100kb long. The genes found within the plasmid can be expressed by the bacterium and so proteins encoded for by the plasmid can be formed in the bacterium.

The most common plasmids are those which encode for antibiotic resistance. They are highly prevalent because of the great advantage they give to bacterial survivability. The are multiple genes found on plasmids which promote antibiotic resistance all with different mechanisms of producing resistance.

For example β-lactam antibiotics (such as penicillin derivatives) work by inhibiting cell wall synthesis, however β-lactamase is a bacterial enzyme which breaks the ring structure of these antibiotics are removes their antibacterial properties. These enzyme is encoded for by genes found within plasmids, which can easily be spread around the population thus increasing the resistance of the population to β-lactam antibiotics.

Other forms of plasmid-conferred attributes include:

  • Bacteriocins – Proteinaceous toxins which inhibit the growth of competitor species a specific example of this is colicin, a bacteriocin produced by Escherichia coli which exerts a cytotoxic effect within competitor species’ cytoplasm.
  • Toxin production – Toxins which affect other species such as the heat-labile enterotoxin produced by E. coli which causes diarrhoea or the Clostridium tetani tetanus toxin, the neurotoxin responsible for causing tetanus.
  • Protein delivery systems – Such as the Yersinia pestis secretion system, which allows them to inject protein materials into other organisms (such as immune cells), these injected proteins are known as YOPs (Yersinia outer proteins).
  • Adhesins – Typically pili (protein surface projections) which allow adherence to bind to cells, which is important in pathogenicity. Such adhesins can be found in E. coli and Shigella.
  • Metabolic activities – For example, the ability of Salmonella species to ferment lactose

Transduction

Transduction is the transfer of DNA which is mediated by bacteriophages (viruses which infect bacteria, combining their DNA with the bacterium host). Many bacteriophages have both lytic and lysogenic cycles of reproduction (typically viruses are one or the other). The lytic cycle is where the virus enters the cell, replicates using the cell’s own machinery and then lyses the cell releases large numbers of virus. The lysogenic cycle is where the virus incorporates its genetic material into the cell where it lies dormant until a later date, the viral genes are replicated when the cell replicates allowing them to reactivate and continue into a lytic cycle (formation of phages).

Bacteriophage-encoded virulence factors include:

  • Streptococcus pyogenes toxin (which causes scarlet fever)
  • Corynebacterium diphtheriae toxin
  • Clostridium botulinum toxin

E. coli O157:H7 contains around 1.4Mb of extra DNA when compared to E. coli K12 (a much less pathogenic strain which is used in laboratories). It is suggested that around 50% of this extra genetic material has been derived from bacteriophages.

Vibrio cholera Toxin

Another bacteriophage encoded virulence factor is the Vibrio cholera toxin, however for this toxin to be produced by the bacterium, genetic material from two bacteriophages must be acquired.

The initial bacteriophage (VPI phage) infects a non-toxic Vibrio cholera bacterium, its transduced genetic material encodes for pili proteins which act as an attachment site for the secondary bacteriophage (CTX phage). When CTX phage binds, the genetic material it incorporates encodes for the production of the cholera toxin and thus the bacterium becomes toxogenic.

Recombination

Transformed and transduced (i.e. non-plasmid DNA) is inserted into the recipient bacterial chromosome by recombination. Recombination is also responsible for the rearrangement of DNA fragments around the genome (genomic plasticity) which enables the bacteria to survive in rapidly changing environments due to the alterations in the genes which are expressed. Recombination can be either homologous (RecA dependent – a DNA repair and maintenance protein) or non-homologous.

Homologous Recombination

DNA inserted into the bacterium can be incorporated into the bacterial chromosome if there are regions of homology expressed on both the foreign transmitted DNA and the bacterial chromosome DNA. The differences between the foreign and bacterial DNA will cause RecA to attempt to ‘repair’ the chromosome. In doing so, the foreign DNA may be incorporated into the bacterial chromosome, (there is also the chance that it will be disregarded).

Non-homologous Recombination

Non-homologous recombination allows genetic material to enter the bacterial chromosome without RecA. Small fragments of DNA known as insertion sequences (ISs) are what enter the chromosome. ISs typically consist of DNA fragments from bacteriophages or elsewhere, however, all that an IS codes for is its motility and insertion. ISs integrate into the bacterial chromosome through self-encoded transposition enzymes (as opposed to RecA) and when within the chromosome they facilitate replication of themselves. The short ISs then become numerous within the bacterial chromosome.

The presence of multiple ISs within the bacterial chromosome is what allows genetic plasticity (The movement or deletion of short fragments of DNA within the chromosome). Genetic plasticity leads to alterations in genes or the removal of genes altogether, this has the potential to increase pathogenicity. For example, Bordetella bronchiseptica is a gram negative bacterium responsible for causing infectious bronchitis, which rarely infects humans. B. pertussis on the hand is the major causative agent of pertussis (whooping cough) and is an obligate human pathogen. It is believed that genetic plasticity caused a reshuffling of the B. bronchiseptica chromosome which lead to the formation of the obligate human pathogen B. pertussis.

Transposons

Transposons are transmitted by similar means to ISs, however they are more structurally complex and much larger, due to the greater amount of genetic information they carry. This extra genetic material encodes for attributes such as antibiotic resistance, toxins and enzymes.

Some transposons (known as conjugative transposons) excise themselves from the bacterial chromosome to form circular intermediates of DNA, much like plasmids. These can also be transmitted around a population, again like plasmids, to spread resistance to antibiotics etc.

Pathogenicity Islands

Pathogenetic islands are also laterally-transmitted. They are large integrated DNA elements which carry multiple virulence-associated genes such as those which encode for adherence factors, secretion systems, toxins and invasion factors. A bacterium may have multiple pathogenicity islands and due to the incorporation of multiple virulence factors on one island, the transmission of one island often causes a non-toxogenic bacterium to become pathogenic.

They may be incorporated into the main bacterial chromosome or any plasmids which may be present in the cytoplasm, the DNA found within these islands may contain remnants from bacteriophages and IS elements. The GC-content (guanine-cytosine content) is often different from the rest of the genome.