The discovery of penicillin by Fleming in 1929 was a breakthrough that seemed to ensure recovery from bacterial infections, in particular S. aureus infections. Unfortunately, resistance was observed almost immediately after the “wonder drug” was released for public use. Ever since, the battle between man and bacteria has continued. However, in the last decade, an increasing numbers of S. aureus strains have appeared, especially the Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Staphylococcus aureus (VRSA). These strains were not common at one time and were basically found in a few hospitals. Nowadays, their spread is endemic. Not only hospitals have them but closed community institutions with elderly people and people with disabilities. Individuals with compromised immune systems are most at risk since they cannot defend themselves against the bacteria. Bacteria like S. aureus have established different levels of resistance, which are mostly based on genetic recombination and the acquisition of Resistance Transformation Factors (RTFs) onto their plasmids. Acquisition can come from other strains of S. aureus or other species of bacteria. It should be very clear that bacterial resistance is a threatening problem that has been caused by the overuse as well as the misuse of antibiotics like penicillin. In addition, antimicrobial products work the same way than antibiotics do, thereby creating a much larger environmnent for mutants to appear, especially when they are overused. Many research laboratories are now designing novel drugs that they hope will replace the failing antibiotics. The main strategy is to target specific components of the resistance pathways and engineer molecules that will interfere with these pathways at different levels.
The Evolution of Bacterial Resistance in Staphylococcus aureus: History, Mechanisms, and Remedial Strategies
In the United States alone, 500,000 patients get a staphylococcal infection every year. Staphylococcus aureus is found on the skin or in the nose of a healthy person. S. aureus infections vary in their gravity. They can go from minor skin infections such as pimples, boils, and cellulitis, even causing abscesses. Severe infections threaten lives: pneumonia, meningitis, endocarditis, Toxic Shock Syndrome, and Septicemia are a few examples of such infections. S. aureus belongs to the Gram-positive bacterial group. Gram-positive indicates that when stained with Gram’s stain, the characteristic color of the cells taking up the dye will be violet blue (Gram-negative gives a red color). It is called a coccus because it is spherical in shape. In fact, it looks like grape-like clusters when viewed under a microscope. When the cells are grown on blood agar, large golden yellow colonies are observed, hence the name “aureus” from the Latin word for gold. The genome for the bacterium possesses approximately 2,600 genes, representing 2.8 million bp of DNA. Plasmids may also be counted as part of its genome. The basic ways an infection can spread is through skin contacts with an infected individual, contact through an infected open wound, as well as contacts with contaminated objects such as clothing, sheets, towels, and even athletic equipment. S. aureus can survive on domesticated animals and is known to cause mastitis in cows. Fundamentally, human to human contacts are the most worrisome reason why a S. aureus infection can spread: nocosomial contaminations and infections can affect different individuals, from babies to older adults, all having a weaker immune system, therefore, being at risk for a potential deadly outcome. (Staphylococcus, 1-3)
Penicillin’s effect on Staphylococci was discovered by Alexander Fleming in 1929 when his agar plate with the cocci became contaminated with a Penicillium mold. By 1946, penicillin became available and proved very effective in killing staphylocci as well as streptocci. The remarkable characteristic of penicillin was its selective toxicity, meaning that the drug was able to kill the bacteria but not the host. At that point, the majority of the medical establishment and the public were convinced that penicillin was a “wonder drug”. Unfortunately, resistance to the antibiotic became apparent a short while after its introduction. Around the late 1940s and into the 1950’s other drugs were discovered: streptomycin, chloroamphenicol, and tetracycline. Even though these drugs had wide spectrum efficiency against both Gram-positive and Gram-negative bacteria, it was short-lived. After a Shigella outbreak in 1953 Japan, these drugs were found to have become useless because this bacterium species was resistant to all of them. By 1950, 40% of hospital S. aureus isolates were resistant to penicillin. By 1960, the percentage had risen to 80%. Scientists needed to understand how resistant strains of bacteria in general were coming into existence. In addition, it became necessary to develop a strategy to eliminate or at least lessen the bacterial resistance problem. (Chambers, 2001)
The basis for general microbial resistance relies on two main categories: a natural resistance and an acquired resistance. A natural resistance may arise due to the presence of a key gene causing the appearance of a resistant phenotype. Additionally, a naturally resistant bacterium may lack a transport system or a viable target to be hit by the drug. The other type of resistance “strategy” involves the bacterium undergoing either a spontaneous mutation and selection or an exchange of genes between strains and/or species. Conjugation (cell to cell contact as DNA is transferred from donor to recipient), transduction (viral gene transfer) by a phage, and transformation (new genes provided by a lysed cell) are the main ways bacteria genetically recombine their DNA. Usually, plasmids contain the Resistance Transfer Factors (RTFs) that confer resistance to a recipient strain or species. (Todar, 2006)
Antibiotic-resistant infections are dangerous because no drug can kill the infectious bacteria, clearly implying that mortality will be high, especially in individuals with compromised immune systems like AIDS patients, for example. One general reason for the emergence of resistant strains or species of bacteria, including Staphylococcus aureus, is the excessive use of antibiotics and their misuse for viral infections. Viruses are not susceptible to antibiotics. Furthermore, patients are told to take the drugs for a specific length of time. However, many people believe (falsely) that if they feel better, they can stop taking the antibiotics and “save some for later”. In that case, all the bacterial cells are not killed if a sufficient dose of antibiotics is not taken. Therefore, surviving cells may include mutants that are now drug-resistant. These mutants can then infect the host or infect someone else through contact with the host. Another danger is the contemporary overuse of antimicrobial products (hand and body soaps, kitchenware, clothes, pillows, baby toys etc…). The multiplication of these types of products creates the perfect environment for mutants for the same reason than that of the overuse of antibiotics.
Penicillins and cephalosporins were used against S. aureus. Their mechanism of action was to inhibit cell wall synthesis due to the beta-lactam central ring structure of the drug. However, S. aureus became resistant to penicillin by using a specific type of enzymes, the beta-lactamases, which opens the beta-lactam ring, rendering the drug useless. So, other drugs were needed to counteract these enzymes. One strategy was to create beta-lactamase-resistant penicillins, such as methicillin, oxacillin, for example, that are able to resist degradation by the enzymes. A second strategy was to synthesize drugs that could be used to bind to beta-lactamases irreversibly, thereby preventing the enzymes to catalyze the opening of the beta-lactam ring of penicillins and cephalosporins. Such drugs are amoxicillin and clavulanic acid. Yet, the bacteria were not out of options. As mentioned above, penicillin is involved in the interruption of cell wall synthesis since beta-lactam antibiotics are structural analogs of the modular components of peptidoglycan. Penicillin Binding Proteins (PBPs) are present in bacterial cells in large numbers and are involved in the final steps of the synthesis of peptidoglycan, the major component of bacterial cell walls. The mechanism is based on the structural analogy of penicillins with modular components of the peptidoglycan. The PBPs bind penicillin irreversibly, inactivating the enzyme. Inhibition of the PBPs triggers the formation of defects within the wall, weakening the cell to lysis and death. Besides using beta-lactamases, S. aureus and other bacteria overcame the inactivation of the PBPs by overproduction and mutation of the enzymes to a lower affinity for penicillins. The resulting resistance then produced MRSA (Methicillin Resistant Staphylococcus aureus, Methicillin is a penicillin derivative) and subsequently, VRSA (Vancomycin Resistant Staphylococcus aureus). Vancomycin is a totally different type of antibiotics. Essentially, it is a glycopeptide that inihibits peptidoglycan synthesis. As discussed previously, genetic recombination allows the bacteria to modify or produce enzymes to combat the drugs. Resistance to Methicillin occurs by the acquisition of mecA, which codes for an altered PBP that has a lower affinity. Resistance to glycopeptides is caused by the acquisition of the vanA gene. Interestingly, the vanA gene originates from another species of bacteria (enterococci) and codes for an enzyme that produces an altered peptidoglycan that vancomycin is unable to bind to. (Blot, Vandewoude, Hoste, Colardyn, 2002; Chang et al., 2003; Walsh, Howe, 2002)
The extent of the resistance problem is increasing. In the past few years, the decrease of vancomycin susceptibility has been prevalent in many epidemic lineages. In fact, reduced vancomycin susceptibility is found in most MRSA lineages as well, amplifying the resistance problem. From a genetic standpoint, the emergence of “distinct pandemic clones” has the potential to lead to more MRSA infections, rendering them almost impossible to treat. S. aureus infections have been reported in hospitals all over the world (U.S. included), in closed communities like institutionalized elderly people and people with physical or mental disabilities. Food-borne infections with S. aureus have been seen as well. (Enright, Robinson, Randle, Feil, Grundmann, Spratt, 2002)
Many academic research laboratories are searching for ways to lessen or stop the resistance problems, with S. aureus and most bacteria. Modern research aims at producing novel molecules to oppose bacterial resistance measures. This implies that drug design is essential to find new drugs that will evade resistance mechanisms. In addition, progress in one bacterial strain may help have more options for another strain or species. The following two publications are the best examples of what biochemists are doing to combat bacterial resistance. Pratt and colleagues (2003) have worked on the design of brand new molecules: the aryl malonamates. The strategy employed was to design isomers of natural substrates for the bacterial enzyme and modify certain structural elements to produce compounds able to inhibit it (the enzymes in this study belong to P99 Enterobacter clocae). (Cabaret, Adediran, Pratt, Wakselman, 2003) Other targets needed to be hit. Pratt and colleagues (2004) designed compounds aimed at inhibiting a peptidoglycan peptidase, an enzyme involved in cell wall synthesis, by using a novel beta-lactam mimetic of a peptidoglycan component. (Josephine, Kumar, Pratt, 2004) In this example, the beta lactam structure is further modified to mimic elements of the peptidoglycan layer, in this case targeting side-chains. These strategies are very hard to work out and may take years to complete. However, more structural studies of key inhibitory components of S. aureus and others may give more ideas on how to create inhibitors.
Strategies need to be implemented to prevent these infections in the first place. Good hygiene is a very simple idea but it has always been effective. The use of antibiotics should be very strictly regulated and the public should always be educated as to how they should take their medication. Finally, as resistant strains are increasing for S. aureus, combining the MRSA lineages with vancomycin/glycopeptides resistance, more research must be done to design new drugs to combat the evolution of resistance.