Ranking of Major Classes of Antibiotics for Activity against Stationary Phase Pseudomonas aeruginosa and Identification of Clinafloxacin + Cefuroxime + Gentamicin Drug Combination that Eradicates Persistent P. aeruginosa Infection in a Murine Cystic Fibrosis Model

2019 ◽  
Author(s):  
Yuting Yuan ◽  
Rebecca Yee ◽  
Naina Gour ◽  
Xinzhong Dong ◽  
Jie Feng ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Stationary Phase ◽  
Drug Combination ◽  
Rna Synthesis ◽  
Bacterial Load ◽  
Sulfa Drugs ◽  
Protein Synthesis Inhibitors ◽  
Persistent Infections ◽  
Biofilm Bacteria ◽  
Dna Synthesis Inhibitors

AbstractPseudomonas aeruginosa can cause serious persistent infections such as ventilator-associated pneumonia, sepsis, biofilm-related infections as in cystic fibrosis (CF) patients. Although CF lung infections can be treated with antibiotics, full clearance is difficult due to P. aeruginosa persistence. While antibiotic activity against growing P. aeruginosa is well documented, their activity against the non-growing persisters enriched in stationary phase cultures has not been well studied. Here, we systematically evaluated and ranked the six major classes of antibiotics, cell wall and cell membrane inhibitors, protein synthesis inhibitors, DNA synthesis inhibitors, RNA synthesis inhibitors, sulfa drugs, and nitrofurantoin, for their activity against both growing and persister forms of P. aeruginosa using colony forming count (CFU) and SYBR Green I/Propidium Iodide (PI) viability assay. Among the six major classes of antibiotics, cell wall and cell membrane inhibitors (Cefuroxime and Colistin), DNA synthesis inhibitors (Clinafloxacin) and sulfa drugs (Sulfamethoxazole) had good activity against stationary phase cells. In contrast, protein synthesis inhibitors (Gentamicin), RNA synthesis inhibitor (Rifampicin) and Nitrofurantoin had relatively poor activity against the stationary phase P. aeruginosa but relatively high activity against log phase P. aeruginosa. Clinafloxacin is the only single drug that could completely kill all (109 CFU) stationary phase cells in a 4 day drug exposure. The Cefuroxime + Gentamicin+ Clinafloxacin combination could kill all biofilm bacteria in 2 days whereas the clinically used drug combination Cefuroxime + Gentamicin + Colistin only partially killed the biofilm bacteria with 103 CFU remaining. In a murine persistent CF lung infection model, only Cefuroxime + Gentamicin+ Clinafloxacin cleared all bacteria in the infected lungs, whereas Clinafloxacin alone, or Cefuroxime + Clinafloxacin, or the current recommended drug combination Cefuroxime + Gentamicin, all failed to completely clear the bacterial load in the lungs. The complete sterilization of the bacterial load is a property of Clinafloxacin combination, as Cefuroxime + Gentamicin+ Levofloxacin combination was unable to clear the bacterial load in the lungs. Our findings demonstrate the importance of persister drug clinafloxacin, offer new therapeutic approaches for more effective treatment of persistent P. aeruginosa infections, and may have implications for treating other persistent infections.

scholarly journals Biofilm/Persister/Stationary Phase Bacteria Cause More Severe Disease Than Log Phase Bacteria – II Infection with Persister Forms of Staphylococcus aureus Causes a Chronic Persistent Skin Infection with More Severe Lesion that Takes Longer to Heal and is not Eradicated by the Current Recommended Treatment in Mice

2018 ◽  
Author(s):  
Rebecca Yee ◽  
Yuting Yuan ◽  
Cory Brayton ◽  
Andreina Tarff Leal ◽  
Jie Feng ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Stationary Phase ◽  
Skin Infection ◽  
Bacterial Load ◽  
Mouse Skin ◽  
Infection Model ◽  
Persister Cells ◽  
Persistent Infections ◽  
Recommended Treatment ◽  
Biofilm Bacteria

AbstractStaphylococcus aureus is an opportunistic pathogen that can cause persistent infections clinically. Treatment for chronic S. aureus infections ranges from at least one week to several months and such infections are prone to relapse likely due to the presence of persistent forms of bacteria such as persister cells. Persister cells, which are bacterial cells that become dormant under stress conditions, can be isolated in vitro but their clinical significance in in vivo infections are largely unclear. Here, we evaluated S. aureus persistent forms using stationary phase cultures and biofilm bacteria (enriched in persisters) in comparison with log phase cultures in terms of their ability to cause disease in a mouse skin infection model. Surprisingly, we found that infection of mice with stationary phase cultures and biofilm bacteria produced a more severe chronic skin infection with more pronounced lesions which took longer to heal than log phase (actively growing) cultures. After two week infection, the bacterial load and skin tissue pathology, as determined by hyperplasia, immune cell infiltration, and crust/lesion formation, of mice infected with the more persistent forms (e.g. stationary phase bacteria and biofilm bacteria) were greater than mice infected with log phase bacteria. Using our persistent infection mouse model, we showed that the clinically recommended treatment for recurrent S. aureus skin infection, doxycycline + rifampin, was not effective in eradicating the bacteria in the treatment study, despite reducing lesion sizes and pathology in infected mice. Analogous findings were also observed in a Caenorhabditis elegans model, where S.aureus stationary phase cultures caused a greater mortality than log phase culture as early as two days post-infection. Thus, we established a new model for chronic persistent infections using persister bacteria that could serve as a relevant model to evaluate therapeutic options for persistent infections in general. Our findings connect persisters with persistent infections, have implications for understanding disease pathogenesis, and are likely to be broadly valid for other pathogens.

scholarly journals A Drug Combination Approach Targeting Both Growing Bacteria and Dormant Persisters Eradicate Persistent Staphylococcus aureus Biofilm Infection

2019 ◽  
Author(s):  
Rebecca Yee ◽  
Yuting Yuan ◽  
Andreina Tarff ◽  
Cory Brayton ◽  
Naina Gour ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mouse Model ◽  
Drug Combination ◽  
Skin Lesions ◽  
Combination Regimen ◽  
Persistent Infections ◽  
Biofilm Bacteria ◽  
Combination Approach ◽  
Biofilm Infection

AbstractStaphylococcus aureus can cause a variety of infections, many of which involve biofilm infections. Inside biofilms, growing and non-growing bacteria such as persisters co-exist, making it challenging to completely eradicate a persistent and recurrent infection with current treatments. Despite the clinical relevance, most of the current antibiotic treatments mainly kill the growing bacteria and have poor activity against non-growing persister bacteria and thus have limited effect on treating persistent infections including biofilm infections. We previously proposed a Yin-Yang model using a drug combination approach targeting both growing bacteria and persister bacteria for more effective clearance of persistent infections. Here, as a proof of principle, we showed that combining drugs that have high activity against growing forms, such as vancomycin or meropenem, with drugs that have robust anti-persister activity, such as clinafloxacin and oritavancin, could completely eradicate S. aureus biofilm bacteria in vitro. In contrast, single or two drugs including the current treatment for persistent S. aureus infection doxycycline plus rifampin failed to kill all biofilm bacteria in vitro. We then developed a chronic persistent skin infection mouse model with biofilm-seeded bacterial inocula demonstrating that biofilm bacteria caused more severe and persistent skin lesions than log phase S. aureus bacteria. More importantly, we found that the drug combination which eradicated biofilm bacteria in vitro is more efficacious than current treatments and completely eradicated S. aureus biofilm infection in mice. The complete eradication of biofilm bacteria is attributed to the unique high anti-persister activity of clinafloxacin, which could not be replaced by other fluoroquinolones such as moxifloxacin, levofloxacin or ciprofloxacin. Our study is the first to demonstrate that the combination of meropenem, daptomycin, plus clinafloxacin completely cleared the persistent infection, healed the lesions, and had less inflammation, while mice treated with doxycycline plus rifampin, the current clinically recommended treatment for chronic tissue infection, failed to do so. We also compared our persister drug combination with other approaches for treating persistent infections including gentamicin+fructose and ADEP4+rifampin in the S. aureus biofilm infection mouse model. Neither gentamicin+fructose nor ADEP4+rifampin could eradicate or cure the persistent biofilm infection in mice. In contrast, our drug combination regimen with persister drug clinafloxacin plus meropenem and daptomycin completely eradicated and cured the persistent biofilm infection in 7 days. An unexpected observation is that ADEP4 treatment group developed worsened skin lesions and caused more extensive pathology than the untreated control mice. Our study demonstrates an important treatment principle for persistent infections by targeting both growing and non-growing heterogeneous bacterial populations utilizing persister drugs for more effective eradication of persistent and biofilm infections. Our findings may have implications for improved treatment of many other persistent infections in general.

scholarly journals Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

2006 ◽  
Vol 80 (1) ◽  
pp. 360-371 ◽  
Author(s):  
Dorothea L. Sawicki ◽  
Silvia Perri ◽  
John M. Polo ◽  
Stanley G. Sawicki
Keyword(s):  
Host Response ◽  
Rna Synthesis ◽  
Late Phase ◽  
Host Cells ◽  
Minus Strand ◽  
Wild Type ◽  
Persistent Infections ◽  
Infected Cells ◽  
Replicative Intermediates ◽  
Transcription Complexes

ABSTRACT In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ∼10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

scholarly journals An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Martina Koeva ◽  
Alina D. Gutu ◽  
Wesley Hebert ◽  
Jeffrey D. Wager ◽  
Lael M. Yonker ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lactate Dehydrogenase ◽  
Stationary Phase ◽  
Persister Cells ◽  
Content Type ◽  
Ph Values ◽  
Potentiation Effect ◽  
Metabolite Concentrations ◽  
Ldh Assay

ABSTRACTBacterial persisters are a quasidormant subpopulation of cells that are tolerant to antibiotic treatment. The combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an antipersister strategy that is aimed at reducing recurrentPseudomonas aeruginosainfections by enhancing the killing ofP. aeruginosapersisters. Stationary-phase cultures ofP. aeruginosawere used to generate persister cells. A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions, including a range of pH values and in the presence of azithromycin or cystic fibrosis (CF) patient sputum. In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing a lactate dehydrogenase (LDH) assay. Enhanced killing of up to 6 orders of magnitude ofP. aeruginosapersisters over a range of CF isolates, including mucoid and nonmucoid strains, was observed for the tobramycin-fumarate combination compared to killing with tobramycin alone. Furthermore, significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum. Fumarate also reduced the cytotoxicity of tobramycin-treatedP. aeruginosato human epithelial airway cells. Finally, in mucoid and nonmucoid CF isolates, complete eradication ofP. aeruginosabiofilm was observed in the colony biofilm assay due to fumarate potentiation. These data suggest that a combination of tobramycin with fumarate as an antibacterial potentiator may be an attractive therapeutic for eliminating recurrentP. aeruginosainfections in CF patients through the eradication of bacterial persisters.

scholarly journals Oral cysteamine as an adjunct treatment in cystic fibrosis pulmonary exacerbations: An exploratory randomized clinical trial

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242945
Author(s):  
Graham Devereux ◽  
Danielle Wrolstad ◽  
Stephen J. Bourke ◽  
Cori L. Daines ◽  
Simon Doe ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cystic Fibrosis ◽  
Randomized Clinical Trial ◽  
Outcome Measures ◽  
Leukocyte Count ◽  
Bacterial Load ◽  
Adjunct Treatment ◽  
Dosing Regimens ◽  
Pulmonary Exacerbations ◽  
Blood Leukocyte

Background Emerging data suggests a possible role for cysteamine as an adjunct treatment for pulmonary exacerbations of cystic fibrosis (CF) that continue to be a major clinical challenge. There are no studies investigating the use of cysteamine in pulmonary exacerbations of CF. This exploratory randomized clinical trial was conducted to answer the question: In future pivotal trials of cysteamine as an adjunct treatment in pulmonary exacerbations of CF, which candidate cysteamine dosing regimens should be tested and which are the most appropriate, clinically meaningful outcome measures to employ as endpoints? Methods and findings Multicentre double-blind randomized clinical trial. Adults experiencing a pulmonary exacerbation of CF being treated with standard care that included aminoglycoside therapy were randomized equally to a concomitant 14-day course of placebo, or one of 5 dosing regimens of cysteamine. Outcomes were recorded on days 0, 7, 14 and 21 and included sputum bacterial load and the patient reported outcome measures (PROMs): Chronic Respiratory Infection Symptom Score (CRISS), the Cystic Fibrosis Questionnaire–Revised (CFQ-R); FEV1, blood leukocyte count, and inflammatory markers. Eighty nine participants in fifteen US and EU centres were randomized, 78 completed the 14-day treatment period. Cysteamine had no significant effect on sputum bacterial load, however technical difficulties limited interpretation. The most consistent findings were for cysteamine 450mg twice daily that had effects additional to that observed with placebo, with improved symptoms, CRISS additional 9.85 points (95% CI 0.02, 19.7) p = 0.05, reduced blood leukocyte count by 2.46x109 /l (95% CI 0.11, 4.80), p = 0.041 and reduced CRP by geometric mean 2.57 nmol/l (95% CI 0.15, 0.99), p = 0.049. Conclusion In this exploratory study cysteamine appeared to be safe and well-tolerated. Future pivotal trials investigating the utility of cysteamine in pulmonary exacerbations of CF need to include the cysteamine 450mg doses and CRISS and blood leukocyte count as outcome measures. Clinical trial registration NCT03000348; www.clinicaltrials.gov.

Total bacterial load, inflammation, and structural lung disease in paediatric cystic fibrosis

2020 ◽  
Vol 19 (6) ◽  
pp. 923-930 ◽  
Author(s):  
Steven L. Taylor ◽  
Lex E.X. Leong ◽  
Kerry L. Ivey ◽  
Steve Wesselingh ◽  
Keith Grimwood ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Bacterial Load

scholarly journals Identification of FDA-Approved Drugs with Activity against Stationary Phase Bartonella henselae

Antibiotics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 50 ◽  
Author(s):  
Tingting Li ◽  
Jie Feng ◽  
Shuzhen Xiao ◽  
Wanliang Shi ◽  
David Sullivan ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Stationary Phase ◽  
Drug Exposure ◽  
Bartonella Henselae ◽  
Drug Candidates ◽  
Persistent Infections ◽  
Viability Assay ◽  
Approved Drugs ◽  
Pyrvinium Pamoate ◽  
Effective Treatments

Bartonella henselae can cause various infections in humans, ranging from benign and self-limiting diseases to severe and life-threatening diseases as well as persistent infections that are difficult to treat. To develop more effective treatments for persistent Bartonella infections, in this study, we performed a high-throughput screen of an FDA-approved drug library against stationary phase B. henselae using the SYBR Green I/propidium iodide (PI) viability assay. We identified 110 drug candidates that had better activity against stationary phase B. henselae than ciprofloxacin, and among the top 52 drug candidates tested, 41 drugs were confirmed by microscopy to have higher activity than the current frontline antibiotic erythromycin. The identified top drug candidates include pyrvinium pamoate, daptomycin, methylene blue, azole drugs (clotrimazole, miconazole, sulconazole, econazole, oxiconazole, butoconazole, bifonazole), aminoglycosides (gentamicin and streptomycin, amikacin, kanamycin), amifostine (Ethyol), antiviral Lopinavir/ritonavir, colistin, nitroxoline, nitrofurantoin, verteporfin, pentamidine, berberine, aprepitant, olsalazine, clinafloxacin, and clofoctol. Pyrvinium pamoate, daptomycin, methylene blue, clotrimazole, and gentamicin and streptomycin at their respective maximum drug concentration in serum (Cmax) had the capacity to completely eradicate stationary phase B. henselae after 3-day drug exposure in subculture studies. While the currently used drugs for treating bartonellosis, including rifampin, erythromycin, azithromycin, doxycycline, and ciprofloxacin, had very low minimal inhibitory concentration (MIC) against growing B. henselae, they had relatively poor activity against stationary phase B. henselae, except aminoglycosides. The identified FDA-approved agents with activity against stationary phase B. henselae should facilitate development of more effective treatments for persistent Bartonella infections.

scholarly journals Stressors and Stress Responses in Cystic Fibrosis

2018 ◽  
Vol 5 (1) ◽  
pp. 11-29 ◽  
Author(s):  
Zsuzsa Bebok ◽  
Lianwu Fu
Keyword(s):  
Cystic Fibrosis ◽  
Stress Response ◽  
Stress Responses ◽  
Genetic Disorder ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Bicarbonate Transport ◽  
Persistent Infections ◽  
Human Disorders ◽  
The Unfolded Protein Response

Abstract Cystic fibrosis (CF) is a life-shortening, genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The primary cause of CF is reduced CFTR-mediated chloride and bicarbonate transport, due to mutations in CFTR. However, inflammation and persistent infections influence clinical outcome. Cellular stress response pathways, such as the unfolded protein response (UPR) and the integrated stress response (ISR), referred to here as cellular stress response pathways (SRPs), contribute to the pathology of human disorders. Multiple studies have indicated activation of SRPs in CF tissues. We review our present understanding of how SRPs are activated in CF and their contribution to pathology. We conclude that reduced CFTR function in CF organs establishes a tissue environment in which internal or external insults activate SRPs. SRPs contribute to CF pathogenesis by reducing CFTR expression, enhancing inflammation with consequent tissue remodeling. Understanding the contribution of SRPs to CF pathogenesis is crucial even in the era of CFTR “modulators” that are designed to potentiate, correct or amplify CFTR function, since there is an urgent need for supportive treatments. Importantly, CF patients with established pathology could benefit from the targeted use of drugs that modulate SRPs to reduce the symptoms.

Toll-like receptor-4 genotype influences the survival of cystic fibrosis mice

2010 ◽  
Vol 299 (2) ◽  
pp. G381-G390 ◽  
Author(s):  
Juan C. Canale-Zambrano ◽  
Meagan L. Auger ◽  
Christina K. Haston
Keyword(s):  
Cystic Fibrosis ◽  
Cell Proliferation ◽  
Double Mutant ◽  
Bacterial Load ◽  
Intestinal Disease ◽  
Intestinal Cell ◽  
Inflammatory Cell Infiltrate ◽  
Toll Like Receptor ◽  
Bacterial Classification ◽  
Crypt Cell Proliferation

Toll-like receptor (Tlr) 4 is a lipopolysaccharide (LPS) receptor that contributes to the regulation of intestinal cell homeostasis, a condition that is altered in the intestines of cystic fibrosis mice. Herein, we assessed whether Tlr4 genotype influences cystic fibrosis intestinal disease by producing and phenotyping 12-wk (adult)- and 4-day (neonate)-old mice derived from BALB cystic fibrosis transmembrane conductance regulator, Cftr+/tm1Uncand C.C3- Tlr4Lps-d/J ( Tlr4−/−), progenitors. Intestinal disease was assayed through mouse survival, crypt-villus axis (CVA) length, cell proliferation, bacterial load, bacterial classification, inflammatory cell infiltrate, and mucus content measures. Of the 77 Cftr−/−(CF) mice produced, only one Cftr/ Tlr4 double-mutant mouse lived to the age of 12 wk while the majority of the remainder succumbed at ∼4 days of age. The survival of CF Tlr4+/−mice exceeded that of both CF Tlr4+/+and Cftr/ Tlr4 double-mutant mice. Adult CF mice presented increased Tlr4 expression, CVA length, crypt cell proliferation, and bacterial load relative to non-CF mice, but no differences were detected in Tlr4+/−compared with Tlr4+/+CF mice. The double-mutant neonates did not differ from Tlr4+/+or Tlr4+/−CF mice by intestinal CVA length or bacterial load, but fewer Tlr4+/−CF neonates presented with luminal mucus obstruction in the distal ileum, and the intestinal mast cell increase of CF mice was not evident in double-mutant neonates. We conclude that Tlr4 deficiency reduces the survival, but does not alter the intestinal phenotypes, of extended CVA or increased bacterial load in BALB CF mice.

scholarly journals Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis.

1987 ◽  
Vol 7 (12) ◽  
pp. 4585-4588 ◽  
Author(s):  
J F Amara ◽  
H F Lodish
Keyword(s):  
Protein Synthesis ◽  
Dictyostelium Discoideum ◽  
Rna Synthesis ◽  
Protein Synthesis Inhibitors ◽  
Protein And Rna Synthesis ◽  
The Common ◽  
Mrna Destabilization ◽  
Actin Protein ◽  
Specific Mrna

We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein.

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