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Objectives: Difficult-to-treat infections caused by antibiotic-susceptible strains have been linked to the occurrence of persisters, a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. These persisters can be identified phenotypically by plating on nutrient agar because of their altered growth dynamics, resulting in colony-size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remains unknown., Methods: We plated bacteria derived from 132 patient samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. We retained 36 Staphylococcus aureus monocultures for further analysis. We investigated clinical factors associated with increased colony growth-delay with regression analyses. We corroborated the clinical findings using in vitro grown static biofilms exposed to distinct antibiotics., Results: The extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients (from no delay to a maximum of 57.6 hours). Increased heterogeneity coincided with increased median colony growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay (13.3 hours; 95% CI 7.13-19.6 hours; p < 0.001). S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with clindamycin or levofloxacin exhibited prolonged growth-delay (p < 0.05 for 11 of 12 comparisons), correlating with a strain-dependent increase in antibiotic tolerance., Discussion: Colony-size heterogeneity upon direct sampling of difficult-to-treat S. aureus infections was frequently observed. Hence, future studies are needed to assess the potential benefit of phenotypic heterogeneity quantification for staphylococcal infection prognosis and treatment guidelines., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus , underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat., Competing Interests: The authors declare no competing interest.

Background: Staphylococcus aureus-infected patients treated with antibiotics that are effective in vitro often experience relapse of infection because the bacteria hide in privileged locations. These locations include abscesses and host cells, which contain low-pH compartments and are sites from which nonstable S. aureus small-colony variants (SCVs) are frequently recovered., Methods: We assessed the effect of low pH on S. aureus colony phenotype and bacterial growth, using in vitro and in vivo models of long-term infection., Results: We showed that low pH induced nonstable SCVs and nonreplicating persisters that are capable of regrowth. Within host cells, S. aureus was located in phagolysosomes, a low-pH compartment. Therapeutic neutralization of phagolysosomal pH with ammonium chloride, bafilomycin A1, or the antimalaria drug chloroquine reduced SCVs in infected host cells. In a systemic mouse infection model, treatment with chloroquine also reduced SCVs., Conclusions: Our results show that the acidic environment favors formation of nonstable SCVs, which reflect the SCVs found in clinics. They also provide evidence that treatment with alkalinizing agents, together with antibiotics, may provide a novel translational strategy for eradicating persisting intracellular reservoirs of staphylococci. This approach may also be extended to other intracellular bacteria., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

The Gram-positive human pathogen Staphylococcus aureus causes a variety of human diseases such as skin infections, pneumonia, and endocarditis. The micrococcal nuclease Nuc1 is one of the major S. aureus virulence factors and allows the bacterium to avoid neutrophil extracellular trap (NET)-mediated killing. We found that addition of the protein synthesis inhibitor clindamycin to S. aureus LAC cultures decreased nuc1 transcription and subsequently blunted nuclease activity in a molecular beacon-based fluorescence assay. We also observed reduced NET degradation through Nuc1 inhibition translating into increased NET-mediated clearance. Similarly, pooled human immunoglobulin specifically inhibited nuclease activity in a concentration-dependent manner. Inhibition of nuclease activity by clindamycin and immunoglobulin enhanced S. aureus clearance and should be considered in the treatment of S. aureus infections., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined., Results: Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa) was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation., Conclusions: The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future.