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Antibiotics, Drug-Resistance and Alternatives
The increase in antibiotic resistance parallels the increase in antibiotic use in humans.
And antibiotic resistance is spreading fast! There are two reasons for it:
a) the increase in infection transmission and
b) inappropriate antibiotic use.
If a bacterial pathogen is able to develop resistance to an antibiotic, the substance becomes useless in the treatment of an infectious disease caused by that pathogen. That means we always have to develop new antibiotics not only against ‘old’ diseases but also against new bacterial diseases which we did not know several decades ago, like Legionnaire’s disease, gastric ulcers, Lyme disease and skin eating Streptococci.
ANTIBIOTICS are substances mostly produced by microorganisms that kill or inhibit other microorganisms. They are soil products and a by-product of secondary cellular metabolism.
The very first antibiotic, penicillin, discovered 1929 by Alexander Fleming, inhibited the growth of Staphylococci by a mould called Penicillium. Marketed in 1946, penicillin was used against infections caused by Staphylococci and Streptococci and was extremely successful against strep throat, pneumonia, septicaemia, skin and wound infections, scarlet fever and toxic shock syndrome. It had the ability to kill the bacterial pathogens without harming the host.
The first drug-resistance to penicillin in some strains of these bacteria was observed immediately after the introduction of the drug. Nowadays the resistance to penicillin happens in 80% of all strains of Staphylococcus aureus.
In the late 40s and 50s streptomycin, chloramphenicol and tetracycline was discovered and it was the age of antibiotic chemotherapy. These antibiotics were successful against many bacterial pathogens and even some intracellular parasites.
Some bacteria always showed an inherent (natural) resistance to certain antibiotics, others which previously were sensitive to antibiotics can develop a resistance. This mostly happens through HORIZONTAL EVOLUTION: the acquisition of genes for resistance from another organism. This happens in nature through conjugation, transduction and transformation.
More people are contracting infections. Sinusitis among adults and ear infections among children is on the rise. Why? Doctors prescribed always antibiotics to treat these infections, although most of the times the illness is viral and antibiotics do NOT work against a virus.
Of course some people clearly need to be treated with antibiotics but the majority of experts are concerned about the inappropriate use of these powerful drugs.
Other factors of resistance: increased use of day-care facilities, immunosuppressant drugs during chemotherapy and organ transplants, breakdown in public health measures (closing down of hospitals, reduction in prevention programmes, ready-meals in hospitals) and antibiotic use in livestock.
Another very important factor in drug resistance is that patients often stop taking the drug too soon, because symptoms improve. This is the most typical way to create drug resistance. If the infection returns a few weeks later, a different drug must be used for treatment.
We have now reached the dangerous point where bacteria show increased resistance against Vancomycin, the last drug which works against hospital-acquired staph infections (first reported in 1987).
This 2005 photograph depicted a cutaneous abscess on the knee of a prison inmate, which had been caused by methicillin-resistant Staphylococcus aureus bacteria, referred to by the acronym MRSA.
S. aureus bacteria are amongst the populations of bacteria normally found existing on ones skin surface. However, over time, various populations of these bacteria have become resistant to a number of antibiotics, which makes them very difficult to fight when attempting to treat infections where MRSA bacteria are the responsible pathogens. These antibiotics include methicillin and other more common antibiotics such as oxacillin, penicillin and amoxicillin.
Staph infections, including MRSA, occur most frequently among persons in hospitals and healthcare facilities such as nursing homes and dialysis centers, who have weakened immune systems, however, the manifestation of MRSA infections that are acquired by otherwise healthy individuals, who have not been recently hospitalized, or had a medical procedure such as dialysis, or surgery, first began to emerged in the mid- to late-1990's. These infections in the community are usually manifested as minor skin infections such as pimples and boils. All pictures CDC, Atlanta
HOW DOES DRUG RESISTANCE WORK?
Some species of bacteria have low permeability membrane and are thereby “intrinsically” resistant to many antibiotics. They are selected out in the multitude of antibiotics present in the hospital environment and thus caused many hospital acquired infections. Some strains of originally antibiotic-susceptible species may also require resistance through decreases in the permeability of membrane barriers. Another mechanism to prevent access of drugs to target is the membrane associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with permeation barrier.
Antibiotics have been highly effective and the general population now expects that any bacterial infection will be cured easily by one of these agents. The emergence of resistance bacteria is changing this situation dramatically.
Resistance bacteria are of two kinds: First, the constant presence of antibiotics in the hospital environment has selected out the unaltered strains of those species that may not possess strong virulence but are intrinsically resistant to a number of antibiotics. These include Pseudomonas aeruginosa and Enterococcus species which infect debilitated patients in hospitals as “opportunistic pathogens”. Second, there are those bacterial species that are well known for their pathogenicity. Many of these “professional pathogens” used to be exquisitely susceptible to antimicrobial agents. But many years of antibiotic usage have selected out drug-resistant strains which either contain alterations in their chromosome or have acquired resistance plasmids (R plasmids) or resistance-conferring transposons from another organism.
Bacteria utilise several ingenious mechanisms to develop resistance, including degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug target. These mechanisms are all quite specific for a single drug or a single class of drugs. BUT there are more general mechanisms of drug resistance in which access of the unaltered agent to the target is prevented by the barrier and active transport functions of the biological membranes. Thus, an organism can surround itself with a barrier of low permeability in order to decrease the influx of the drug into the cell and can also pump out the drug in an energy-dependant fashion.
The major permeability barrier in any membrane is the lipid bilayer structure and its barrier property is inversely correlated with its fluidity. It is not possible to make the cytoplasmic membrane much less permeable because this would require decreasing the membrane fluidity and interfering with proper functioning of the membrane proteins. Thus, some bacteria protect themselves by constructing an additional structure that surrounds the cell, outside the cytoplasmic membrane.
Most Gram-positive bacteria are surrounded by a thick peptidoglycan cell wall. This structure, although mechanically strong, appears to offer little resistance to the diffusion of small molecules, such as antibiotics, because its meshwork is too coarse. In contrast, Gram negative bacteria like E. coli surround themselves with a second membrane, the outer membrane, which functions as an effective barrier.
The outer leaflet of the outer bilayer is composed of an unusual lipid, lipopolysaccharide (LPS) rather than the usual glycerophospholipid found in most other biological membranes. Fatty acid chains present in LPS are all saturated. Because unsaturated fatty acid residues make the interior of the lipid bilayer fluid by preventing the right packing of the hydrocarbon chains, the absence of unsaturated fatty acid is expected to make the interior of the LPS leaflet much less fluid. In addition, an LPS molecule contains six or seven covalently linked fatty acid chains in contrast to the glycerophospholipid, that contains only two fatty acid residues…
The vast majority of clinically important antibiotics and chemotherapeutic agents show some hydrophobicity, which allow them to diffuse across the lipid bilayers of the cytoplasmic membrane (Prominent exceptions include fosfomycin and aminoglycerides such as streptomycin). Clearly, the LPS-containing asymmetric bilayer of the bacterial outer membrane serves as an efficient barrier against rapid penetration by these lipophilic antibiotics and chemotherapeutic agents. Bacteria,surrounded by these effective bilayers must have a special way to bring in nutrients. For this purpose the outer membrane has special proteins, PORINS, which produce non-specific aqueous diffusion channels across the membrane. The properties of the porin channels contribute to the exclusion of antibiotics in several ways: In E. coli the most constricted portion has an opening of only 7-1o A(Angstroem), which makes the influx of many antibiotics nearly impossible or extreme slow. Furthermore the constriction is lined with a number of charged amino acids residues, which orient the water molecules in a fixed direction. This makes entrance of lipophilic molecules into the constriction difficult, because it disturbes this energetically favourable orientation of water.
In spite of this arrangement, relatively hydrophilic agents, such as certain semi-synthetic Beta-lactams can readily penetrate through the porin channels of enteric bacteria. A different strategie is adopted by a Gram negative soil bacteria P. aeruginosa. This organism totally lacks the ‘classical’ high permeability porins that are present in most other Gram negative bacteria and is left only with a low efficiency porin that allows the diffusion of small molecules at about one hundredth the rate through the classical porin channels. Thus, hydrophilic antibiotics diffuse across the outer membrane of this organism only very slowly and the organism is thus ‘intrinsically resistant’ (resistant without any chromosomal mutation or the acquisition of an R plasmid) to a wide variety of commonly used antibiotics. For essential nutrient uptake, P. aeruginosa’s outer membrane contains of special channels, each facilitating the diffusion of a specific class of compounds. Mycobacteria, Gram-positive bacteria phylogenetically remote from P. aeruginosa, have adapted a similar strategy of surrounding with a barrier of generally low permeability. These bacteria are again intrinsically resistant to most antibiotics and pose a major public health problem. The mycobacterial barrier also appears to consist of a lipid bilayer of unusually high order and thus presumably low fluidity. As with the outer membrane of Gram negative bacteria, one of the leaflets consist of an unusual lipid. The fluidity seems to be even lower than in LPS, because the main fatty acid in these mycobacterial cell wall is mycolic acid which contains more than 70 carbon atoms with only a few double bonds. Hundreds of mycolic acid residues are covalently linked to a common head group, an arabinogalactan polysaccharide which in turn is covalently linked to the underlying peptidoglycan structure. The influx is apparently carried out by the mycobacterial porin , which is present in very small amounts and allows only very slow diffusion of small molecules through the channels. The low permeability of both, the lipid matrix and the porin results in the very slow penetration by the antibiotics. The diffusion rate of beta-lactam antibiotics, for example, is even slower than in P. aeruginosa.
Even organisms normally surrounded by a cell envelope of relatively high permeability can develop resistance by decreasing the permeability of the envelope. When an agent mainly diffuses across the barrier through a specific channel, mutational loss of this channel can be an efficient mechanism of resistance. A non-classical beta-lactam compound, imipenem, shows an exceptional activity against P. aeruginosa mainly because this agent diffuses through a specific channel, OprD, whose physiological function appears to be that of a transport of an amino acid. But this meant P. aeruginosa could become resistant to imipenem by just loosing this channel. (which really happens)…
In a similar manner, beta-lactam compounds designed to mimic iron-chelating compounds during their transports through the outer membrane are known to select mutants that are defective in these specific transports.
Surprisingly, mutants with decreased expression of porins are not very resistant to certain antibiotics. F.e. in E.coli, the minimal inhibitory concentration (MIC) for tetracycline increasing by only 50% after the loss of porin and that for cefotaxime (a beta-lactam compound only slowly hydrolysed by the beta-lactamase commonly found in Gram negative bacteria) by only 100%.
This occurs, because even the most effective permeability barrier cannot shut out the influx of small molecules. Experimental data show that the half-equilibration time for hydrophilic beta-lactams across the outer membrane is less than 1 second in E. coli. Typhus, decreasing the outer membrane further, the drug is still able to penetrate the cell within minutes (rapidly enough to exert its action). Similarily, even the low permeability outer membrane of P. aeruginosa cannot prolong the half-equilibration of most hydrophilic antibiotics beyond several minutes.
All this (the half equilibration time) suggests, that there must be a second contributor in addition to the permeability barrier! E. coli does not inactivate tetracycline and its hydrolyte cefotaxime only slowly; for these agents the second contributor is effectively absent, and thus decreased outer membrane permeability has little effect on MIC. In contrast the hydrolytic inactivation of the agents by beta-lactamase located in the periplasm (the space between the outer and cytoplasmic membrane) function as an effective second factor for redily hydrolized beta-lactams, such as cephatholin in E. coli. The two contributors, the outer membrane and beta-lactamase, work together synergistically because under this condition the enzyme can produce significant resistance by hydrolysing only the small number of beta-lactam molecules that slip through the outer membrane barrier. When the hydrolysis occur efficiently, the permeability barrier becomes a limiting step in the overall degradation of drugs by intact bacteria, and thus decreases in porin levels have pronouncing effects on MIC.
Resistance by the active pumping out (transmembrane efflux) of noxious agents began to attract scientists in the 1980s/ S. Levy and co-workers showed that plasmid-coded tetracycline resistance of e-coli is based on energy-dependent efflux. This was followed shortly afterward by the demonstration that the plasmid-coded cadmium resistance of Staphylococcus aureus was also based on an efflux mechanism.
Active grug efflux systems can be divided into four (4) different families:
1.
Major facilitator (MF) family, which shows sequent homology to the glucose facilitators of mammalian cells and also includes drug efflux proteins of eukaryotic microbes.
2.
Resistant nodulation division (RND), which also includes transporters that pump out cadmium, cobalt and nickel ions.
3.
Smr, Staphylococcus multidrug resistance family, consisting of small transporters that presumably contain on four transmembrane helices
4.
ABC (adenosine triphosphate) binding cassette transporters which are composed of two transmembrane and two ATP binding subunits or domains.
Each member of the first three families is a single cytoplasmic membrane protein that extrudes drugs by using proton-motive force. There is little sequence homology between MF and RND proteins although both are composed of two homologue halves each usually containing six transmembrane alpha helices…
It is increasingly recognized that active efflux plays a major role in the resistance of many organisms to many agents.
Streptomyces coelifactor protects itself from the methylenomycin it produces a number of antibiotic efflux genes have been identified. (Some MF family and some ABC family.) Haemophilus influenzae contains a homologous chromosomal gene and thus the non-enzymatic resistance of this species is very likely due to active efflux.
An exciting development in this field has been the discovery of bacterial efflux systems that can handle a wide variety of drugs, reminiscent of the mdr system in mammalian cells. Systems such as QacA, Smr, QacE or McvR pump out quaternany amine compounds as well as basic dyes and are often called multidrug efflux systems. However the substrates of these systems are at least physically similar, being amphiphilic molecules with positive charges. NorA in S. aureus turned out to be a Bmr homologue was shown to pump out cationic dyes, puromycin and chloramphenicol, solutes that are unrelated not only in chemical structure, but also in physical properties.
The acrA mutation of E. coli K12 which had been thought to produce drug hypersusceptibility by increasing the outer membrane permeability, was shown to inactivate a multidrug efflux complex. An alteration at a chromosomal gene of E. coli, marRAB, also produces significant resistance to a wide range of antibiotics including fluoroquinolones, chloramphenicol, tetracycline and beta-lactams.
Efflux transporters are located in the cytoplasmic membrane and thus in Gram-negative bacteria maybe assumed to be pumped out into the periplasm. If so, the efflux does not make the bacteria more resistant, because it is still difficult to get rid of the antibiotics. (Because of the presence of the outer membrane barrier). One possibility is the presence of accessory proteins that occur together with many efflux transporters of both MF and MFD families. These proteins are thought to bridge the cytoplasmic transporter AND an outer membrane channel so that the drugs can be extruded directly into the surrounding medium rather than into the periplasm.
It is likely that these accessory proteins form complexes with some channel proteins in the outer membrane.
As mentioned, the low permeability of the outer membrane alone is not likely to produce clinically significant levels of resistance and a second contributor is needed. In many systems the efflux system is this contributor, but this does not mean that the outer membrane barrier is not important.
Because the intracellular concentration of any drug is a result of the balane between influx and efflux, it is likely that the slow influx of various agents through the low-permeability outer membrane makes efflux an especially effective mechanism in P. aeruginosa. That is, even if organisms with a high permeability outer membrane, such as E. coli, had an efflux machinery of similar efficiency, it would not produce a significant level of resistance unless the agent has the size or the structure that slows down its permeation through the outer membrane.
E.coli mutants produce smaller amounts of OmpF porin (which produce a larger channel and therefore plays a majpor role for the penetration of antibiotics). With the influx thus decreased, active efflux can create a much higher resistance.
Increased expression of efflux transporters often accompanied by the expression of the OmpF porin synthesis, may occur without any genetic alteration.
Because specific mechanisms of antibiotic resistance were thought to be more important, efforts to produce more effective antibiotics have usually involved modification of specific groups on antibiotic molecules in order to make them inert as potential substrates.
The intrinsic resistance to a wide variety of antibiotics seen in the important opportunistic pathogen P. aeruginosa, is indeed due to a combination of a multidrug efflux transporter and an effective permeability barrier AND increased expression of the efflux transporter … is the most probable cause of resistance.
This will be a bi challenge for scientists and the pharma-industry, because some of the multidrug efflux systems seem to pump out any amphiphilic compound.
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