Staphylococcus aureus persisters are associated with reduced clearance in a catheter-associated biofilm infection.

Background: Staphylococcus aureus causes a wide variety of infections, many of which are chronic or relapsing in nature. Antibiotic therapy is often ineffective against S. aureus biofilm-mediated infections. Biofilms are difficult to treat partly due to their tolerance to antibiotics, however the underlying mechanism responsible for this remains unknown. One possible explanation is the presence of persister cells-dormant-like cells that exhibit tolerance to antibiotics. Recent studies have shown a connection between a fumC (fumarase C, a gene in the tricarboxylic acid cycle) knockout strain and increased survival to antibiotics, antimicrobial peptides, and in a Drosophila melanogaster model. Objective: It remained unclear whether a S. aureus high persister strain would have a survival advantage in the presence of innate and adaptive immunity. To further investigate this, a fumC knockout and wild type strains were examined in a murine catheter-associated biofilm model. Results: Interestingly, mice struggled to clear both S. aureus wild type and the fumC knockout strains. We reasoned both biofilm-mediated infections predominantly consisted of persister cells. To determine the persister cell population within biofilms, expression of a persister cell marker (Pcap5A::dsRED) in a biofilm was examined. Cell sorting of biofilms challenged with antibiotics revealed cells with intermediate and high expression of cap5A had 5.9-and 4.5-fold higher percent survival compared to cells with low cap5A expression. Based on previous findings that persisters are associated with reduced membrane potential, flow cytometry analysis was used to examine the metabolic state of cells within a biofilm. We confirmed cells within biofilms had reduced membrane potential compared to both stationary phase cultures (2.5-fold) and exponential phase cultures (22.4-fold). Supporting these findings, cells within a biofilm still exhibited tolerance to antibiotic challenge following dispersal of the matrix through proteinase K. Conclusion: Collectively, these data show that biofilms are largely comprised of persister cells, and this may explain why biofilm infections are often chronic and/or relapsing in clinical settings.

Interruption of the tricarboxylic acid cycle in Staphylococcus aureus leads to increased tolerance to innate immunity.

Staphylococcus aureus is widely known for its resistance and virulence causing public health concerns. However, antibiotic tolerance is also a contributor to chronic and relapsing infections. Previously, it has been demonstrated that persister formation is dependent on reduced tricarboxylic acid (TCA) cycle activity. Persisters have been extensively examined in terms of antibiotic tolerance but tolerance to antimicrobial peptides (AMPs) remains largely unexplored. AMPs are a key component of both the human and Drosophila innate immune response. TCA cycle mutants were tested to determine both antibiotic and AMP tolerance. Challenging with multiple classes of antibiotics led to increased persister formation (100- to 1,000-fold). Similarly, TCA mutants exhibited AMP tolerance with a 100- to 1,000-fold increase in persister formation when challenged with LL-37 or human ß-defensin 3 (hßD3). The ability of TCA cycle mutants to tolerate the innate immune system was further examined with a D. melanogaster model. Both males and females infected with TCA cycle mutants exhibited increased mortality and had higher bacterial burdens (1.5 log) during the course of the infection. These results suggest increasing the percentage of persister cells leads to increased tolerance to components of the innate immune system.

Polymicrobial Interactions Induce Multidrug Tolerance in Staphylococcus aureus Through Energy Depletion.

Staphylococcus aureus is responsible for a high number of relapsing infections, which are often mediated by the protective nature of biofilms. Polymicrobial biofilms appear to be more tolerant to antibiotic treatment, however, the underlying mechanisms for this remain unclear. Polymicrobial biofilm and planktonic cultures formed by S. aureus and Candida albicans are 10- to 100-fold more tolerant to oxacillin, vancomycin, ciprofloxacin, delafloxacin, and rifampicin compared to monocultures of S. aureus. The possibility of C. albicans matrix components physically blocking antibiotic molecules from reaching S. aureus was ruled out as oxacillin, ciprofloxacin, delafloxacin, and rifampicin were able to diffuse through polymicrobial biofilms. Based on previous findings that S. aureus forms drug tolerant persister cells through ATP depletion, we examined nutrient deprivation by determining glucose availability, which indirectly correlates to ATP production via the tricarboxylic acid (TCA) cycle. Using an extracellular glucose assay, we confirmed that S. aureus and C. albicans polymicrobial cultures depleted available glucose faster than the respective monocultures. Supporting this finding, S. aureus exhibited decreased TCA cycle activity, specifically fumarase expression, when grown in the presence of C. albicans. In addition, S. aureus grown in polymicrobial cultures displayed 2.2-fold more cells with low membrane potential and a 13% reduction in intracellular ATP concentrations than in monocultures. Collectively, these data demonstrate that decreased metabolic activity through nutrient deprivation is a mechanism for increased antibiotic tolerance within polymicrobial cultures.

Stochastic Variation in Expression of the Tricarboxylic Acid Cycle Produces Persister Cells.

Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and ß-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.IMPORTANCE Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.

Protease-Mediated Growth of Staphylococcus aureus on Host Proteins Is opp3 Dependent.

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (ΔpckA) and glutamate dehydrogenase (ΔgudB), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment.IMPORTANCEStaphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources.

Amino Acid Catabolism in Staphylococcus aureus and the Function of Carbon Catabolite Repression.

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kinase, also abrogated growth of JE2 in CDM, suggesting that ATP production from pyruvate-producing amino acids is also critical for growth. In addition, although a functional respiratory chain was absolutely required for growth, the oxygen consumption rate and intracellular ATP concentration were significantly lower during growth in CDM than during growth in glucose-containing media. Finally, transcriptional analyses demonstrated that expression levels of genes coding for the enzymes that synthesize glutamate from proline, arginine, and histidine are repressed by CcpA and carbon catabolite repression. These data show that pathways important for glutamate catabolism or ATP generation via Pta/AckA are important for growth in niches where glucose is not abundant, such as abscesses within skin and soft tissue infections.IMPORTANCES. aureus is a significant cause of both morbidity and mortality worldwide. This bacterium causes infections in a wide variety of organ systems, the most common being skin and soft tissue. Within a staphylococcal abscess, levels of glucose, a preferred carbon source, are limited due to the host immune response. Therefore, S. aureus must utilize other available carbon sources such as amino acids or peptides to proliferate. Our results show that glutamate and amino acids that serve as substrates for glutamate synthesis, particularly proline, function as major carbon sources during growth, whereas other amino acids that generate pyruvate are important for ATP synthesis via substrate-level phosphorylation in the Pta-AckA pathway. Our data support a model whereby certain amino acid catabolic pathways, and acquisition of those particular amino acids, are crucial for growth in niches where glucose is not abundant.

Persister formation in Staphylococcus aureus is associated with ATP depletion.

Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics(1). Persisters are associated with chronic infections and antibiotic treatment failure(1-3). In Escherichia coli, toxin-antitoxin modules have been linked to persister formation(4-6). The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting toxin-antitoxin modules in S. aureus did not affect the level of persisters. Here, we show that S. aureus persisters are produced due to a stochastic entrance into the stationary phase accompanied by a drop in intracellular adenosine triphosphate. Cells expressing stationary-state markers are present throughout the growth phase, and increase in frequency with cell density. Cell sorting revealed that the expression of stationary markers is associated with a 100-1,000-fold increase in the likelihood of survival to antibiotic challenge. The adenosine triphosphate level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

Persister formation in Staphylococcus aureus is associated with ATP depletion.

Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic infections and antibiotic treatment failure1-3. In Escherichia coli, toxin/antitoxin (TA) modules have been linked to persister formation4-6. The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance into stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers is associated with a 100-1000 fold increase in the likelihood of survival to antibiotic challenge. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

Use of electroporation and conjugative mobilization for genetic manipulation of Staphylococcus epidermidis.

To perform mechanistic studies on the biology of bacteria including metabolism, physiology, and pathogenesis, it is essential to possess the tools required for genetic manipulation. Introduction of plasmid DNA into Staphylococcus epidermidis for subsequent genetic manipulation, including allelic replacement and complementation experiments, is typically performed by either electroporation or conjugative mobilization. Herein, standard protocols and tips for the transfer of plasmid DNA to S. epidermidis by these two methods are provided.

The Ktr potassium transport system in Staphylococcus aureus and its role in cell physiology, antimicrobial resistance and pathogenesis.

Potassium (K(+) ) plays a vital role in bacterial physiology, including regulation of cytoplasmic pH, turgor pressure and transmembrane electrical potential. Here, we examine the Staphylococcus aureus Ktr system uniquely comprised of two ion-conducting proteins (KtrB and KtrD) and only one regulator (KtrA). Growth of Ktr system mutants was severely inhibited under K(+) limitation, yet detectable after an extended lag phase, indicating the presence of a secondary K(+) transporter. Disruption of both ktrA and the Kdp-ATPase system, important for K(+) uptake in other organisms, eliminated regrowth in 0.1 mM K(+) , demonstrating a compensatory role for Kdp to the Ktr system. Consistent with K(+) transport mutations, S. aureus devoid of the Ktr system became sensitive to hyperosmotic conditions, exhibited a hyperpolarized plasma membrane, and increased susceptibility to aminoglycoside antibiotics and cationic antimicrobials. In contrast to other organisms, the S. aureus Ktr system was shown to be important for low-K(+) growth under alkaline conditions, but played only a minor role in neutral and acidic conditions. In a mouse competitive index model of bacteraemia, the ktrA mutant was significantly outcompeted by the parental strain. Combined, these results demonstrate a primary mechanism of K(+) uptake in S. aureus and a role for this system in pathogenesis.

Inactivation of the Pta-AckA pathway causes cell death in Staphylococcus aureus.

During growth under conditions of glucose and oxygen excess, Staphylococcus aureus predominantly accumulates acetate in the culture medium, suggesting that the phosphotransacetylase-acetate kinase (Pta-AckA) pathway plays a crucial role in bacterial fitness. Previous studies demonstrated that these conditions also induce the S. aureus CidR regulon involved in the control of cell death. Interestingly, the CidR regulon is comprised of only two operons, both encoding pyruvate catabolic enzymes, suggesting an intimate relationship between pyruvate metabolism and cell death. To examine this relationship, we introduced ackA and pta mutations in S. aureus and tested their effects on bacterial growth, carbon and energy metabolism, cid expression, and cell death. Inactivation of the Pta-AckA pathway showed a drastic inhibitory effect on growth and caused accumulation of dead cells in both pta and ackA mutants. Surprisingly, inactivation of the Pta-AckA pathway did not lead to a decrease in the energy status of bacteria, as the intracellular concentrations of ATP, NAD(+), and NADH were higher in the mutants. However, inactivation of this pathway increased the rate of glucose consumption, led to a metabolic block at the pyruvate node, and enhanced carbon flux through both glycolysis and the tricarboxylic acid (TCA) cycle. Intriguingly, disruption of the Pta-AckA pathway also induced the CidR regulon, suggesting that activation of alternative pyruvate catabolic pathways could be an important survival strategy for the mutants. Collectively, the results of this study demonstrate the indispensable role of the Pta-AckA pathway in S. aureus for maintaining energy and metabolic homeostasis during overflow metabolism.

CcpA regulates arginine biosynthesis in Staphylococcus aureus through repression of proline catabolism.

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced (13)C(5) labeled arginine when grown with (13)C(5) proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.