Antibiotics are drugs that have been developed to destroy or show interference with the growth of microorganisms. The first antibiotic properties revealed were synthetic chemicals, particularly drugs containing arsenic, which were discovered by Ehrlich at the beginning of the 20th century. Sulphonamide inhibitors of folate metabolism were developed by Domagz in the 1930s which then followed with the development of the first true antibiotic, penicillin in the 1940s. (Wright 2011) Upon this discovery, production and distribution of antibiotic drugs increased and within five years antibiotic resistance was distinguished.
Not only do antibiotics help save lives but incorrect use of antibiotics exposes the microbes to the drug where a small number of bacteria are able to adapt and survive, producing antibiotic resistant microorganisms. Evidence suggests that the main cause of this problem is the misuse and over prescription of antibiotics both in humans and livestock animals. Majority of fatal nosocomial infections are caused by antibiotic resistant bacteria. Mechanisms A bacteria may produce a resistance gene by inheriting a mutilated gene or through gene exchange. Homer, Ritchie-Dunham et al. 2000) Bacteria that produce antibiotics under external antibiotic pressure and in absence of resistance mechanisms, undergo biological stress developed from membrane/cell wall damage, threatened protein synthesis or transformed DNA supercoiling. The increase of biological stress is known to cause mutations. (Baquero and Blazquez 1997) Target modification is an important mechanism relating to resistance. Multiple mutations may increase the level of resistance while single mutations deliver a high level of resistance.
The change in the amino acid sequence modifies the structure of the protein enough to interfere with antibiotic binding and action. Target modification can also occur through highly efficient and region specific modification catalysed by enzymes. An example of this method is ribosome methyltransferase. The ribosomal ribonucleic acid (rRNA) resistance gene, erythromycin-resistant methylase (Erm) transforms the 23S rRNA of the ribosome creating resistance to 3 different classes of antibiotics; macrolides e. g. erythromycin, lincosamides eg. Clindamycin and type B streptogramins e. g. quinupristin.
Although these antibiotics differ in molecular structure, they all bind to a specific site on the peptide exit tunnel of the large subunit of the ribosome and block the antibiotic binding site. Another important mechanism is chemical modification, this was first recorded in 1940 with the discovery of penicillin which was found to inactivate ? -lactamase activity. ?-lactamase enzymes are considered the most important and widespread resistance enzyme. Chemical modifications operate by forming a covalent enzyme intermediate followed by hydrolysis, or metal-activation of a nucleophilic water molecule. Wright 2011)
Gram negative bacteria that produce extended spectrum ? -lactamase enzymes (ESBL) have been noted to be multidrug resistant pathogens that cause serious illness. (Lee, Bae et al. 2012) Microbes have also evolved to find ways to evade antibiotic action through molecular bypass such as resistance to glycopeptide antibiotics. The glycopeptide antibiotic binds to the acyl-D-Alanyl-D-Analine terminus of the peptidoglycan of the bacteria cell wall which is a substrate for enzymes that join nearby chains of peptidoglycan through a transpeptidation reaction.
Substitution of the amide bond of D-Ala-D-Ala with an ester linkage removes a donor H-bond and prevents binding of an antibiotic. (Wright 2011) Efflux was first defined as a mechanism of resistance to tetracycline in Escherichia coli, with the plasmid encoded single component protein export of tetracycline across the membrane but as further studies were conducted it was noted that numerous plasmid and chromosome encoded mechanisms exist and are vital determinants of resistance.
The five classes of bacterial efflux systems are the major facilitator (MF) superfamily, the ATP-binding cassette (ABC) family, the resistance-nodulation-division (RND) family, the small multidrug resistance (SMR) family and the multidrug and toxic compound extrusion (MATE) family. RND family transporters are frequently found in Gram-negative bacteria and usually operate as part of a tripartite system that contains a periplasmic membrane fusion protein (MFP) and an outer membrane factor (OMF).
This organisation is also seen on occurrence with ABC and MF family exporters. All members but the ABC family function as econdary transporters, catalysing drug-ion antiport. Efflux mediated resistance to biocides exhibit a wide range of substrate specificity. Quaternary ammonium compounds (QAC) have been defined in Gram-positive bacteria majority being in the Staphylococcus spp. Most of the determinants are plasmid-encoded SMR family exporters e. g. QacG, and QacH, although QacA/B is a MF family efflux system, through plasmid acquisition resistance occurs.
Chromosomal efflux determinants of QAC resistance although rare in Gram-positive, have been defined and consist of Staphylococcus aureus NorA multidrug transporter implicated in fluoroquinolone resistance. Poole 2005) Research developments Resistance to sulphonamides came to surface while treating the military in the 1940’s. The antibiotics used today are mainly synthetic derivatives of the core classes of antibiotics that were developed between the 1940’s and 1960’s. Although using derivatives may temporarily combat resistance it may also lead to a higher level of resistance to a certain class of antibiotics. As stated in the Review of antimicrobial resistance between May and August 2014, two new glycopeptides and one new oxazolidinone became available to tackle gram positive bacteria.
And in December 2014, two new cephalosporin/ beta-lactamase inhibitor combinations were licensed which have activity against selected Gram-negatives. Although producing more antibiotics may help the problem at present, it is not a long term solution as the bacteria will evolve to resist the new chemicals. Not only do we need antibiotics to help fight infection but we also need to reduce the effects of resistance. To do so we need to comprise strict policies and procedures on antibiotic use and prescriptions, veterinary use and agricultural use as well as educating the population on the importance of proper personal hygiene.
Sensitivity tests were developed and used to test antibiotic sensitivity/resistance including the antibiotic disc. By the 1960’s the Kirby-Bauer test was in place to determine the degree of resistance by measuring the zone around the disc. (Gradmann 2013) Tedizolid is a derivative of oxazolidinone that has shown activity against multidrug resistant gram positive organisms including Methicillin resistant Staphylococcus aureus (MRSA), Vancomycin resistant Enterococci (VRE) and penicillin resistant Streptococcus pneumoniae (including linezolid resistant strains).
Tedizolid binds to the 23S rRNA of the 50S subunit, avoiding formation of the 70S initiating complex and preventing protein synthesis. According to this study, tedizolid seems to be a potential contender for the treatment of multidrug resistant gram positive infections and has many other benefits including that the drug only needs to be administered once daily due to its long half-life and has shown no signs of interaction with serotonergic agents or any severe adverse effects. (Kisgen April 2014)
Biofilms are multicellular microorganism colonies that are occur on surfaces. They are known to cause over 50% of human infections and are problematic due to their resistant properties. Bacteria respond to environmental changes by activating the stringent response (SR). This response synthesises two signalling nucleotides, guanosine 5’ -diphosphate 3’ –diphosphate (ppGpp) and guanosine 5’ –triphosphate 3’ –diphosphate (pppGpp). These nucleotides act as second messengers, are induced by stress and are thought to be involved in the formation of biofilms in certain species.
Synthetic cationic peptides have been recognized to show inhibition against the pppGpp nucleotides in biofilms by blocking the stress response that is responsible for biofilm development. The study uncovered that the peptide, innate defence regulator (IDR)-1018 can be used as a broad-spectrum anti-biofilm agent as it specifically targets and kills already existing biofilm cells and prevents biofilm formation in Gram positive and Gram negative species.
Overproduction of pppGpp was achieved by adding an analogue of L-serine which induced the stringent response by inhibiting charging of seryl-tRNA synthetase and by induction of the cloned RelA gene. (de la Fuente-Nunez 2014) Phage therapy is a technique that has been studied for many years; the CRISPR/Cas system was discovered after recent studies of bacterial phage-resistant mechanisms and is able to modify/edit gene sequence. Clustered, regularly interspaced, short palindromic repeats/CRISPR-associated systems protect microbes and act like an immunity memory for prokaryotes.
Bacteriophages have been isolated from every bacterial environment that exists and is believed that at one or more type of bacteriophage infects every strain of bacteria. The main interest in the defence against resistance is using lytic phages as they will kill the host rather than mix with them. CRISPR/Cas system is used to re-sensitise the bacteria to the drug by targeting a specific sequence. (Keen 2015) The latest drug to be produced claiming to avoid developing resistance is called teixobactin.
Teixobactin prevents the formation of the cell wall by binding to a conserved type of lipid II, a precursor of peptidoglycan and lipid III, a precursor of cell wall teichoic acid. As most antibiotics are made by using bacteria found in soil, it is difficult to mimic their natural environment in laboratory testing. This study explains how they used a multichannel device (iChip) to simultaneously isolate and grow uncultured microbes. As soon as a colony was formed, a number of uncultured isolates were able to grow.
Extracts from the isolates were screened for antimicrobial properties on plates cultured with S. aureus, an extract from a new species was found to show good activity. The genome was then sequenced based on DNA/DNA hybridisation and reported to belong to a new genus related to Aquabacteria. A molar mass of 1242Da was determined by mass spectrophotometry but was not located in the databases available. The compound was then isolated and officially named teixobactin. This drug showed remarkable activity against Gram positive pathogens including strains known to be multidrug resistant.
Teixobactin showed to have no toxicity against mammalian cells tested, and showed no haemolytic activity. No mutant cells were formed when plating low doses. It was noted that teixobactin strongly repressed the production of peptidoglycan and is now the first member of a new class of antibiotics called the class of lipid II binding antibiotics. (Ling, Schneider et al. 2015) Although this drug sounds promising, this alone will not prevent future resistance as everything evolves to adapt and survive.