Solving the Puzzle of the Super Bug

Solving the Puzzle of the Super Bug

"We have applied the latest genome sequencing technology to show that Staph can readily become vancomycin (antibiotic) resistant by acquiring a single mutation in its DNA. When the bacteria mutate, they are reprogramming themselves, changing their cell walls to resist the action of our antibiotics"-Dr. Stinear.

 

                The treatment of mild to serious infections from Staphylococcus aureus (Golden Staph) is severely hindered by the development of antibiotic resistance. This antimicrobial resistance is a major public health threat which is further worsened by the development of strains of Staph bacteria with resistance to strong antibiotics such as vancomycin and daptomycin, which are considered last line antimicrobials. Patients in hospitals are more susceptible to staph infections because their immune systems are already compromised.  In hospitals around the world, infections with methicillin-resistant Staph aureus (MRSA) continue to cause a significant number of unnecessary deaths.  Therefore developing treatments to fight staph resistant strains as well as reduce the number of cases in hospitals is a major topic of study.  Recently research has added a new piece to the puzzle of elucidating the mechanism by which Staph evades the immune response to develop resistance to these last-line antibiotics.  

Frighteningly so, a small number of clones of staph account for the large number of hospital acquired infections.  In Australia, multi-locus sequence type (MLST) 239 termed ST239 comprises the major clone MRSA and has been infecting patients for over 30 years.  Unfortunately this clone is resistant to almost all antibiotic types therefore the current treatment for such an infection is the strong antibiotic vancomycin. Generally speaking, vancomycin is only prescribed after treatment with other antibiotics has failed; therefore it is administered as a last resort. However, recently strains have evolved to develop a low resistance to this antibiotic as well.  These strains partially resistant to vancomycin are named vancomycin-intermediate S. aureus (VISA).  The genetics of these strains that enable them to resist vancomycin antibiotics are the topic of a recently published study in the journal PLoS Pathogens titled Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalkR.

Previously conducted research has suggested that many cases of VISA emerge from fully-vancomycin susceptible S. aureus (VSSA) parental strains during persistent infection or in some cases from the evolution of daptomycin non-susceptibility despite the fact that the patient is not exposed to daptomycin. Daptomycin is an antibiotic that exerts its effects on the cell membrane and the link to VISA is not well understood but has only recently been demonstrated. Preliminary research has also revealed that VISA strains are likely to appear due to sequential point mutations in key staphylococcal regulatory genes.  Phenotypic differences between VISA and the parental strain VSSA show increased cell wall thickness and reduce autolytic activity. Autolytic activity is the destruction of cells and tissues of an organism by enzymes produced by the cells themselves. It is known to occur widely in bacteria where it is needed for the hydrolysis of peptidoglycan, a component of the bacteria cell wall. The authors of this paper focused their attention on identifying the potential mutations in the walKR two-component regulatory locus involved in control of cell wall metabolism thought to be involved in the development of this phenotype. 

To identify the genetic mechanism underlying the development of the VISA strain, five pairs of VSSA and VISA were selected where the resistant VISA strain isolated was from the susceptible parental VSSA strain during failed vancomycin therapy.  All of the strains analyzed had been treated with vancomycin without daptomycin meaning that any changes in reactions to daptomycin were from vancomycin exposure only. All of the VISA strains showed increases in daptomycin minimum inhibitory concentrations (MICs). MICs are the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.  All of the clinical pairs were from Australia and New Zealand where ST239 MRSA accounts for the dominant hospital clone of MRSA in the region. Comparison of the JKD6009 and JKD6008, a VSSA and VISA pair of MRSA, revealed that there were 6 nucleotide substitutions in the JKD6008 of the VISA and the mutations were shown to be located in the sensor region of the graS gene which partially explains the reduced vancomycin susceptibility of the strain.  Using a read-mapping technique the authors examined genetic differences between the JKD6009 and JKD6008 which have been shown to be closely linked to the ST239 Australian strains of interest as well as compared the 4 other VSSA/VISA pairs.  The authors discovered a striking single nucleotide polymorphism (SNP) in the walk gene (walKR) of JKD6008. A SNP is a DNA sequence variation occurring when a single nucleotide is different between the two chromosomes.  Furthermore, 3 out of the 4 other clinical pairs also had single mutations within the walKR locus. 

The mutations in this locus were common to strains of S. aureus showing how the parental strain developed intermediate vancomycin resistance by acquiring mutations in a key regulatory region.  The mutations in this locus were found to also cause daptomycin non-susceptibility even though the strains were never exposed to this antibiotic. When the authors replaced the mutated walk or WalR into the parent strain using bi-directional allelic replacement experiments, the walk mutations from the VISA strain JKD6008 was introduced into the complementary parent VSSA JKD6009 generating TPS3130. This procedure was also performed on the other pairs of VSSA/VISA strains. From the data collected, they determined that the mutations were in fact sufficient to cause antibiotic resistance. The authors then examined the impact of the walKR mutations on the formation of characteristic VISA phenotypes. In particular they measured autolytic activity and cells wall thickness in the allele-swapped strains compared to their VSSA and VISA parents. Interestingly, these replacements led to reductions in autolytic activity, increases in cell wall thickness and regulation of metabolism in the organisms with the introduction of the walKR allele from the VISA strain into the VSSA parent. The opposite phenotypes were observed for the reverse substitution of VSSA walKR allele substitution into VISA strains.  

In addition, the authors performed a microarray analysis to explore the global regulatory effects of the walKR mutants compared to the parental strains. They discovered 73 genes that were up-regulated and 90 genes that were down-regulated in the walKR mutants compared to the parent strains. The mutations to the walKR region revealed a down-regulation of the major autolysin expression which could explain the changes in autolytic activity observed in WalKR mutants.  The authors found mutations in the walKR led to significant reductions in biofilm formation that mirrored changes seen in the VISA strains. The authors propose that there may be an absence of extracellular DNA  (eDNA) released in the formation of biofilm which is a result of autolysis. Therefore, maybe VISA strains which are deficient in autolysis may be deficient in biofilm production.  Also the authors found an up-regulation of genes responsible for pyrimidine biosynthesis which could explain the observed increase in cell wall thickness and decreased capsular polysaccharide in walKR mutant strains.

The study highlights the high adaptability of Staph in the presence of antimicrobial treatment. Above all, it suggests that there are more pieces of the puzzle that need to be solved in order for us to improve the way we use antibiotics to treat bacterial infections. Single nucleotide changes in S. aureus are capable of producing dramatically altered bacterial behavior and antimicrobial resistance.  Mutations in the regulatory locus, walKR, reveal a common mechanism for the evolution of multi-drug resistance in these bacteria which leads to daptomycin cross-resistance and impacting the virulence of the pathogen.  The study shows the sensitivity of this pathogen to single mutations and therefore stresses the importance of developing therapeutic strategies that avoid potentially impacting the organism to induce mutations in this locus which could increase its virulence. 

References

Howden BP, McEvoy CRE, Allen DL, Chua K, Gao W, et al. (2011) Evolution of Multidrug Resistance    during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR. PLoS Pathog 7(11): e1002359. doi:10.1371/journal.ppat.1002359