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

Vestergaard, Martin; Paulander, Wilhelm; Leng, Bingfeng; Nielsen, Julius; Westh, Henrik; Ingmer, Hanne

doi:10.3389/fmicb.2016.01866

Cited by 15 publications

(17 citation statements)

References 45 publications

(78 reference statements)

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“…Since the atpA mutant has a hyperpolarized membrane 25 , we also assessed hBD4 susceptibility of the menD transposon mutant, which has a depolarized membrane 37 . The menD mutant was indeed more tolerant to hBD4 compared with WT, as no reduction in viable cell count was observed following 2 h exposure to hBD4 at 5 µM ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Liu

Beck

Nøhr-Meldgaard

et al. 2020

Sci Rep

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Antimicrobial peptides (AMps) are an important part of the human innate immune system for protection against bacterial infections, however the AMps display varying degrees of activity against Staphylococcus aureus. previously, we showed that inactivation of the Atp synthase sensitizes S. aureus towards the AMp antibiotic class of polymyxins. Here we wondered if the Atp synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the Wt, even when the oxidative burst is inhibited.these results show that the staphylococcal Atp synthase enhance tolerance of S. aureus towards some human AMps and this indicates that inhibition of the Atp synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMps of the innate immune system. Bacterial pathogens that cause disease in humans remain a serious threat to public health and antibiotics are still our primary weapons in treating many bacterial diseases. The ability to eradicate bacterial infections is however challenged by development of resistance for every type of antibiotic introduced to the clinic 1. The majority of the new small molecule antibiotics in clinical development are however inhibiting the same targets as already marketed antibiotics 2. As an alternative to small molecule antibiotics, antimicrobial peptides (AMPs) are also explored in clinical trials, however most of the AMPs are only tested for topical applications due to toxicity issues and low metabolic stability 3. Here we propose a new strategy to combat bacterial infections, namely to sensitize bacteria to the naturally occurring antimicrobial peptides of the human body and hence boosting the antibacterial capabilities of the innate immune system to eradicate bacterial infections. Humans are continuously exposed to numerous, and potentially pathogenic, microorganisms, where the innate immune system provides the first line of defense. AMPs constitute an important defense mechanism of the innate immune system against invading microorganisms, due to their antimicrobial and immune stimulatory properties 4,5. In humans, several classes of AMPs have been identified, such as α-and β-defensins, the cathelicidin LL-37 and histatins 5. The α-defensins consist of six members, which are divided into human neutrophil peptides (HNP1-4) and human α-defensin 5 and 6 (HD5 and HD6). HNP1-4 are highly concentrated in the granules of neutrophils, but are also expressed in monocytes, lymphocytes and natural killer cells. HD5 and HD6 are primarily ...

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Section: Resultsmentioning

confidence: 99%

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Liu

Beck

Nøhr-Meldgaard

et al. 2020

Sci Rep

Self Cite

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“…Also, small colony variants of S . aureus are resistant to aminoglycosides due to mutations in TCA cycle or electron transport chain genes and they display reduced membrane potential 38 , 39 . Recently, ATP content was reported to be a determining factor for whether or not exponentially growing cells become persisters 4 , 14 .…”

Section: Discussionmentioning

confidence: 99%

Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase

Wang

Bojer

George

et al. 2018

Sci Rep

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Persister cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of cells in stationary phase. By enrichment from a transposon mutant library in S. aureus we identified mutants that in this growth phase displayed enhanced persister cell formation. We found that inactivation of either sucA or sucB, encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10–100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F1F0 ATPase. In S. aureus, TCA cycle activity and gene expression are de-repressed in stationary phase but single cells with low expression may be prone to form persisters. While ATP levels were not consistently affected in high persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that persister cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.

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“…While, in most of the cases, CFU counts and metabolic activity were affected to a similar extent by antibiotics, a noticeable exception concerned tobramycin: it caused a marked reduction in the number of CFU for ATCC 25923 in biofilms grown in ASM but only a marginal reduction in metabolic activity. Aminoglycosides are known to induce the formation of small-colony variants of S. aureus both in vivo and in vitro (7,(44)(45)(46)(47). We demonstrate here the presence of small colonies, which strongly suggests that tobramycin selects for SCVs specifically in biofilms cultivated in ASM.…”

Section: Discussionmentioning

confidence: 99%

“…Identification and metabolic activity of small-colony variants. When appropriate, the presence of SCVs was determined by spreading the bacteria recovered from biofilms on 5 types of agar plates, namely, TSA, TSA supplemented with 1 mg/liter hemin (TSAH) or menadione (TSAM), TSA supplemented with both 1 mg/liter hemin and menadione (TSAMH), and Columbia blood agar (CBA) plates (44,52). The colonies were counted after 24 h of incubation at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Activity of Antibiotics against Staphylococcus aureus in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium

Iglesias

Wilms

Vanbever

et al. 2019

Antimicrob Agents Chemother

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Staphylococcus aureus is a highly prevalent pathogen in the respiratory tract of young patients with cystic fibrosis (CF) and causes biofilm-related infections. Here, we set up an in vitro model of a biofilm grown in Trypticase soy broth supplemented with glucose and NaCl (TGN) or in artificial sputum medium (ASM) and used it to evaluate on a pharmacodynamic basis the activity of antibiotics used in CF patients and active on staphylococci (meropenem, vancomycin, azithromycin, linezolid, rifampin, ciprofloxacin, tobramycin). Rheological studies showed that ASM was more elastic than viscous, as was also observed for sputa from CF patients, with elastic and viscous moduli being, respectively, similar to and slightly lower than those of CF sputa. Biofilms formed by methicillin-sensitive S. aureus strain ATCC 25923 and methicillin-resistant S. aureus strain ATCC 33591 reached maturity after 24 h, with biomass (measured by crystal violet staining) and metabolic activity (assessed by following resazurin metabolization) being lower in ASM than in TGN and viability (assessed by bacterial counts) being similar in both media. Full concentration-response curves of antibiotics obtained after 24 h of incubation of biofilms showed that all antibiotics were drastically less potent and less efficient in ASM than in TGN toward viability, metabolic activity, and biomass. Tobramycin selected for small-colony variants, specifically in biofilms grown in ASM; the auxotrophism of these variants could not be established. These data highlight the major influence exerted by the culture medium on S. aureus responsiveness to antibiotics in biofilms. The use of ASM may help to determine effective drug concentrations or to evaluate new therapeutic options against biofilms in CF patients.

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

Cited by 15 publications

References 45 publications

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Inactivation of TCA cycle enhances Staphylococcus aureus persister cell formation in stationary phase

Activity of Antibiotics against Staphylococcus aureus in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium

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