scholarly journals RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
C. J. Kovacs ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Critical Function ◽  
Content Type ◽  
The Impact

ABSTRACT Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans. IMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

scholarly journals BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans

2012 ◽  
Vol 78 (8) ◽  
pp. 2914-2922 ◽  
Author(s):  
J. P. Bitoun ◽  
S. Liao ◽  
X. Yao ◽  
S.-J. Ahn ◽  
R. Isoda ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Stress Responses ◽  
Antimicrobial Agents ◽  
Galleria Mellonella ◽  
Cell Envelope ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Lipid Ii ◽  
The Impact

ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo

2019 ◽  
Vol 63 (3) ◽  
Author(s):  
Stefanie Gerson ◽  
Jonathan W. Betts ◽  
Kai Lucaßen ◽  
Carolina Silva Nodari ◽  
Julia Wille ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Galleria Mellonella ◽  
Resistance Mechanism ◽  
Resistant Isolate ◽  
Infection Model ◽  
Colistin Resistance ◽  
Strain Specificity ◽  
Content Type

ABSTRACT Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

scholarly journals Generic Vancomycin Enriches Resistant Subpopulations of Staphylococcus aureus after Exposure in a Neutropenic Mouse Thigh Infection Model

2011 ◽  
Vol 56 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Carlos A. Rodriguez ◽  
Maria Agudelo ◽  
Andres F. Zuluaga ◽  
Omar Vesga
Keyword(s):  
Staphylococcus Aureus ◽  
Population Analysis ◽  
Recovery Process ◽  
Control Group ◽  
Infection Model ◽  
Content Type ◽  
Sterile Saline ◽  
Generic Products ◽  
The Impact

ABSTRACTPrevious studies have shown that “bioequivalent” generic products of vancomycin are less effectivein vivoagainstStaphylococcus aureusthan the innovator compound. Considering that suboptimal bactericidal effect has been associated with emergence of resistance, we aimed to assessin vivothe impact of exposure to innovator and generic products of vancomycin onS. aureussusceptibility. A clinical methicillin-resistantS. aureus(MRSA) strain from a liver transplant patient with persistent bacteremia was used for which MIC, minimum bactericidal concentration (MBC), and autolytic properties were determined. Susceptibility was also assessed by determining a population analysis profile (PAP) with vancomycin concentrations from 0 to 5 mg/liter. ICR neutropenic mice were inoculated in each thigh with ∼7.0 log10CFU. Treatment with the different vancomycin products (innovator and three generics; 1,200 mg/kg of body weight/day every 3 h) started 2 h later while the control group received sterile saline. After 24 h, mice were euthanized, and the thigh homogenates were plated. Recovered colonies were reinoculated to new groups of animals, and the exposure-recovery process was repeated until 12 cycles were completed. The evolution of resistance was assessed by PAP after cycles 5, 10, 11, and 12. The initial isolate displayed reduced autolysis and higher resistance frequencies thanS. aureusATCC 29213 but without vancomycin-intermediateS. aureus(VISA) subpopulations. After 12 cycles, innovator vancomycin had significantly reduced resistant subpopulations at 1, 2, and 3 mg/liter, while the generic products had enriched them progressively by orders of magnitude. The great capacity of generic vancomycin to select for less susceptible organisms raises concerns about the role of therapeutic inequivalence of any antimicrobial on the epidemiology of resistance worldwide.

scholarly journals Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

2012 ◽  
Vol 12 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Yeissa Chabrier-Roselló ◽  
Kimberly J. Gerik ◽  
Kristy Koselny ◽  
Louis DiDone ◽  
Jennifer K. Lodge ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Cryptococcus Neoformans ◽  
Nitrosative Stress ◽  
Galleria Mellonella ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Oxidative And Nitrosative Stress ◽  
Independent Manner ◽  
Content Type

ABSTRACTCryptococcus neoformansPKH2-01andPKH2-02are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied inS. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show thatPKH2-02but notPKH2-01is required forC. neoformansto tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion ofPKH2-02leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress.PKH2-02function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using aGalleria mellonellamodel of low-temperature cryptococcosis, we found thatPKH2-02is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of thepkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion ofPKH2-02affects the interaction betweenC. neoformansand phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling inC. neoformansis crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.

scholarly journals The Novel β-Lactam Enhancer Zidebactam Augments theIn VivoPharmacodynamic Activity of Cefepime in a Neutropenic Mouse LungAcinetobacter baumanniiInfection Model

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
S. S. Bhagwat ◽  
H. Periasamy ◽  
S. S. Takalkar ◽  
S. R. Palwe ◽  
H. N. Khande ◽  
...  
Keyword(s):  
Bactericidal Effect ◽  
Multidrug Resistant ◽  
Mouse Lung ◽  
Infection Model ◽  
Bactericidal Action ◽  
Content Type ◽  
Time Kill ◽  
The Impact

ABSTRACTWCK 5222 is a combination of cefepime and the high-affinity PBP2-binding β-lactam enhancer zidebactam. The cefepime-zidebactam combination is active against multidrug-resistant Gram-negative bacteria, including carbapenemase-expressingAcinetobacter baumannii. The mechanism of action of the combination involves concurrent multiple penicillin binding protein inhibition, leading to the enhanced bactericidal action of cefepime. The aim of the present study was to assess the impact of the zidebactam-mediated enhancedin vitrobactericidal action in modulating the percentage of the time that the free drug concentration remains above the MIC (percentfT>MIC) for cefepime required for thein vivokilling ofA. baumannii. Cefepime and cefepime-zidebactam MICs were comparable and ranged from 2 to 16 mg/liter for theA. baumanniistrains (n = 5) employed in the study. Time-kill studies revealed the improved killing of these strains by the cefepime-zidebactam combination compared to that by the constituents alone. Employing a neutropenic mouse lung infection model, exposure-response analyses for all theA. baumanniistrains showed that the cefepimefT>MIC required for 1-log10kill was 38.9%. In the presence of a noneffective dose of zidebactam, the cefepimefT>MIC requirement dropped significantly to 15.5%, but it still rendered a 1-log10kill effect. Thus, zidebactam mediated the improvement in cefepime’s bactericidal effect observed in time-kill studies, manifestedin vivothrough the lowering of cefepime’s pharmacodynamic requirement. This is a first-ever study demonstrating a β-lactam enhancer role of zidebactam that helps augment thein vivoactivity of cefepime by reducing the magnitude of its pharmacodynamically relevant exposures againstA. baumannii.

scholarly journals Minimal Inhibitory Concentration (MIC)-Phenomena in Candida albicans and Their Impact on the Diagnosis of Antifungal Resistance

2019 ◽  
Vol 5 (3) ◽  
pp. 83 ◽  
Author(s):  
Ulrike Binder ◽  
Maria Aigner ◽  
Brigitte Risslegger ◽  
Caroline Hörtnagl ◽  
Cornelia Lass-Flörl ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Treatment Response ◽  
Minimal Inhibitory Concentration ◽  
Antifungal Therapy ◽  
Inhibitory Concentration ◽  
Galleria Mellonella ◽  
Antifungal Susceptibility ◽  
Infection Model ◽  
The Impact

Antifungal susceptibility testing (AFST) of clinical isolates is a tool in routine diagnostics to facilitate decision making on optimal antifungal therapy. The minimal inhibitory concentration (MIC)-phenomena (trailing and paradoxical effects (PXE)) observed in AFST complicate the unambiguous and reproducible determination of MICs and the impact of these phenomena on in vivo outcome are not fully understood. We aimed to link the MIC-phenomena with in vivo treatment response using the alternative infection model Galleria mellonella. We found that Candida albicans strains exhibiting PXE for caspofungin (CAS) had variable treatment outcomes in the Galleria model. In contrast, C. albicans strains showing trailing for voriconazole failed to respond in vivo. Caspofungin- and voriconazole-susceptible C. albicans strains responded to the respective antifungal therapy in vivo. In conclusion, MIC data and subsequent susceptibility interpretation of strains exhibiting PXE and/or trailing should be carried out with caution, as both effects are linked to drug adaptation and treatment response is uncertain to predict.

scholarly journals Postantibiotic and Sub-MIC Effects of Exebacase (Lysin CF-301) Enhance Antimicrobial Activity againstStaphylococcus aureus

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Jun Taek Oh ◽  
Cara Cassino ◽  
Raymond Schuch
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
The United States ◽  
Infection Model ◽  
Postantibiotic Effect ◽  
Content Type ◽  
Wall Ultrastructure ◽  
Bacterial Fitness

ABSTRACTCF-301 (exebacase) is a recombinantly produced bacteriophage-derived lysin (cell wall hydrolase) and is the first agent of this class to enter clinical development in the United States for treating bacteremia including endocarditis due toStaphylococcus aureus. Whereas rapid bactericidal activity is the hallmarkin vitroandin vivoresponse to CF-301 at exposures higher than the MIC, prolonged antimicrobial activity, mediated by cell wall damage, is predicted at concentrations less than the MIC. In the current study, a series ofin vitropharmacodynamic parameters, including the postantibiotic effect (PAE), postantibiotic sub-MIC effect (PA-SME), and sub-MIC effect (SME), were studied to determine how short-duration and sub-MIC CF-301 exposures affect the growth of surviving staphylococci and extend its antimicrobial activity. Mean PAE, PA-SME, and SME values up to 4.8, 9.3, and 9.8 h, respectively, were observed against 14 staphylococcal strains tested in human serum; growth delays were extended by 6 h in the presence of daptomycin. Exposures to CF-301 at sub-MIC levels as low as 0.001× to 0.01× MIC (∼1 to 10 ng/ml) resulted in aberrant cell wall ultrastructure, increased membrane permeability, dissipation of membrane potential, and inhibition of virulence phenotypes, including agglutination and biofilm formation. A mouse thigh infection model designed to study the PAE was used to confirm our findings and demonstratein vivogrowth delays of ≥19.3 h. Our findings suggest that at CF-301 concentrations less than the MIC during therapeutic use, sustained reductions in bacterial fitness and virulence may substantially enhance efficacy.

scholarly journals Chitosan Ameliorates Candida auris Virulence in a Galleria mellonella Infection Model

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Laís Salomão Arias ◽  
Mark C. Butcher ◽  
Bryn Short ◽  
Emily McKloud ◽  
Chris Delaney ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Survival Rates ◽  
Health Care Facilities ◽  
Antifungal Drugs ◽  
Infection Model ◽  
Candida Auris ◽  
Gene Expression Response ◽  
Content Type

ABSTRACT Candida auris has emerged as a multidrug-resistant nosocomial pathogen over the last decade. Outbreaks of the organism in health care facilities have resulted in life-threatening invasive candidiasis in over 40 countries worldwide. Resistance by C. auris to conventional antifungal drugs such as fluconazole and amphotericin B means that alternative therapeutics must be explored. As such, this study served to investigate the efficacy of a naturally derived polysaccharide called chitosan against aggregative (Agg) and nonaggregative (non-Agg) isolates of C. auris in vitro and in vivo. In vitro results indicated that chitosan was effective against planktonic and sessile forms of Agg and non-Agg C. auris. In a Galleria mellonella model to assess C. auris virulence, chitosan treatment was shown to ameliorate killing effects of both C. auris phenotypes (NCPF 8973 and NCPF 8978, respectively) in vivo. Specifically, chitosan reduced the fungal load and increased survival rates of infected Galleria, while treatment alone was nontoxic to the larvae. Finally, chitosan treatment appeared to induce a stress-like gene expression response in NCPF 8973 in the larvae likely arising from a protective response by the organism to resist antifungal activity of the compound. Taken together, results from this study demonstrate that naturally derived compounds such as chitosan may be useful alternatives to conventional antifungals against C. auris.

scholarly journals In VivoActivities of Ceftolozane, a New Cephalosporin, with and without Tazobactam against Pseudomonas aeruginosa and Enterobacteriaceae, Including Strains with Extended-Spectrum β-Lactamases, in the Thighs of Neutropenic Mice

2012 ◽  
Vol 57 (4) ◽  
pp. 1577-1582 ◽  
Author(s):  
W. A. Craig ◽  
D. R. Andes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Infection Model ◽  
Wild Type ◽  
Gram Negative ◽  
Content Type ◽  
Peak Dose ◽  
Extended Spectrum ◽  
The Impact ◽  
Primary Index

ABSTRACTCeftolozane is a new cephalosporin with potent activity againstPseudomonas aeruginosaandEnterobacteriaceae. A neutropenic murine thigh infection model was used to determine which pharmacokinetic/pharmacodynamic index and magnitude drives the efficacy of ceftolozane with Gram-negative bacilli, to compare the rates ofin vivokilling ofP. aeruginosaby ceftolozane and ceftazidime, and to determine the impact of different ratios of ceftolozane plus tazobactam onEnterobacteriaceaecontaining extended-spectrum β-lactamases (ESBLs). Neutropenic mice had 106.2-7.1CFU/thigh when treated with ceftolozane for 24 h with (i) various doses (3.12 to 1,600 mg/kg) and dosage intervals (3, 6, 12, and 24 h) against twoEnterobacteriaceaestrains, (ii) 0.39 to 800 mg/kg every 6 h for fourEnterobacteriaceaeand fourP. aeruginosastrains, and (iii) 400 or 800 mg/kg with 2:1. 4:1, and 8:1 ratios of tazobactam against fiveEnterobacteriaceaestrains with ESBLs. The pharmacokinetics of ceftolozane at 25, 100, and 400 mg/kg were linear with peak/dose values of 1.0 to 1.4 and half-lives of 12 to 14 min. T>MIC was the primary index driving efficacy. For stasis (1 log kill), T>MIC was 26.3% ± 2.1% (31.6% ± 1.6%) for wild-typeEnterobacteriaceae, 31.1% ± 4.9% (34.8% ± 4.4%) forEnterobacteriaceaewith ESBLs, and 24.0% ± 3.3% (31.5% ± 3.9%) forP. aeruginosa. At 200 mg/kg every 3 h, the rate ofin vivokilling ofP. aeruginosawas faster with ceftolozane than with ceftazidime (−0.34 to −0.41 log10CFU/thigh/h versus −0.21 to −0.24 log10CFU/thigh/h). The 2:1 ratio of ceftolozane with tazobactam was the most potent combination studied. The T>MIC required for ceftolozane is less than with other cephalosporins and may be due to more rapid killing.

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