Alternative Evolutionary Pathways for Drug-Resistant Small Colony Variant Mutants in Staphylococcus aureus

ABSTRACT Staphylococcus aureus is known to generate small colony variants (SCVs) that are resistant to aminoglycoside antibiotics and can cause persistent and recurrent infections. The SCV phenotype is unstable, and compensatory mutations lead to restored growth, usually with loss of resistance. However, the evolution of improved growth, by mechanisms that avoid loss of antibiotic resistance, is very poorly understood. By selection with serial passaging, we isolated and characterized different classes of extragenic suppressor mutations that compensate for the slow growth of small colony variants. Compensation occurs by two distinct bypass mechanisms: (i) translational suppression of the initial SCV mutation by mutant tRNAs, ribosomal protein S5, or release factor 2 and (ii) mutations that cause the constitutive activation of the SrrAB global transcriptional regulation system. Although compensation by translational suppression increases growth rate, it also reduces antibiotic susceptibility, thus restoring a pseudo-wild-type phenotype. In contrast, an evolutionary pathway that compensates for the SCV phenotype by activation of SrrAB increases growth rate without loss of antibiotic resistance. RNA sequence analysis revealed that mutations activating the SrrAB pathway cause upregulation of genes involved in peptide transport and in the fermentation pathways of pyruvate to generate ATP and NAD+, thus explaining the increased growth. By increasing the growth rate of SCVs without the loss of aminoglycoside resistance, compensatory evolution via the SrrAB activation pathway represents a threat to effective antibiotic therapy of staphylococcal infections. IMPORTANCE Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance. Small colony variants (SCVs) of Staphylococcus aureus are a significant clinical problem, causing persistent and antibiotic-resistant infections. However, SCVs are unstable and can rapidly evolve growth-compensated mutants. Previous data suggested that growth compensation only occurred with the loss of antibiotic resistance. We have used selection with serial passaging to uncover four distinct pathways of growth compensation accessible to SCVs. Three of these paths (reversion, intragenic suppression, and translational suppression) increase growth at the expense of losing antibiotic resistance. The fourth path activates an alternative transcriptional program and allows the bacteria to produce the extra ATP required to support faster growth, without losing antibiotic resistance. The importance of this work is that it shows that drug-resistant SCVs can evolve faster growth without losing antibiotic resistance.

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Having your cake and eating it - Staphylococcus aureus small colony variants can evolve faster growth rate without losing their antibiotic resistance

Microbial Cell ◽

10.15698/mic2017.08.587 ◽

2017 ◽

Vol 4 (8) ◽

pp. 275-277 ◽

Cited By ~ 1

Author(s):

Gerrit Brandis ◽

Sha Cao ◽

Douglas L. Huseby ◽

Diarmaid Hughes

Keyword(s):

Staphylococcus Aureus ◽

Growth Rate ◽

Antibiotic Resistance ◽

Small Colony Variants ◽

Small Colony

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Novel Research Models for Staphylococcus aureus Small Colony Variants (SCV) Development: Co-pathogenesis and Growth Rate

Frontiers in Microbiology ◽

10.3389/fmicb.2020.00321 ◽

2020 ◽

Vol 11 ◽

Author(s):

James Lee ◽

Peter S. Zilm ◽

Stephen P. Kidd

Keyword(s):

Staphylococcus Aureus ◽

Growth Rate ◽

Small Colony Variants ◽

Small Colony

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Joint antibiotic and phage therapy: addressing the limitations of a seemingly ideal phage for treating Staphylococcus aureus infections

10.1101/2020.04.24.060335 ◽

2020 ◽

Cited By ~ 1

Author(s):

Brandon A. Berryhill ◽

Douglas L. Huseby ◽

Ingrid C. McCall ◽

Diarmaid Hughes ◽

Bruce R. Levin

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Infections ◽

Phage Therapy ◽

Receptor Site ◽

Clinical Isolates ◽

Broad Host Range ◽

Antibiotic Resistant ◽

Small Colony Variants ◽

Small Colony

AbstractIn response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. (i) This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. (ii) Because of the mode of action of PYOSaS. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. (iii) PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear the populations of S. aureus. Due to the ascent of a phenotypically diverse array of small colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.Significance StatementThe increasing frequency of antibiotic-resistant pathogens has fostered a quest for alternative means to treat bacterial infections. Prominent in this quest is a therapy that predates antibiotics: bacteriophage. This study explores the potential of a phage, PYOSa, for treating Staphylococcus aureus infections in combination with antibiotics. On first consideration, this phage, isolated from a commercial therapeutic cocktail, seems ideal for this purpose. The results of this population dynamic and genomic analysis study identify a potential liability of using PYOSa for therapy. Due to the production of potentially pathogenic atypical small colony variants, PYOSa alone cannot eliminate S. aureus populations. However, we demonstrate that by following the administration of PYOSa with bactericidal antibiotics, this limitation and potential liability can be addressed.

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Virulence of Staphylococcus aureus Small Colony Variants in the Caenorhabditis elegans Infection Model

Infection and Immunity ◽

10.1128/iai.74.2.1091-1096.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1091-1096 ◽

Cited By ~ 45

Author(s):

Costi D. Sifri ◽

Andrea Baresch-Bernal ◽

Stephen B. Calderwood ◽

Christof von Eiff

Keyword(s):

Staphylococcus Aureus ◽

Caenorhabditis Elegans ◽

Infection Model ◽

Alternative Host ◽

Antibiotic Resistant ◽

Small Colony Variants ◽

C Elegans ◽

Small Colony ◽

Morphological Variants ◽

Slow Growing

ABSTRACT Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.

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Staphylococcus aureus small-colony variants exhibit genotype specific hyperinflammation

10.1101/2020.08.21.261750 ◽

2020 ◽

Author(s):

Qing Tang ◽

Mimi R. Precit ◽

Maureen K. Thomason ◽

Fariha Ahmed-Qadri ◽

Adelle P. McFarland ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Neutrophil Infiltration ◽

Host Responses ◽

Macrophage Infection ◽

Independent Manner ◽

Antibiotic Resistant ◽

Small Colony Variants ◽

Small Colony ◽

Pharmacological Blockade ◽

Respiratory Outcomes

SUMMARYStaphylococcus aureus small colony variants (SCVs) cause a variety of persistent, antibiotic resistant infections. Respiratory infection with thymidine-dependent SCVs is common among children with cystic fibrosis and is associated with worse respiratory outcomes, although the underlying pathophysiological mechanisms remain to be defined. Here, we characterized normal colony (NC) S. aureus and two phenotypically distinct SCVs, hemin-dependent (HD-SCV) and thymidine-dependent (TD-SCV), for their capacities to affect host responses. Among these, TD-SCVs produced elevated levels of c-di-AMP in a thymidine-dependent fashion. TD-SCVs elicited increased IFN-I production in a STING-dependent and cGAS-independent manner during macrophage infection. However, murine lung infection with TD-SCVs revealed both STING-dependent increases in G-CSF and STING-independent elevation of CCL3, leading to higher airway neutrophil infiltration and activation, which were reduced by pharmacological blockade of CCL3 signaling. Our results suggest the elevated inflammatory capacity of TD-SCVs may contribute to their pathogenesis, and that targeting CCL3-mediated inflammation may improve outcomes.

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Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study

Journal of Medical Microbiology ◽

10.1099/jmm.0.009720-0 ◽

2009 ◽

Vol 58 (8) ◽

pp. 1067-1073 ◽

Cited By ~ 147

Author(s):

Rachna Singh ◽

Pallab Ray ◽

Anindita Das ◽

Meera Sharma

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Wild Type ◽

Planktonic Cells ◽

Intrinsic Resistance ◽

Persister Cells ◽

Small Colony Variants ◽

Small Colony ◽

Wild Type Parent

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of Staphylococcus aureus biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0–100 μg ml−1 at 37 °C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells – most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of S. aureus biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with S. aureus biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

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