scholarly journals Genetic dissection of the fermentative and respiratory contributions supporting Vibrio cholerae hypoxic growth

2020 ◽  
Author(s):  
Emilio Bueno ◽  
Brandon Sit ◽  
Matthew K. Waldor ◽  
Felipe Cava
Keyword(s):  
Vibrio Cholerae ◽  
Respiratory Chain ◽  
Antimicrobial Agents ◽  
Infection Model ◽  
Genetic Dissection ◽  
Low Oxygen ◽  
Pathogen Fitness ◽  
Antimicrobial Interventions

ABSTRACTBoth fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors such as nitrate (NO3−). During canonical NO3− respiration, NO3− is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, only requires a single periplasmic NO3− reductase (NapA) to undergo NO3− respiration, suggesting that the pathogen possesses a non-canonical NO3− respiratory chain. Here, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3− respiration in V. cholerae. We found that while the V. cholerae NO3− respiratory chain is primarily composed of homologues of established NO3− respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic bases for interactions between disparate energy production pathways that support pathogen fitness in shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions.IMPORTANCEBacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways-fermentation and respiration. Here, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3− that consists largely of components found in other NO3− respiratory systems, but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3− respiration were required for efficient pathogen growth in both laboratory conditions and in an animal infection model. These observations provide genetic evidence for fermentative-respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

scholarly journals Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Emilio Bueno ◽  
Brandon Sit ◽  
Matthew K. Waldor ◽  
Felipe Cava
Keyword(s):  
Vibrio Cholerae ◽  
Respiratory Chain ◽  
Antimicrobial Agents ◽  
Specific Interaction ◽  
Infection Model ◽  
Genetic Dissection ◽  
Low Oxygen ◽  
Content Type

ABSTRACT Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors, such as nitrate (NO3−). During canonical NO3− respiration, NO3− is reduced in a stepwise manner to N2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, requires only a single periplasmic NO3− reductase (NapA) to undergo NO3− respiration, suggesting that the pathogen possesses a noncanonical NO3− respiratory chain. In this study, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3− respiration in V. cholerae. We found that while the V. cholerae NO3− respiratory chain is primarily composed of homologues of established NO3− respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic basis of a specific interaction between disparate energy production pathways that supports pathogen fitness under shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions. IMPORTANCE Bacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways: fermentation and respiration. In this study, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3− that consists largely of components found in other NO3− respiratory systems but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3− respiration were required for efficient pathogen growth under both laboratory conditions and in an animal infection model. These observations provide a specific example of fermentative respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.

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 Fusidic Acid-Resistant Mutants of Salmonella enterica Serovar Typhimurium with Low Fitness In Vivo Are Defective in RpoS Induction

2003 ◽  
Vol 47 (12) ◽  
pp. 3743-3749 ◽  
Author(s):  
Mirjana Macvanin ◽  
Johanna Björkman ◽  
Sofia Eriksson ◽  
Mikael Rhen ◽  
Dan I. Andersson ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fusidic Acid ◽  
Salmonella Enterica ◽  
Antimicrobial Agents ◽  
Relative Fitness ◽  
Infection Model ◽  
Macrophage Infection ◽  
Serovar Typhimurium

ABSTRACT Mutants of Salmonella enterica serovar Typhimurium resistant to fusidic acid (Fusr) have mutations in fusA, the gene encoding translation elongation factor G (EF-G). Most Fusr mutants have reduced fitness in vitro and in vivo, in part explained by mutant EF-G slowing the rate of protein synthesis and growth. However, some Fusr mutants with normal rates of protein synthesis still suffer from reduced fitness in vivo. As shown here, Fusr mutants could be similarly ranked in their relative fitness in mouse infection models, in a macrophage infection model, in their relative hypersensitivity to hydrogen peroxide in vivo and in vitro, and in the amount of RpoS production induced upon entry into the stationary phase. We identify a reduced ability to induce production of RpoS (σs) as a defect associated with Fusr strains. Because RpoS is a regulator of the general stress response, and an important virulence factor in Salmonella, an inability to produce RpoS in appropriate amounts can explain the low fitness of Fusr strains in vivo. The unfit Fusr mutants also produce reduced levels of the regulatory molecule ppGpp in response to starvation. Because ppGpp is a positive regulator of RpoS production, we suggest that a possible cause of the reduced levels of RpoS is the reduction in ppGpp production associated with mutant EF-G. The low fitness of Fusr mutants in vivo suggests that drugs that can alter the levels of global regulators of gene expression deserve attention as potential antimicrobial agents.

scholarly journals Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS− strains generate small colony variants in vivo that are distinct from in vitro variants

Microbiology ◽  
2010 ◽  
Vol 156 (12) ◽  
pp. 3699-3709 ◽  
Author(s):  
Lisa K. Nelson ◽  
M. Mark Stanton ◽  
Robyn E. A. Elphinstone ◽  
Janessa Helwerda ◽  
Raymond J. Turner ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phenotypic Variation ◽  
Antimicrobial Agents ◽  
Mucosal Surface ◽  
Infection Model ◽  
Bacterial Survival ◽  
Small Colony Variants ◽  
Small Colony

Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS− strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS− did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS− parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.

scholarly journals Blocking the alternative sigma factor RpoN reduces virulence ofPseudomonas aeruginosaisolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

2018 ◽  
Author(s):  
MG Lloyd ◽  
JL Vossler ◽  
CT Nomura ◽  
JF Moffat
Keyword(s):  
Cystic Fibrosis ◽  
Sigma Factor ◽  
Antimicrobial Agents ◽  
Laboratory Strain ◽  
Infection Model ◽  
Alternative Sigma Factor ◽  
Beta Lactam ◽  
Cis Acting

AbstractMultidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria.Pseudomonas aeruginosais an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections.P. aeruginosais inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment.P. aeruginosahas virulence factors that are regulated by sigma factors in response to the tissue microenvironment. The alternative sigma factor, RpoN (σ54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a “molecular roadblock”, binding RpoN consensus promoters at the −24 site and blocking transcription. RpoN* reduces virulence ofP. aeruginosalaboratory strains bothin vitroandin vivo,but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically variedP. aeruginosastrains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in aP. aeruginosa – C. elegansinfection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strainP. aeruginosaPA19660Xen5. Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

scholarly journals Pyocin efficacy in a murine model of Pseudomonas aeruginosa sepsis

2020 ◽  
Author(s):  
Anne Six ◽  
Khedidja Mosbahi ◽  
Madhuri Barge ◽  
Colin Kleanthous ◽  
Thomas Evans ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Murine Model ◽  
Antimicrobial Agents ◽  
Galleria Mellonella ◽  
Bloodstream Infections ◽  
Infection Model ◽  
Antibiotic Resistant ◽  
Naturally Occurring

SynopsisBackgroundBloodstream infections with antibiotic resistant Pseudomonas aeruginosa are common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced by P. aeruginosa that have potential for human use.ObjectivesTo determine if pyocin treatment is effective in a murine model of sepsis with P. aeruginosa.MethodsRecombinant pyocins S5 and AP41 were purified tested for efficacy in a Galleria mellonella infection model and a murine model of P. aeruginosa sepsis.ResultsBoth pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis using Galleria mellonella, both pyocins significantly prolonged survival. Following injection into mice, both showed extensive distribution into different organs. When administered 5 hours after infection, both pyocins reduced mortality, with pyocin S5 being more effective than AP41.ConclusionsPyocins S5 and AP41 show in vivo biological activity and can improve survival in a murine model of P. aeruginosa infection. They hold promise as novel antimicrobial agents for treatment of multi-drug resistant infections with this microbe.

scholarly journals Dietary Isothiocyanates, Sulforaphane and 2-Phenethyl Isothiocyanate, Effectively Impair Vibrio cholerae Virulence

2021 ◽  
Vol 22 (19) ◽  
pp. 10187
Author(s):  
Klaudyna Krause ◽  
Agnieszka Pyrczak-Felczykowska ◽  
Monika Karczewska ◽  
Magdalena Narajczyk ◽  
Anna Herman-Antosiewicz ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Galleria Mellonella ◽  
Stringent Response ◽  
In Vitro Assays ◽  
Infection Model ◽  
Phenethyl Isothiocyanate ◽  
Oral Rehydration ◽  
Dna And Rna

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.

scholarly journals Activity of β-lactam antibiotics against Metallo-β-lactamase-producing Enterobacterales in Animal Infection Models: A Current State of Affairs

2021 ◽  
Author(s):  
Tomefa E. Asempa ◽  
Kamilia Abdelraouf ◽  
David P. Nicolau
Keyword(s):  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Infection Model ◽  
Model Studies ◽  
Animal Infection ◽  
Zinc Concentrations ◽  
State Of Affairs ◽  
Reported Data

Metallo-β-lactamases (MBL) result in resistance to nearly all β-lactam antimicrobial agents as determined by currently employed susceptibility testing methods. However, recently reported data demonstrating that variable and supra-physiologic zinc concentrations in conventional susceptibility testing media compared with physiologic (bioactive) zinc concentrations may be mediating discordant in vitro-in vivo MBL-resistance. While treatment outcomes in patients appear suggestive of this discordance, these limited data are confounded by comorbidities and combination therapy. To that end, the goal of this review is to evaluate the extent of β-lactam activity against MBL-harboring Enterobacterales in published animal infection model studies and provide contemporary considerations to facilitate the optimization of current antimicrobials and development of novel therapeutics.

scholarly journals Postantibiotic and Sub-MIC Effects of Azithromycin and Isepamicin against Staphylococcus aureus and Escherichia coli

1998 ◽  
Vol 42 (2) ◽  
pp. 414-418 ◽  
Author(s):  
F. Fuentes ◽  
J. Izquierdo ◽  
M. M. Martín ◽  
M. L. Gomez-Lus ◽  
J. Prieto
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Infection Model ◽  
Continuous Administration ◽  
E Coli ◽  
Previously Treated ◽  
In Vitro Data

ABSTRACT Investigations of pharmacodynamic parameters (postantibiotic effect [PAE], sub-MIC effects [SMEs], etc.) have been progressively employed for the design of dosing schedules of antimicrobial agents. However, there are fewer in vivo than in vitro data, probably because of the simplicity of the in vitro procedures. In this study, we have investigated the in vitro PAE, SME, and previously treated (postantibiotic [PA]) SME (1/2 MIC, 1/4 MIC and 1/8 MIC) of azithromycin and isepamicin against standard strains ofStaphylococcus aureus and Escherichia coliby using centrifugation to remove the antibiotics. In addition, the in vivo PAE and SME have been studied with the thigh infection model in neutropenic mice. Finally, in vivo killing curves with two dosing schedules were determined to examine whether the PAE can cover the time that antimicrobial agents are below the MIC. The two antimicrobial agents induced moderate-to-high in vitro PAEs, SMEs, and PA SMEs against S. aureus (>8 h) andE. coli (3.38 to >7.64 h). The in vivo PAEs were also high (from 3.0 to 3.6 h), despite the fact that isepamicin had lower times above the MIC in serum. Only azithromycin showed a high in vivo SME against the two strains (1.22 and 1.75 h), which indicated that the in vivo PAEs were possibly overestimated. In the killing kinetics, no great differences (<0.5 log10) were observed between the schedule that took the PAE into account and the continuous administration of doses. These results are comparable with those of other authors and suggest that these antimicrobial agents could be administered at longer intervals without losing effectiveness.

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