The Cold War was a tough time to live in. However, unlike our society where such tension tends to ultimately resolve (peacefully or not), in the microbial world, it’s a constant tug-of-war. The study by Koch et al (2014) on Staphylococcus aureus shed some light on the mechanism of this microbial arms race and its consequences.
Staphylococcus aureus, first discovered in 1880, is a gram-positive cocci (Masalha et al., 2001). Though S. aureus is usually innocuous, most strains can cause severe infections in bone and soft tissue, and ultimately lead to fatal diseases and complications. In addition, S. aureus infections can be difficult to treat due to strong resistance to antibiotics like methicillin (Kreiswirth et al, 1993). These strains are known as methicillin-resistant S. aureus (MRSA) and are often categorized by means of acquisition, for example, from community (CA-MRSA) or hospitals (HA-MRSA), due to its epidemic nature (Gardete and Tomasz, 2014). Now, MRSA is one of the most lethal infectious agents with a fatality rate of 20%, and is responsible for more deaths in the United States than HIV (Klevens et al., 2007).
Vancomycin and Vancomycin-intermediate S. aureus (VISA)
Vancomycin is an antibiotic often used to treat MRSA. Vancomycin (red) binds and inactivates precursors (light green), which are broadly located in the peptidoglycan (PG) layer (light blue), at the septum--the site of new PG synthesis (Pereira et al, 2007). S. aureus can acquire resistance by gaining mutations in cell wall synthesis regulatory genes that thicken its cell wall (Atilano et al, 2010). With a thicker cell wall, it’s harder for vancomycin to diffuse and reach cell wall-synthesis sites (Koch et al, 2014). This resistant phenotype is called Vancomycin-intermediate S. aureus (VISA).
Spontaneous diversification of S. aureus under Mg2+-rich conditions
Studies have shown that chronic S. aureus infections occur frequently in locations with high Mg2+ concentration (Gunther, 2011). Therefore, Koch et al (2014) adopted tryptone soy broth (TSB) medium + 100 mM MgCl2 to culture CA-MRSA clinical isolate SC01. As shown in Fig. 3&4, they found that the original colony (O) would spontaneously differentiate into two strains, white (W) and yellow (Y). It is remarkable that W strain emerged first at day 2 while Y strain only appeared after day 3 (Fig 4). This delay suggests that W has some role to play in the development of Y.
As shown in Figure 5, O, W, and Y display distinct physiology and gene expression. Staphyloxanthin is a yellow pigment in O strain and Y strain. Koch et al (2014) used LAC, another clinical isolate of CA-MRSA, as control wild type C+, and LAC Δσ B mutant as C- (note that this is flipped in hemolysis in Fig 5: wild type is C- and Δσ B mutant is C+). σ B determines specific binding of RNA polymerase to DNA and directly regulates gene expression. W strain, unlike the O or the Y strain, has very high hemolytic toxin secretion and has phenotype similar to that of the LAC ΔσB mutant. Genome sequencing and other experiments confirmed that W strain, unlike O or Y strain, has hyperactive agr quorum sensing pathway (agr QS). A simplified pathway is shown in Fig 6. As O and Y strain are pigmented, σB has to be expressed to activate crt which encodes pigment production. This fact suggests that neither O or Y has activated agr QS pathway because the expression of σB inhibits agr QS pathway (Fig 6).
The advantageous agr QS pathway
The agr QS pathway provides great advantage to W (Fig 6). Agr QS up-regulates Psm, a type of surfactant that helps W to spread. In a spread assay, W formed a ring around the O colony and restricted the growth of O. Another important gene also up-regulated by agr is the bsa gene, which encodes toxins and antibiotics. Supernatants of the three cultures (O, W, and Y) were added to B. subtilis cultures to test antimicrobial activity. Supernatants from W inhibited the growth of B. subtilis, and this confirmed that agr QS offers W an edge in the competition for resources. Koch et al (2014) then hypothesized that W out-competes O using surfactants and toxins; in response, O generates a new strain Y, that stands a better chance winning the fight.
Y strain exhibit a VISA-like phenotype resistant to Bsa
The next questions would be: which part (if any), of the agr QS pathway, could exert inhibitory influence on O strain and shape the development of Y? If W strain is capable of fast dispersal and toxin production, can Y strain “out-run” W and resist these toxins? To answer these questions, Koch et al (2014) tested many different possibilities. After a few rounds of elimination, bsa seemed to be the most promising candidate in the tug-of-war between O, W, and Y. When the bsa gene was mutated, only W phenotype appeared and Y did not develop (Fig 7). However, when Bsa protein was added to O strain, Y still developed while W strain did not (Fig 8).
Bsa, encoded by the gene bsa, is an antibiotic that targets lipid II at the site of bacterial cell wall synthesis, the same precursor that Vancomycin acts on (Fig 2). To compare and contrast the effects of the two antibiotics, Bsa and Vancomycin had been added to the cultures of the three phenotypes, a VSSA strain and a VISA strain (Fig 9). It is obvious that the O strain resembled the VSSA strain in both Bsa and Vancomycin conditions: growth curves quickly plummeted to zero at comparatively low concentrations of Bsa and Vancomycin. More interestingly, the Y strain seemed very similar to VISA with significantly greater success tolerating higher concentrations of Bsa and Vancomycin. This resistance to both Bsa and Vancomycin was not found in O or W strain, suggesting that resistance is specific to Y strain.
These evidence indicated that Y acquires resistance to compete with Bsa-secreting W strain. Because Bsa and Vancomycin have similar targets and mechanisms of action, Y strain is fortunately resistant to both. Whole genome sequencing experiments have also detected gene mutations commonly found in clinical isolates of VISA, such as graRS and walKR, in Y strain; both genes regulates cell wall synthesis.
Conclusions, consequences and future directions
The above experiments focused primarily on the relationships between spontaneously differentiated phenotypes of Community-associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) and the evolution of Vancomycin-Intermediate Staphylococcus aureus (VISA) in vitro. Spontaneous phenotype differentiation was also found in vivo in mouse models and clinical settings. The W phenotype utilizes the Δσ B mutation to maintain a hyperactive agr QS pathway and outcompete the Original strain. Bsa toxin downstream inhibits the growth of O, which responds by generating the Y strain with acquired resistance to Bsa and Vancomycin. Y strain resistance is gained via mutations in regulatory genes controlling cell wall synthesis.
These findings have shed new light on microbial resistance and how it can be acquired without human interference, such as antibiotic misuse and overuse. Intra-strain arms race like this could be going on long before Homo sapiens ever set foot on earth. The pressure for new ways to treat infections is ever more pressing and vital. For microbes, acquiring resistance to antibiotic drugs and medications almost seem like a by-product instead of the ultimate goal. Without effective treatments against microbial infections, public health conditions would inevitably fall back to times when small wounds and cuts could be fatal.
This study also suggests a new potential approach to antimicrobial treatment. Traditionally, whenever confronted with microbial infections, we tend to simply use antibiotics and wipe out the entire microbial community. This type of treatment creates many problems, especially in diseases where a healthy microbiome could be vital in recovery. Killing off the entire microbial community makes it easier for pathogens to spread unchecked and unchallenged, and harder for patients to re-establish a healthy microbiome. The findings of Koch et al (2014) inspire another approach: instead of decimating every microbe at the infection site, if we could target the most resistant/ “evolved” phenotypes, like the Y strain (VISA) in MRSA, and leave the rest of the microbiome in tact, curing the infection could be easier and more effective.
More research on Y strain physiology and genesis could shed new light on new antibiotic candidates and benefit patients with chronic MRSA or VISA infections. It would be curious to know more about the role Mg2+ plays in this arms race. Perhaps Y strain requires Mg2+ to generate more mutations to deal with stress (i.e. Vancomycin or Bsa) or maybe Mg2+ acts on the agr QS pathway using an yet unknown mechanism.
The next questions would be: which part (if any), of the agr QS pathway, could exert inhibitory influence on O strain and shape the development of Y? If W strain is capable of fast dispersal and toxin production, can Y strain “out-run” W and resist these toxins? To answer these questions, Koch et al (2014) tested many different possibilities. After a few rounds of elimination, bsa seemed to be the most promising candidate in the tug-of-war between O, W, and Y. When the bsa gene was mutated, only W phenotype appeared and Y did not develop (Fig 7). However, when Bsa protein was added to O strain, Y still developed while W strain did not (Fig 8).
Bsa, encoded by the gene bsa, is an antibiotic that targets lipid II at the site of bacterial cell wall synthesis, the same precursor that Vancomycin acts on (Fig 2). To compare and contrast the effects of the two antibiotics, Bsa and Vancomycin had been added to the cultures of the three phenotypes, a VSSA strain and a VISA strain (Fig 9). It is obvious that the O strain resembled the VSSA strain in both Bsa and Vancomycin conditions: growth curves quickly plummeted to zero at comparatively low concentrations of Bsa and Vancomycin. More interestingly, the Y strain seemed very similar to VISA with significantly greater success tolerating higher concentrations of Bsa and Vancomycin. This resistance to both Bsa and Vancomycin was not found in O or W strain, suggesting that resistance is specific to Y strain.
These evidence indicated that Y acquires resistance to compete with Bsa-secreting W strain. Because Bsa and Vancomycin have similar targets and mechanisms of action, Y strain is fortunately resistant to both. Whole genome sequencing experiments have also detected gene mutations commonly found in clinical isolates of VISA, such as graRS and walKR, in Y strain; both genes regulates cell wall synthesis.
Conclusions, consequences and future directions
The above experiments focused primarily on the relationships between spontaneously differentiated phenotypes of Community-associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) and the evolution of Vancomycin-Intermediate Staphylococcus aureus (VISA) in vitro. Spontaneous phenotype differentiation was also found in vivo in mouse models and clinical settings. The W phenotype utilizes the Δσ B mutation to maintain a hyperactive agr QS pathway and outcompete the Original strain. Bsa toxin downstream inhibits the growth of O, which responds by generating the Y strain with acquired resistance to Bsa and Vancomycin. Y strain resistance is gained via mutations in regulatory genes controlling cell wall synthesis.
These findings have shed new light on microbial resistance and how it can be acquired without human interference, such as antibiotic misuse and overuse. Intra-strain arms race like this could be going on long before Homo sapiens ever set foot on earth. The pressure for new ways to treat infections is ever more pressing and vital. For microbes, acquiring resistance to antibiotic drugs and medications almost seem like a by-product instead of the ultimate goal. Without effective treatments against microbial infections, public health conditions would inevitably fall back to times when small wounds and cuts could be fatal.
This study also suggests a new potential approach to antimicrobial treatment. Traditionally, whenever confronted with microbial infections, we tend to simply use antibiotics and wipe out the entire microbial community. This type of treatment creates many problems, especially in diseases where a healthy microbiome could be vital in recovery. Killing off the entire microbial community makes it easier for pathogens to spread unchecked and unchallenged, and harder for patients to re-establish a healthy microbiome. The findings of Koch et al (2014) inspire another approach: instead of decimating every microbe at the infection site, if we could target the most resistant/ “evolved” phenotypes, like the Y strain (VISA) in MRSA, and leave the rest of the microbiome in tact, curing the infection could be easier and more effective.
More research on Y strain physiology and genesis could shed new light on new antibiotic candidates and benefit patients with chronic MRSA or VISA infections. It would be curious to know more about the role Mg2+ plays in this arms race. Perhaps Y strain requires Mg2+ to generate more mutations to deal with stress (i.e. Vancomycin or Bsa) or maybe Mg2+ acts on the agr QS pathway using an yet unknown mechanism.
References
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