Role of Metal Availability in Modulating Antibiotic Efficacy

EGTA, a calcium chelator with limited zinc affinity, does not change antibiotic killing kinetics compared with the chelator-absent control.

“When EGTA, a potent chelator of calcium with limited affinity for zinc, was used, no change to antibiotic killing kinetics as compared to the chelator absent control was observed (Fig. 2D; Fig. S5D).”

The increased survival against antibiotics in the presence of CP is not associated with a change in the minimum inhibitory concentration (MIC).

“Additionally, the observed increased survival against antibiotics in the presence of CP was not associated with a change in the minimum inhibitory concentration (MIC) of the antibiotic (Table S2), demonstrating that antibiotics remain unbound and capable of inhibiting their target in the presence of CP.”

Addition of TPEN inhibits Atl-mediated cell wall clearance in zymography.

“Comparison of gels with and without TPEN, showed that the addition of TPEN inhibited Atl-mediated cell wall clearance (Fig. S7A-B).”

In zinc-replete environments, manganese limitation potentiates β-lactam antibiotic killing.

“On the other hand, in zinc-replete environments, manganese limitation was found to potentiate β-lactam antibiotic killing.”

A CP variant lacking both metal binding sites cannot induce oxacillin tolerance.

“The mutant protein was unable to induce tolerance to oxacillin, strongly implicating metal limitation as the driver of b-lactam tolerance.(Fig 2A ; Fig. S5A).”

The CFU recovered in the presence of 60 μg/mL CP is similar to media lacking CP.

“Consistent with this, the CFU recovered in the presence of 60 µg/mL CP was similar to media lacking CP (Fig. 3A).”

CP induces similar antibiotic tolerance in multiple clinical isolates of S. aureus.

“To explore if changes in antibiotic efficacy were specific to the MSSA strain Newman, multiple clinical isolates of S. aureus (Table S1) were tested and a similar induction of antibiotic tolerance in all isolates was observed (Fig. S4A).”

Supplementation with exogenous ZnSO4 restores antibiotic efficacy in media containing both TPEN and ZnSO4.

“Furthermore, supplementation of the media containing both TPEN and exogenous ZnSO4 was sufficient to restore antibiotic efficacy (Fig. 2C; Fig. S5C).”

CP increases tolerance of a clinical MRSA bacteremia isolate and the MRSA laboratory strain JE2 to ceftobiprole.

“Additionally, the presence of CP similarly increased tolerance of a clinical MRSA bacteremia isolate and the MRSA laboratory strain JE2 (Table S1) to ceftobiprole, a b-lactam approved for the treatment of MRSA bacteremia (Fig. S4B).”

CP addition does not inhibit cell wall synthesis, as shown by HADA accumulation comparable to untreated cells.

“These results showed that the addition of CP did not result in the inhibition of cell wall synthesis, as seen by the accumulation of HADA signal comparable to that of untreated cells (Fig. 3B and Fig. S6).”

In the presence of CP, complete loss of autolytic activity is observed, represented by little change in OD.

“In the presence of CP, complete loss of autolytic activity was observed as represented by little change in OD (Fig. 4A).”

Supernatant collected from Δatl cultures shows a lack of autolytic activity, as evident by limited change in OD600.

“Clearance in OD600 is due to the activity of autolysins, particularly that of Atl, as supernatant collected from Datl cultures shows a lack of activity, as evident by limited change in the OD600 (Fig. S7C).”

In the presence of 60 μg/mL CP, approximately 50-fold more S. aureus survived treatment with oxacillin and nafcillin.

“In the presence of 60 µg/mL CP, approximately 50-fold more S. aureus survived treatment with both oxacillin and nafcillin (Fig. 1B-C; Fig. S1B-C).”

Bovine serum albumin (BSA) at similar protein concentrations to CP has no impact on S. aureus survival after oxacillin treatment.

“Our results showed that BSA had no impact on survival post oxacillin treatment (Fig. S4C).”

Measurement of cellular ADP/ATP ratios with and without CP shows no reductions to the energy equilibrium of the cell.

“Measurement of cellular ADP/ATP ratios with and without the addition of CP showed no reductions to the energy equilibrium of the cell (Fig. 3C).”

In a murine infection model, the efficacy of oxacillin is significantly enhanced in CP-deficient mice.

“In a murine infection model, the efficacy of oxacillin was significantly enhanced in CP-deficient mice, demonstrating that CP impairs βlactam activity in vivo.”

TPEN decreases β-lactam efficacy in a dose-dependent manner, culminating in a 1000-fold increase in S. aureus survivors relative to the metal-replete control.

“Similar to CP, the addition of TPEN resulted in decreased efficacy of blactam antibiotics in a dose-dependent response, culminating in a 1000-fold increase in S. aureus survivors relative to the metal replete control (Fig. 2B; Fig. S5B).”

Reduced antibiotic efficacy occurred for cultures treated with 1 mM MV.

“Results showed potentiation of antibiotic treatment for cultures treated with 50 uM MV (Fig. 4B) and conversely reduced antibiotic efficacy for cultures treated with 1 mM MV (Fig. 4C).”

CP significantly inhibits oxacillin efficacy in a mouse model of bacteremia.

“Furthermore, CP significantly inhibits oxacillin efficacy in a mouse model of bacteremia.”

• AssumptionRests on an assumption: The mouse bacteremia model is predictive of human S. aureus infection and antibiotic treatment outcomes.

The addition of methyl viologen to the Mn-replete environment increases autolytic activity.

“Furthermore, our results show that the addition of methyl viologen, a ROS producing compound, to the Mn-replete environment increases autolytic activity (Fig. 4A).”

Manganese limitation increases the expression and activity of autolysins by S. aureus.

“Our data demonstrates manganese limitation increases the expression and activity of autolysins by S. aureus, bacterial enzymes required for cell wall degradation and bacterial lysis following β-lactam treatment.”

By removing environmental manganese from the media through Chelex-treatment, increased autolytic activity and expression were observed under conditions of Mn limitation.

“By removing environmental manganese from the media through Chelex-treatment, we observed increased autolytic activity and expression under conditions of Mn limitation.”

In the absence of atl, the addition of CP failed to exhibit a significant effect on antibiotic survival.

“In the absence of atl, the addition of CP failed to exhibit a significant effect on antibiotic survival (Fig. 4D).”

• AssumptionRests on an assumption: The genetic deletion of atl does not have pleiotropic effects that confound the interpretation of CP-induced tolerance.

The addition of wildtype CP, or DS2 CP, resulted in around 4-fold increased expression of autolysins Atl and Sle1 for S. aureus Newman.

“Our results show that the addition of wildtype CP, or DS2 CP, resulted in around 4-fold increased expression of autolysins Atl and Sle1 for S. aureus Newman (Fig. 3A-B).”

The addition of DS1 CP or DS1/DS2 CP resulted in similar autolysin expression levels relative to cultures grown in the absence of CP.

“Conversely, the addition DS1 CP or DS1/DS2 CP, resulted in similar autolysin expression levels relative to cultures grown in the absence of CP.”

No significant differences in oxacillin treatment between wild-type and S100A9-/- mice were observed in kidney and heart tissue.

“These effects were not observed in the kidney and heart tissue where no significant differences in oxacillin treatment between wild-type and S100A9-/-mice were observed (Fig. S9A-B).”

Treatment outcome can either be significantly improved or reduced depending on the concentration and duration of ROS exposure.

“Additionally, our results show that treatment outcome can either be significantly improved or reduced depending on the concentration and duration of ROS exposure (Fig. 4B-C).”

Similar results were observed with the addition of CP, where autolysin expression increased as a result of CP-driven Mn chelation.

“Similar results were also observed with the addition of CP, where autolysin expression increased as a result of CP-driven Mn chelation.”

Challenging cells with 1 mM methyl viologen at the time of antibiotic treatment results in the reduction of antibiotic efficacy.

“This contrasts the challenge of cells with 1 mM methyl viologen at the time of antibiotic treatment, which results in the reduction of antibiotic efficacy (Fig. 4C).”

CP-treated cells showed regions of increased peptidoglycan compared to cross-sections.

“Comparison between cross-sections showed regions of increased peptidoglycan for CP-treated cells (black arrows).”

The addition of 60 μg/mL CP showed noticeable changes to the structure of the cell walls after 2 hours.

“The addition of 60 µg/mL CP showed noticeable changes to the structure of the cell walls after 2 hours (Fig. S8).”

Potentiation of antibiotic treatment occurred for cultures treated with 50 uM MV.

“Results showed potentiation of antibiotic treatment for cultures treated with 50 uM MV (Fig. 4B) and conversely reduced antibiotic efficacy for cultures treated with 1 mM MV (Fig. 4C).”

Oxacillin showed significantly improved bacterial clearance in the liver of CP-deficient mice with approximately 20-fold less bacterial survivors relative to wild-type mice.

“Oxacillin showed significantly improved bacterial clearance in the liver of CP-deficient mice with approximately 20-fold less bacterial survivors relative to wild-type mice (Fig. 6B).”

When cultures of S. aureus are grown in the presence of 50 μM methyl viologen and then challenged with oxacillin, treatment efficacy is improved.

“When cultures of S. aureus are grown in the presence of 50 µM methyl viologen and then challenged with oxacillin, treatment efficacy is improved (Fig. 4B).”

Addition of methyl viologen influences treatment efficacy.

“Figure 2.4 Addition of methyl viologen influences treatment efficacy.”

Manganese availability influences autolysin activity across multiple strains of S. aureus.

“Figure 2.2 Manganese availability influences autolysin activity across multiple strains of S. aureus.”

Addition of wild-type or ΔS2 calprotectin increases expression of autolysin Atl.

“Addition of wild-type or ΔS2 calprotectin increases expression of autolysins Atl and Sle1”

Addition of calprotectin does not reduce HADA signal intensity.

“Addition of calprotectin does not reduce HADA signal intensity”

The addition of calprotectin results in loss of S. aureus Atl activity.

“The addition of CP was found to result in loss of S. aureus Atl activity”

• Also stated in: § Additional information · plain

  “Autolytic activity is decreased by the zinc limiting activity of calprotectin”

Zinc limitation induces antibiotic tolerance in a mechanism independent of target site activity or metabolic state.

“Figure 1.3 Zinc limitation induces antibiotic tolerance in a mechanism independent of target site activity or metabolic state.”

Manganese limitation sensitizes Staphylococcus aureus to β-lactam antibiotics.

“Manganese limitation sensitizes Staphylococcus aureus to b-lactam antibiotics.”

Mutation in Atl results in loss of calprotectin-induced antibiotic tolerance.

“mutation in Atl resulted in loss of CP-induced antibiotic tolerance.”

TPEN decreases Atl signal in zymogram.

“TPEN decreases Atl signal in zymogram”

In the presence of calprotectin, regions of peptidoglycan accumulation are observed in the S. aureus cell wall.

“Black arrows within the CP-treated condition highlight regions of peptidoglycan accumulation.”

Loss of autolytic activity induces antibiotic tolerance.

“Chelation of zinc or the loss of autolytic activity, induces antibiotic tolerance”

The addition of calprotectin significantly increased S. aureus survival against several β-lactam antibiotics.

“We observed that the addition of CP significantly increased S. aureus survival against several b-lactam antibiotics.”

Addition of wild-type or ΔS2 calprotectin increases expression of autolysin Sle1.

“Addition of wild-type or ΔS2 calprotectin increases expression of autolysins Atl and Sle1”

Zinc limitation induces antibiotic tolerance.

“Figure 1.2 Zinc limitation induces antibiotic tolerance.”

The addition of calprotectin does not change the antibiotic minimum inhibitory concentration of select β-lactams.

“The addition of calprotectin does not change the antibiotic minimum inhibitory concentration of select b-lactams”

Calprotectin negatively impacts the efficacy of cell-wall acting antibiotics against multiple MSSA and MRSA clinical and laboratory strains.

“Calprotectin negatively impacts the efficacy of cell-wall acting antibiotics against multiple MSSA and MRSA clinical and laboratory strains”

Zinc availability dramatically influences the activity of the main S. aureus autolysin Atl via its Zn-dependent amidase domain.

“Our results showed that the activity of the main S. aureus autolysin, Atl, is dramatically influenced by zinc availability due to the Zndependent amidase domain.”

This potentiation was not correlated with a change to the antibiotic MIC.

“This potentiation was not correlated with a change to the antibiotic MIC (Table S1).”

The efficacy of ciprofloxacin is unchanged with the addition of calprotectin.

“The efficacy of ciprofloxacin is unchanged with the addition of calprotectin”

Manganese availability influences antibiotic response.

“Manganese availability influences antibiotic response, autolysin activity and expression”

Autolytic activity is decreased by the zinc limiting activity of calprotectin.

“Figure 1.4 Autolytic activity is decreased by the zinc limiting activity of calprotectin.”

Mn has an indirect effect by increasing autolysin expression in Mn-deplete medium relative to Mn-replete medium.

“Instead, Mn was found to have an indirect effect by increasing autolysin expression in the Mn-deplete medium relative to Mn-replete medium (Fig. 1C).”

Manganese availability influences autolysin expression.

“Manganese availability influences antibiotic response, autolysin activity and expression”

The presence of calprotectin negatively impacts the efficacy of oxacillin at all concentrations examined above the MIC.

“The presence of calprotectin negatively impacts the efficacy of oxacillin at all concentrations examined above the MIC”

Calprotectin-induced antibiotic tolerance is independent of target site activity and metabolic state.

“Because common causes of antibiotic tolerance, i.e. target site activity and metabolic activity, were not inhibited by the presence of CP, bacterial autolytic activity was then considered as a possible driver of antibiotic tolerance.”

• Also stated in: § Additional information · plain

  “Zinc limitation induces antibiotic tolerance in a mechanism independent of target site activity or metabolic state”

Manganese availability has no direct effect on bacterial growth.

“Manganese availability can be influenced in vitro but has no direct effect on the activity of isolated autolysins or bacterial growth”

The supplementation of manganese does not change the antibiotic minimum inhibitory concentration.

“The supplementation of manganese does not change the antibiotic minimum inhibitory concentration”

The presence of calprotectin negatively impacts the efficacy of cell-wall acting antibiotics in vivo.

“Figure 1.5 The presence of calprotectin negatively impacts the efficacy of cell-wall acting antibiotics in vivo.”

• Also stated in: § Additional information · plain

  “The presence of calprotectin negatively impacts the efficacy of cellwall acting antibiotics in vivo”

Manganese availability has no direct effect on the activity of isolated autolysins.

“Manganese availability can be influenced in vitro but has no direct effect on the activity of isolated autolysins or bacterial growth”

Zinc limitation induces antibiotic tolerance in S. aureus.

“Comparison of multiple chemical chelators and use of single site mutants of CP demonstrated this effect was primarily mediated by zinc limitation.”

• Also stated in: § Additional information · plain

  “Zinc limitation induces antibiotic tolerance”

Manganese availability influences autolysin activity.

“Manganese availability influences antibiotic response, autolysin activity and expression”

CP impairs β-lactam activity in vivo.

“In a murine infection model, the efficacy of oxacillin was significantly enhanced in CP-deficient mice, demonstrating that CP impairs βlactam activity in vivo.”

Zinc and manganese are depleted in an S. aureus liver abscess environment.

“with zinc and manganese found to be depleted in an S. aureus liver abscess environment [11].”

• cites [11]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

CP tightly binds first-row transition metals, including zinc, manganese, iron, copper, and nickel.

“A heterodimer of S100A8 and S100A9, CP tightly binds first-row transition metals, including zinc, manganese, iron, copper, and nickel [13][14][15][16].”

• cites [13]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [14]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

CP-induced tolerance, particularly at lower concentrations, is not attributable simply to slowed growth.

“This strongly suggests that CP-induced tolerance, particularly at lower concentrations, is not attributable simply to slowed growth.”

Deletion of autolysin genes enhances bacterial survival following β-lactam treatment.

“Supporting this model, multiple studies have shown that deletion of autolysin genes enhances bacterial survival following β-lactam treatment [41,45,46].”

• cites [41]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [45]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [46]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

CP-induced tolerance is independent of a reduction in metabolic activity.

“These results suggest that CP induced tolerance is independent of a reduction in metabolic activity.”

Zinc limitation is responsible for the lack of autolytic activity in the presence of CP.

“Since both sites can bind zinc, recovery of autolytic activity for only DS1/DS2 implies that zinc limitation is responsible for the lack of autolytic activity in the presence of CP.”

• AssumptionRests on an assumption: Factors within the host environment that influence autolytic activity remain to be determined.

Limitation of zinc and manganese has opposing effects on β-lactam antibiotic efficacy against Staphylococcus aureus.

“Here, we demonstrate that the limitation of zinc and manganese has opposing effects on β-lactam antibiotic efficacy against Staphylococcus aureus.”

• AssumptionRests on an assumption: The host conditions and factors that lead to β-lactam tolerance during infection are poorly understood.

CP decreases antibiotic efficacy primarily through limitation of zinc.

“Comparison between the two site mutants suggested CP decreases antibiotic efficacy primarily through the limitation of zinc as both variants significantly increase bacterial survival post cefepime treatment (Fig. 2A; Fig. S5A).”

Bacterial adaptation to intracellular oxidative stress under Mn-limiting conditions results in increased expression and activity of bacterial autolysins, sensitizing S. aureus to antibiotic treatment.

“Our data suggests that bacterial adaptation to intracellular oxidative stress under Mn-limiting conditions results in the increased expression and activity of bacterial autolysins, ultimately sensitizing S. aureus to antibiotic treatment.”

Zinc chelation by calprotectin directly inactivates bacterial autolysins, zinc-dependent enzymes required for cell wall degradation and bacterial lysis following β-lactam treatment.

“Our results show that zinc chelation by the host immune protein calprotectin directly inactivates bacterial autolysins, zinc-dependent enzymes required for cell wall degradation and bacterial lysis following β-lactam treatment.”

• Also stated in: § CHAPTER 2. THE INNATE IMMUNE PROTEIN CALPROTECTIN ABLATES THE BACTERICIDAL ACTIVITY OF b-LACTAM ANTIBIOTICS 1 · plain

  “Mechanistic studies revealed that CP inhibits the autolytic activity of Atl, the major S. aureus autolysin, by depriving the enzyme of its zinc cofactor.”

• AssumptionRests on an assumption: The host conditions and factors that lead to β-lactam tolerance during infection are poorly understood.
• AssumptionRests on an assumption: Factors within the host environment that influence autolytic activity remain to be determined.

Zinc limitation induces antibiotic tolerance by a mechanism independent of cell wall synthesis inhibition.

“This suggests that zinc limitation induces antibiotic tolerance by a mechanism independent of cell wall synthesis inhibition.”

Zinc limitation leads to the development of tolerance to β-lactam antibiotics.

“Together, these data demonstrate that zinc limitation leads to the development of tolerance to b-lactam antibiotics.”

• AssumptionRests on an assumption: The host conditions and factors that lead to β-lactam tolerance during infection are poorly understood.

Metal limitation is the driver of CP-induced β-lactam tolerance.

“The mutant protein was unable to induce tolerance to oxacillin, strongly implicating metal limitation as the driver of b-lactam tolerance.(Fig 2A ; Fig. S5A).”

CP can potently inhibit the zinc-dependent protease activity of P. aeruginosa extracellular virulence factors LasA and LasB.

“In support of this, CP can potently inhibit the zinc-dependent protease activity of P. aeruginosa extracellular virulence factors LasA and LasB [66].”

• cites [66]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Increased manganese in the diet increases the availability of this metal during S. aureus infection.

“Increased manganese in the diet has been found to increase the availability of this metal during S. aureus infection [73].”

• cites [73]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Loss of CP increases the availability of zinc, particularly in the liver.

“Despite these additional mechanisms, loss of CP does increase the availability of zinc [54,55], particularly in the liver, as evidenced by the increased virulence of an A. baumannii zinc-transporter mutant in the liver of CP-deficient mice [19].”

• cites [54]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [55]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [19]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Mn limitation enhances the efficacy of the primary class of antibiotics used in the clinical treatment of S. aureus infections.

“In summary, these findings demonstrate that Mn limitation enhances the efficacy of the primary class of antibiotics used in the clinical treatment of S. aureus infections.”

The effect of calprotectin on the activity of Zn-dependent autolysins highlights the susceptibility of extracellular Zn-dependent enzymes to host-driven metal sequestration.

“However, the effect of calprotectin on the activity of Zn-dependent autolysins, as detailed in Chapter 2, highlights the susceptibility of extracellular Zn-dependent enzymes to the action of host-driven metal sequestration.”

The ablation of autolysin activity is sufficient to induce significant tolerance to b-lactams.

“Although the ablation of autolysin activity is sufficient to induce significant tolerance to b-lactams, CP may also impact antibiotic tolerance through inhibition of other zinc and manganese dependent processes that impact cell growth, particularly at higher concentrations.”

Zinc limitation induces tolerance to b-lactam antibiotics.

“While CP binds multiple first row transition metals tightly [13][14][15][16], the current results strongly support that zinc limitation induces tolerance to blactam antibiotics.”

• cites [13]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [14]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [16]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

CP dramatically increases survival against the main class of antibiotics used in the treatment of S. aureus infections.

“In summary, these results show that CP dramatically increases survival against the main class of antibiotics used in the treatment of S. aureus infections.”

Numerous autolysins over a broad range of Gram-positive and Gram-negative species are extracellular metalloenzymes requiring zinc for autolytic activity.

“Numerous autolysins over a broad range of Gram-positive and Gram-negative species are extracellular metalloenzymes requiring zinc for autolytic activity, including Clostridioides difficile Cwp6, Streptococcus pneumoniae LytA, Helicobacter pylori Csd2, and Escherichia coli AmiA/B/C, among many others [68][69][70][71].”

• cites [68]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [69]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [70]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [71]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

CP induces increased autolysin expression as a result of Mn limitation.

“As only wildtype CP and DS2 CP chelate Mn from the media, these results suggest that CP induces increased autolysin expression as a result of Mn limitation.”

Excess Mn decreases neutrophil antibacterial activity against S. aureus by impairing suicidal neutrophil extracellular trap (NET) formation.

“Additionally, excess Mn was found to decrease neutrophil antibacterial activity against S. aureus by impairing suicidal neutrophil extracellular trap (NET) formation [83].”

• cites [83]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Mice that are S100A9-/- are essentially deficient in CP.

“As CP is a heterodimeric S100 protein formed by association of monomers S100A8 and S100A9, mice that are S100A9-/-are essentially deficient in CP [57].”

• cites [57]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Tolerance to cell-wall acting antibiotics has uniquely been shown to develop from disruptions in peptidoglycan turnover.

“While multiple genotypic and environmental causes of tolerance are mediated through decreased metabolic activity or the adoption of a slow or nongrowing state, tolerance to cell-wall acting antibiotics has uniquely been shown to develop from disruptions in peptidoglycan turnover [41].”

• cites [41]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Antibiotic efficacy is dramatically modulated by the host.

“Several studies have shown that antibiotic efficacy is dramatically modulated by the host [39,[60][61][62][63].”

• cites [39,[60][61][62][63]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Increased autolytic activity under manganese limitation is mediated by elevated levels of reactive oxygen species, normally detoxified by bacterial Mn-dependent superoxide dismutases.

“This increased autolytic activity was found to be mediated by elevated levels of reactive oxygen species, normally detoxified by bacterial Mn-dependent superoxide dismutases.”

Inhibition of autolysin activity may contribute to the development of tolerance in the presence of CP.

“This suggests that inhibition of autolysin activity may contribute to the development of tolerance in the presence of CP.”

This study provides the first demonstration of a host-induced state of antibiotic tolerance mediated by targeted inactivation of a bacterial enzyme.

“This study provides the first demonstration of a host-induced state of antibiotic tolerance mediated by targeted inactivation of a bacterial enzyme.”

Antibiotic resistance alone is insufficient to account for all cases of treatment failure.

“While antibiotic resistance is a common cause of treatment failure, resistance alone is insufficient to account for all cases.”

The contribution of CP to metal limitation varies by tissue.

“The contribution of CP to metal limitation varies by tissue.”

Aureolysin is an extracellular metalloprotease produced by S. aureus that requires zinc for catalytic activity.

“the metalloprotease aureolysin is an extracellular enzyme produced by S. aureus which requires zinc for catalytic activity [86].”

• cites [86]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Extracellular zinc-dependent enzymes are vulnerable to CP sequestration of zinc.

“However, extracellular zincdependent enzymes are vulnerable to CP sequestration of zinc.”

The increased efficacy of b-lactams observed in the liver of CP-deficient mice in this study is driven by increased zinc availability.

“This suggests that the increased efficacy of b-lactams observed in the liver of CP-deficient mice in this study is driven by increased zinc availability.”

Our findings suggest that a broader spectrum of host-pathogen interactions can substantially alter antibiotic efficacy.

“While antibiotic tolerance has traditionally been attributed to metabolic downregulation or growth arrest, our findings suggest that a broader spectrum of host-pathogen interactions can substantially alter antibiotic efficacy.”

Even in the presence of high levels of CP, S. aureus is well-equipped to transport zinc and maintain sufficient levels for intracellular functionality.

“Even in the presence of high levels of CP, S. aureus is well-equipped to transport zinc and maintain sufficient levels for intracellular functionality [28,57].”

• cites [28]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [57]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Oxidative stress induces multi-drug tolerance in S. aureus through inactivation of TCA cycle enzymes containing iron-sulfur clusters.

“However, oxidative stress is also found to induce multi-drug tolerance in S. aureus through inactivation of the TCA cycle enzymes containing iron-sulfur clusters [39].”

• cites [39]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Manganese limitation potentiates the antibiotic killing of S. aureus through increased autolytic activity.

“These findings suggest that manganese limitation potentiates the antibiotic killing of S. aureus through increased autolytic activity and may serve as a strategy to enhance the effectiveness of antibiotic treatment.”

The presence of calprotectin negatively impacts the efficacy of cell-wall acting antibiotics during bacteremia.

“The presence of calprotectin negatively impacts the efficacy of cell-wall acting antibiotics during bacteremia.”

• AssumptionRests on an assumption: The S100A9-/- mouse model specifically isolates the effect of CP deficiency on antibiotic efficacy.
• AssumptionRests on an assumption: The mouse bacteremia model is predictive of human S. aureus infection and antibiotic treatment outcomes.

Copper levels in urine increase in response to UTI caused by uropathogenic Escherichia coli or Proteus mirabilis.

“copper levels in the urine increase in response to UTI caused by uropathogenic Escherichia coli or Proteus mirabilis [100].”

• cites [100]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Calprotectin can induce antibiotic tolerance through the ablation of autolysin activity.

“Together these results demonstrate that calprotectin can induce antibiotic tolerance through the ablation of autolysin activity.”

Manipulation of the rodent diet is sufficient to alter the concentrations of metals within tissues.

“Multiple studies have shown that manipulation of the rodent diet is sufficient to alter the concentrations of metals within tissues [73,113].”

• cites [73]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [113]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Chemical chelators of iron, deferasirox and DIBI, improve the antibacterial activity of antibiotics against S. aureus.

“these results show improved antibacterial activity of antibiotics with the addition of an iron chelator [95,96].”

• cites [95]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [96]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Chelation of Zn using EDTA inhibits the formation of cell-cell adhesions important to S. aureus biofilm formation.

“the chelation of Zn using the chemical chelator EDTA was found to inhibit the formation of cell-cell adhesions important to S. aureus biofilm formation [90].”

• cites [90]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• AssumptionRests on an assumption: SasG is a Zn-dependent cell-wall anchored surface protein involved in biofilm formation.
• AssumptionRests on an assumption: Cell adhesion plays a critical role in biofilm formation.
• AssumptionRests on an assumption: SasG contains multiple G5 domains involved in Zn-dependent adhesion.

Comparison of strains using a murine wound infection model showed improved S. aureus survival for strains producing Spm during co-infection with P. aeruginosa.

“Comparison of strains using a murine wound infection model showed improved S. aureus survival for strains producing Spm during co-infection with P. aeruginosa [112].”

• cites [112]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The phagolysosome environment is restricted of iron, zinc, and manganese through the action of NRAMP transporters.

“the phagolysosome environment is also restricted of iron, zinc, and manganese through the action of NRAMP transporters [104].”

• cites [104]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Zinc-limiting conditions could have major impacts on the downstream proteolytic activity of S. aureus.

“zinc-limiting conditions could have major impacts on the downstream proteolytic activity of S. aureus.”

Addition of lactoferrin improves antibiotic killing.

“studies examining the effects of lactoferrin on antibiotic efficacy, have found that the addition of lactoferrin improves antibiotic killing [93].”

• cites [93]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Calprotectin ablates the bactericidal activity of β-lactam antibiotics.

“The innate immune protein calprotectin ablates the bactericidal activity of b-lactam antibiotics.”

• Also stated in: § Additional information · plain

  “Calprotectin negatively impacts the efficacy of cell-wall acting antibiotics”

Copper accumulation within the phagolysosome is an important antibacterial mechanism employed by macrophages for the elimination of internalized pathogens.

“copper accumulation within the phagolysosome is an important antibacterial mechanism employed by macrophages for the elimination of internalized pathogens [101-103].”

• cites [101]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [102]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [103]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The study of metal stress on internalized pathogens presents a complex environment with the potential to dramatically influence cell physiology and response to antibiotic treatment, particularly for pathogens which survive and replicate intracellularly.

“Thus, the study of metal stress on internalized pathogens presents a complex environment with the potential to dramatically influence cell physiology and response to antibiotic treatment, particularly for pathogens which survive and replicate intracellularly.”

S. aureus is not limited intracellularly of copper by the action of calprotectin.

“While S. aureus is not limited intracellularly of copper by the action of calprotectin [57]”

• cites [57]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Calprotectin is capable of sequestering additional first row transition metals such as iron, copper, cobalt, and nickel.

“CP is also capable of sequestering additional first row transition metals such as iron, copper, cobalt, and nickel [13][14][15][16]57].”

• cites [13]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [14]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [15]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Using a murine wound infection model, E. coli abundance within the wound environment was significantly increased during co-infection with E. faecalis.

“using a murine wound infection model, researchers observed that E. coli abundance within the wound environment was significantly increased during co-infection with E. faecalis.”

• cites [110]Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.