Wilhelm Paulander

Antibiotic-Mediated Selection of Quorum-Sensing-Negative Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a human commensal that at times turns into a serious bacterial pathogen causing life-threatening infections. For the delicate control of virulence, S. aureus employs the agr quorum-sensing system that, via the intracellular effector molecule RNAIII, regulates virulence gene expression. We demonstrate that the presence of the agr locus imposes a fitness cost on S. aureus that is mediated by the expression of RNAIII. Further, we show that exposure to sublethal levels of the antibiotics ciprofloxacin, mupirocin, and rifampin, each targeting separate cellular functions, markedly increases the agr-mediated fitness cost by inducing the expression of RNAIII. Thus, the extensive use of antibiotics in hospitals may explain why agr-negative variants are frequently isolated from hospital-acquired S. aureus infections but rarely found among community-acquired S. aureus strains. Importantly, agr deficiency correlates with increased duration of and mortality due to bacteremia during antibiotic treatment and with a higher frequency of glycopeptide resistance than in agr-carrying strains. Our results provide an explanation for the frequent isolation of agr-defective strains from hospital-acquired S. aureus infections and suggest that the adaptability of S. aureus to antibiotics involves the agr locus. IMPORTANCE Staphylococcus aureus is the most frequently isolated pathogen in intensive care units and a common cause of nosocomial infections, resulting in a high degree of morbidity and mortality. Surprisingly, a large fraction (15 to 60%) of hospital-isolated S. aureus strains are agr defective and lack the main quorum-sensing-controlled virulence regulatory system. This is a problem, as agr-defective strains are associated with a mortality level in bacteremic infections and a probability of glycopeptide resistance greater than those of other strains. We show here that agr-negative strains have a fitness advantage over agr-positive strains in the presence of sublethal concentrations of some antibiotics and that the fitness defect of agr-positive cells is caused by antibiotic-mediated expression of the agr effector molecule RNAIII. These results offer an explanation of the frequent isolation of agr-defective S. aureus strains in hospitals and will influence how we treat S. aureus infections. Staphylococcus aureus is the most frequently isolated pathogen in intensive care units and a common cause of nosocomial infections, resulting in a high degree of morbidity and mortality. Surprisingly, a large fraction (15 to 60%) of hospital-isolated S. aureus strains are agr defective and lack the main quorum-sensing-controlled virulence regulatory system. This is a problem, as agr-defective strains are associated with a mortality level in bacteremic infections and a probability of glycopeptide resistance greater than those of other strains. We show here that agr-negative strains have a fitness advantage over agr-positive strains in the presence of sublethal concentrations of some antibiotics and that the fitness defect of agr-positive cells is caused by antibiotic-mediated expression of the agr effector molecule RNAIII. These results offer an explanation of the frequent isolation of agr-defective S. aureus strains in hospitals and will influence how we treat S. aureus infections.

Antibiotic combination therapy can select for broad-spectrum multidrug resistance in Pseudomonas aeruginosa

Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms.

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions

Colistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.

Bactericidal antibiotics increase hydroxyphenyl fluorescein signal by altering cell morphology.

It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH•) formation. Flow cytometric detection of OH• by hydroxyphenyl fluorescein (HPF) probe oxidation was used to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS) concentration. Independently of antibiotics, increased fluorescence was seen for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP). Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin such relationships, methods for detecting bacterial metabolites at a cellular level are needed.

β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) has acquired the mecA gene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control of mecA expression, as the cellular levels of PBP2a were unaltered in the clp mutants. An analysis of the cell envelope properties of the clpX and clpP mutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in the clp mutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance.

Copresence of tet(K) and tet(M) in Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Is Associated with Increased Fitness during Exposure to Sublethal Concentrations of Tetracycline

ABSTRACT The tetracycline resistance gene tet(K) was shown to be integrated within the predominant staphylococcal cassette chromosome mec (SCCmec) element of Danish livestock-associated methicillin-resistant Staphylococcus aureus CC398 (LA-MRSA CC398). These LA-MRSA CC398 isolates already possessed tet(M), but the acquisition of tet(K) significantly improved their fitness at sublethal concentrations of tetracycline. Because tet(K) is genetically linked to SCCmec, the use of tetracycline in food animals may have contributed to the successful spread of LA-MRSA CC398.

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

ABSTRACT Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.

Novel Pathways for Ameliorating the Fitness Cost of Gentamicin Resistant Small Colony Variants

Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g., gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived from S. aureus JE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (WT; 0.59–0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500 generations with or without sub-lethal concentrations of gentamicin. Amelioration of the fitness cost followed three evolutionary trajectories and was dependent on the initial mutation type (point mutation vs. deletion) and the passage condition (absence or presence of gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intra-codonic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to WT levels. For the SCVs carrying deletions, all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels. By whole genome sequencing, we identified compensatory mutations in genes related to the σB stress response (7 out of 10 lineages). Inactivation of rpoF that encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. For all lineages passaged in the presence of gentamicin, fitness compensation via membrane potential restoration was suppressed, however, selected for secondary mutations in fusA and SAUSA300_0749. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamicin.

Activation of the SOS response increases the frequency of small colony variants

BackgroundIn Staphylococcus aureus sub-populations of slow-growing cells forming small colony variants (SCVs) are associated with persistent and recurrent infections that are difficult to eradicate with antibiotic therapies. In SCVs that are resistant towards aminoglycosides, mutations have been identified in genes encoding components of the respiratory chain. Given the high frequencies of SCVs isolated clinically it is vital to understand the conditions that promote or select for SCVs.ResultsIn this study we have examined how exposure to sub-inhibitory concentrations of antibiotics with different mechanism of action influence the formation of SCVs that are resistant to otherwise lethal concentrations of the aminoglycoside, gentamicin. We found that exposure of S. aureus to fluoroquinolones and mitomycin C increased the frequency of gentamicin resistant SCVs, while other antibiotic classes failed to do so. The higher proportion of SCVs in cultures exposed to fluoroquinolones and mitomycin C compared to un-exposed cultures correlate with an increased mutation rate monitored by rifampicin resistance and followed induction of the SOS DNA damage response.ConclusionOur observations suggest that environmental stimuli, including antimicrobials that reduce replication fidelity, increase the formation of SCVs through activation of the SOS response and thereby potentially promote persistent infections that are difficult to treat.

The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all β-lactam antibiotics is of major clinical concern. β-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of β-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37°C, and we reasoned that a better understanding of this growth defect could provide novel insights into how β-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless β-lactams are added to the growth medium. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, β-lactams restored septum synthesis and prevented premature autolytic splitting of clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that β -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that β -lactams do not kill S. aureus simply by weakening the cell wall. Author Summary The bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all β-lactam antibiotics has made treatment increasingly difficult. β-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of β-lactams. Without β-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to β-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of β-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that β-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.