What is MRSA?
MRSA is a methicillin resistant form of the bacterium Staphylococcus aureus. In some ways this name is a little misleading because while it is resistant to methicillin, MRSA can become resistant to almost all antibiotics including penicillin, fluoroquinolone and vancomycin. Staphylococcus aureus is a gram-positive rod-shaped bacterium that is commonly found on/in humans and animals. It can be commensal or pathogenic with its pathogenic forms using protein toxins to lead to diseases such as pneumonia, meningitis and more.
What is antibiotic resistance?
Antibiotic resistance is a way in which bacteria render antibiotics ineffective through some form of change. These changes can range from differing protein shape/structure, changes in DNA sequence/interaction, changes in metabolic pathways and many more. Antibiotic resistance comes about when a bacterium has a random mutation that allows the bacterium to resist the effects of an antibiotic better than other bacteria which do not have that mutation. If this bacterium is exposed to a sublethal dosage of an antibiotic it will continue to live and be able to pass its mutated resistance gene(s) into future generations. After multiple occurrences of this phenomenon some antibiotics could be ineffective at killing the resistant bacteria.
History of MRSA and how it became resistant to varying antibiotics:
When Alexander Fleming accidentally stumbled upon the antibiotic penicillin in 1928, doctors began using the drug to cure a variety of diseases. It has been estimated that by the year of 2000 the discovery of penicillin has saved over 200 million lives (Alexander Fleming). In the years before penicillin, infections from bacteria such as Staphylococcus aureus had a fatality rate of 70-80% (Lowy, 2003). With penicillin being termed a “miracle drug”, doctors began to prescribe the drug for any kind of sickness (Lowy, 2003). This, however, lead many bacteria to evolve mechanisms in which they could successfully live and reproduce in the presence of the penicillin. Even as early as 1942, doctors began to recognize penicillin resistant bacteria and by 1960 more than 80% of staphylococci infections had penicillin resistance (Lowy, 2003). Penicillin works by permanently binding to penicillin binding proteins (PBP), also known as DD-transpeptidase (Goffin and Ghuysen, 2002). The active site of PBP works to cross link peptidoglycans (a key part of the cell wall of gram-positive bacteria) by using a serine to facilitate a nucleophilic substitution that ultimately links two separate peptidoglycan monomers with a D-alanine leaving group (Goffin and Ghuysen, 2002). Penicillin works by having the active site serine perform a nucleophilic attack on the ketone group in the β-lactam ring (an irreversible reaction) and rendering PBP dysfunctional (Lowy, 2003).
Penicillin resistance is mediated by the blaZ gene which encodes for a β-lactamase (enzyme that denatures β-lactam rings) (Lowy, 2003). In MRSA this β-lactamase is called penicillinase because it specifically denatures penicillin (Lowy, 2003). Penicillinase works by hydrolyzing the β-lactam ring, which is done by allowing water to perform a nucleophilic attack on the ketone in the ring (Lowy, 2003). The blaZ gene is controlled by the two other genes BlaR1 and BlaI, blaR1 codes for an anti-repressor while the BlaI gene encodes for a repressor of BlaZ operon (Lowy, 2003). When the BlaR1 is exposed to a β-lactam it cleaves itself which then allows for it interfere with the BlaI repressor and activate the BlaZ operon. This allows for the BlaZ gene to make penicillinase and ultimately denature penicillin (Lowy, 2003).
Image depicting the process of Penicillin Resistance. Provided by (Lowy, 2003)
Mechanism for the formation of Peptidoglycans. Provided by (DD-Transpeptidase, Wikipedia.com)
Methicillin is a β-lactam antibiotic that is within the penicillin family tree (Methicillin). It was first synthesized in 1959 to counter the effects of Staphylococcus bacterium infections that were resistant to penicillin. By 1961 scientists realized that many of these bacteria had also become resistant to methicillin (Lowy, 2003). With the discovery of these new kinds of bacteria scientists noticed the mortality rates of bacterial infections to slowly start increasing. In a study done in 2002, scientists found a significant increase in mortality rate in people with MRSA when compared to the non-resistant form of the bacteria (Cosgrove et al. 2003). Methicillin resistance is attained using the mecA gene (Lowy, 2003); The mecA gene is “part of a genomic island designated staphylococcal cassette chromosome mec” (SCCmec) which are genes that encode for multiple proteins that lead to the production of penicillin-binding protein 2A (PBP2A) (Lowy, 2003). PBP2A is a type of PBP that has a very low affinity for penicillin-based antibiotics including methicillin that allows the bacterial cell to make peptidoglycan bonds even in very high concentrations of these antibiotics (Lowy, 2003).
Once bacteria became resistant to penicillin and many of its derivatives, scientists needed a new antibiotic to try and fight MRSA infections. In the 1980’s fluoroquinolone antibiotics began to be used because they used different mechanisms to kill the bacteria (Lowy, 2003). The alarming result from this was the ability for MRSA to readily to become resistant to these antibiotics (Methicillin). Although unknown exactly how it occurred, these MRSA bacteria were able to very quickly become resistant to fluoroquinolone-based antibiotics (Methicillin). Fluoroquinolone resistance occurs through primary sequence changes in topoisomerase II and topoisomerase IV (Lowy, 2003). Topoisomerase II works to stabilize DNA by not allowing it to over/under coil when in its double helix state (Aldred K. et al, 2014). Topoisomerase IV helps to stabilize DNA when DNA transcription is occurring by making staggered cuts every 4 base pairs and on opposite strands which relieves steric strain on the DNA strands (Aldred K. et al, 2014). DNA Topoisomerase also makes double stranded cuts of bacterial DNA if there is too much torsional strain on the DNA and then rebinds it when the helix unwinds to a stable state (Aldred K. et al, 2014). Fluoroquinolone takes advantage of these two enzymes by making them overcut the DNA in the bacterial genome (Aldred K. et al, 2014). Fluoroquinolones interact with the serine, Mg2+ and glutamic acid in the active site to increase the stability between DNA and the active sites of the topoisomerases (Aldred K. et al, 2014). This in turn increases the steady state concentration of cleavage complexes and leads to irreversible DNA damage (Aldred K. et al, 2014). Although not fully proven, most experts agree that primary sequence changes in topoisomerase II, VI in MRSA have led to fluoroquinolone not being able to non-covalently bind with the active site and therefore not kill the cells (Aldred K. et al, 2014).
The final form of antibiotic resistance I will discuss is Vancomycin, which to current date is our most effective and one of our last resorts in trying to fight MRSA infections. Vancomycin is known as an empiric antibiotic (an antibiotic used by doctors when they are not sure what kind of bacterium is causing an infection) because of its ability to effectively kill a wide variety of gram-positive bacteria. Vancomycin was first used in the 1950’s after many bacteria had developed resistance to penicillin (Levine, 2006). This antibiotic was only used as a last resort because it commonly leads to allergic reactions and is toxic to the body if administered incorrectly (Levine, 2006). After being substituted for other antibiotics because of its perceived toxicity, vancomycin saw a lowered use from 1960 to 1980 (Levine, 2006). In the beginning of 1980’s vancomycin use increased dramatically because it was once again seen as one of the only effective ways to kill MRSA (Levine, 2006). In 1997, however, scientists in Japan isolated the first case of MRSA resistance to vancomycin (Lowy, 2003). Shortly after, many scientists around the world had confirmed that vancomycin resistant MRSA was common in many healthcare settings (Lowy, 2003).
Vancomycin kills bacteria by blocking cross-linkage bonds between peptidoglycans. Unlike penicillin, Vancomycin binds to the peptidoglycans to block the cross linkage instead of targeting the protein that forms them, as seen in penicillin-based mechanisms (Akul, 2011). The vancomycin binds to N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) by creating hydrogen bonds with its D-alanines; these D-alanines are crucial leaving groups in the formation of peptidoglycan bonds (Akul, 2011). As autolysins (natural proteins in bacteria that break peptidoglycan bonds, so the cell can grow) come in they break peptidoglycan bonds allowing vancomycin to bind to more D-alanines, this complex sterically blocks DD-transpeptidase from forming bonds and rebuilding the cell wall (Akul, 2011). Ultimately, the cell loses its structural integrity due to the lack of a sturdy cell wall and cell contents bursts through the cell membrane killing the cell.
MRSA has formed two known methods in which it can resist vancomycin. The first kind is called vancomycin intermediate-resistant S. aureus (VISA) whom have thicker cell walls and therefore more peptidoglycan (Lowy, 2003). More peptidoglycan existing within the cell increases the amount of vancomycin needed to block peptidoglycan linkages. A minimum inhibitory concentration of 8-16 ug/ml of vancomycin is needed to kill VISA cells (Lowy, 2003). The second kind of resistance stems from a vanA operon gene that originally came from Enterococcus faecalis (Showsh, 2001). This operon codes for peptidoglycans that have a D-lactate as the leaving group instead of a D-alanine (Lowy, 2003). This stops the vancomycin from binding to the peptidoglycan because it can no longer form hydrogen bonds. This allows the cell to live in extremely high concentrations of the antibiotic with it living at levels greater than MIC>128 ug/ml (Lowy, 2003).
Mechanism for Vancomycin. Provided by (Vancomycin, Wikipedia.com)
References can be found here