AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.

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1375. In vitro Activity of Cefiderocol and Comparator Agents against Gram-Negative Isolates from Cancer Patients

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1206 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S421-S422 ◽

Cited By ~ 2

Author(s):

Kenneth V I Rolston ◽

Bahgat Gerges ◽

Issam Raad ◽

Samuel L Aitken ◽

Ruth Reitzel ◽

...

Keyword(s):

Cancer Patients ◽

Active Agent ◽

Vitro Activity ◽

Significant Activity ◽

In Vitro Activity ◽

Research Support ◽

Gram Negative ◽

E Coli ◽

Research Grant

Abstract Background Gram-negative bacilli (GNB) are now the predominant cause of bacterial infection in cancer patients (CP). Many GNB are problematic because they have become resistant to commonly used antibiotics. Cefiderocol (CFDC), a novel siderophore cephalosporin, is active against a wide spectrum of GNB. We evaluated its in vitro activity and that of eleven comparator agents against GNB isolated from CP. Methods A total of 341 recent GNB blood isolates from CP were tested using CLSI approved methods for MIC determination by broth microdilution. Comparator agents were amikacin (A), aztreonam (AZ), ceftazidime (CZ), ceftazidime/avibactam (CAV), cefepime (CEF), ciprofloxacin (CIP), colistin (CL), meropenem (MR), ceftolozane/tazobactam (C/T), tigecycline (TG), and trimethoprim/sulfamethoxazole (T/S). Results CFDC MIC90s as mg/L were: S. maltophilia [50 isolates] 0.25, E. coli (ESBL−) [50 isolates] 0.5, E. coli (ESBL+) [51 isolates] 2.0, K. pneumoniae (ESBL− and +) [60 isolates] 0.5; K. pneumoniae (CRE) [22 isolates] 2.0; P. aeruginosa (MDR) [32 isolates] 1.0; E. cloacae [27 isolates] 4.0; Achromobacter spp. [15 isolates] 0.12. CFDC inhibited P. agglomerans, Burkholderia spp., Sphingomonas spp., Ochrobactrum spp. at ≤1 mg/L [23 total isolates] and Elizabethkingia spp. and R. radiobacter at ≤8 mg/L [11 total isolates]. Among comparator agents, only T/S had consistent activity against S. maltophilia. For E. coli (ESBL− and +) MR, TG, CAV, CL were most active. For K. pneumoniae (ESBL–and +) MR, CAV were most active. For K. pneumoniae (CRE) and P. aeruginosa (MDR), none of the comparators had significant activity. For E. cloacae, MR, A, CAV, TG were most active. Among the uncommon organisms, MR and TG had the greatest activity. Conclusion Although susceptibility breakpoints have yet to be determined, CFDC has significant activity (≤4 mg/L) against most problematic Gram-negative organisms causing infections in CP based on available pharmacokinetic/pharmacodynamic data. In particular, its activity against S. maltophilia was superior to the comparators. Also, it was the most active agent against P. aeruginosa (MDR) and K. pneumoniae (CRE). Based on our results, CFDC warrants clinical evaluation for the treatment of blood stream infections caused by GNB in CP. Disclosures K. V. I. Rolston, Merck: Investigator, Research grant; JMI Laboratories: Investigator, Research grant; Shionogi (Japan): Investigator, Research grant. B. Gerges, Shionogi: Collaborator, Research support. S. L. Aitken, Shionogi: Scientific Advisor, Consulting fee; Merck: Scientific Advisor, Consulting fee; Medicines Co: Scientific Advisor, Consulting fee; Achaogen: Scientific Advisor, Consulting fee; Zavante: Scientific Advisor, Consulting fee; R. Prince, Shionogi: Investigator, Research support. Merck: Investigator, Research support.

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Host DNA Repair Proteins in Response toPseudomonas aeruginosain Lung Epithelial Cells and in Mice

Infection and Immunity ◽

10.1128/iai.00815-10 ◽

2010 ◽

Vol 79 (1) ◽

pp. 75-87 ◽

Cited By ~ 35

Author(s):

Min Wu ◽

Huang Huang ◽

Weidong Zhang ◽

Shibichakravarthy Kannan ◽

Andrew Weaver ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Epithelial Cells ◽

Critical Role ◽

Lung Epithelial Cells ◽

Host Cells ◽

Myeloperoxidase Activity ◽

Gram Negative ◽

Lung Epithelial ◽

Dna Repair Proteins

ABSTRACTAlthough DNA repair proteins in bacteria are critical for pathogens' genome stability and for subverting the host defense, the role of host DNA repair proteins in response to bacterial infection is poorly defined. Here, we demonstrate, for the first time, that infection with the Gram-negative bacteriumPseudomonas aeruginosasignificantly altered the expression and enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1) in lung epithelial cells. Downregulation of OGG1 by a small interfering RNA strategy resulted in severe DNA damage and cell death. In addition, acetylation of OGG1 is required for host responses to bacterial genotoxicity, as mutations of OGG1 acetylation sites increased Cockayne syndrome group B (CSB) protein expression. These results also indicate that CSB may be involved in DNA repair activity during infection. Furthermore, OGG1 knockout mice exhibited increased lung injury after infection withP. aeruginosa, as demonstrated by higher myeloperoxidase activity and lipid peroxidation. Together, our studies indicate thatP. aeruginosainfection induces significant DNA damage in host cells and that DNA repair proteins play a critical role in the host response toP. aeruginosainfection, serving as promising targets for the treatment of this condition and perhaps more broadly Gram-negative bacterial infections.

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Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01553-05 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2478-2486 ◽

Cited By ~ 47

Author(s):

Andrea Giacometti ◽

Oscar Cirioni ◽

Roberto Ghiselli ◽

Federico Mocchegiani ◽

Fiorenza Orlando ◽

...

Keyword(s):

Septic Shock ◽

Antimicrobial Peptide ◽

Gram Negative Bacteria ◽

Amphibian Skin ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Rat Models ◽

E Coli ◽

Tnf Α

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

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In Vitro and In Vivo Antibacterial Activities of a Novel Glycylcycline, the 9-t-Butylglycylamido Derivative of Minocycline (GAR-936)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.4.738 ◽

1999 ◽

Vol 43 (4) ◽

pp. 738-744 ◽

Cited By ~ 283

Author(s):

P. J. Petersen ◽

N. V. Jacobus ◽

W. J. Weiss ◽

P. E. Sum ◽

R. T. Testa

Keyword(s):

Anaerobic Bacteria ◽

Tetracycline Resistance ◽

Protective Effects ◽

Gram Negative ◽

E Coli ◽

Resistance Determinants ◽

Aerobic And Anaerobic ◽

Ribosomal Protection

ABSTRACT The 9-t-butylglycylamido derivative of minocycline (TBG-MINO) is a recently synthesized member of a novel group of antibiotics, the glycylcyclines. This new derivative, like the first glycylcyclines, theN,N-dimethylglycylamido derivative of minocycline and 6-demethyl-6-deoxytetracycline, possesses activity against bacterial isolates containing the two major determinants responsible for tetracycline resistance: ribosomal protection and active efflux. The in vitro activities of TBG-MINO and the comparative agents were evaluated against strains with characterized tetracycline resistance as well as a spectrum of recent clinical aerobic and anaerobic gram-positive and gram-negative bacteria. TBG-MINO, with an MIC range of 0.25 to 0.5 μg/ml, showed good activity against strains expressing tet(M) (ribosomal protection), tet(A), tet(B),tet(C), tet(D), and tet(K) (efflux resistance determinants). TBG-MINO exhibited similar activity against methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant streptococci, and vancomycin-resistant enterococci (MICs at which 90% of strains are inhibited, ≤0.5 μg/ml). TBG-MINO exhibited activity against a wide diversity of gram-negative aerobic and anaerobic bacteria, most of which were less susceptible to tetracycline and minocycline. The in vivo protective effects of TBG-MINO were examined against acute lethal infections in mice caused by Escherichia coli, S. aureus, andStreptococcus pneumoniae isolates. TBG-MINO, administered intravenously, demonstrated efficacy against infections caused byS. aureus including MRSA strains and strains containingtet(K) or tet(M) resistance determinants (median effective doses [ED50s], 0.79 to 2.3 mg/kg of body weight). TBG-MINO demonstrated efficacy against infections caused by tetracycline-sensitive E. coli strains as well asE. coli strains containing either tet(M) or the efflux determinant tet(A), tet(B), ortet(C) (ED50s, 1.5 to 3.5 mg/kg). Overall, TBG-MINO shows antibacterial activity against a wide spectrum of gram-positive and gram-negative aerobic and anaerobic bacteria including strains resistant to other chemotherapeutic agents. The in vivo protective effects, especially against infections caused by resistant bacteria, corresponded with the in vitro activity of TBG-MINO.

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Activity of Ceftaroline against Aerobic Gram-Positive and Gram-Negative Pathogens: Effect of Test Method Variability

ISRN Microbiology ◽

10.5402/2011/787290 ◽

2011 ◽

Vol 2011 ◽

pp. 1-5

Author(s):

Diane M. Citron ◽

Yumi A. Warren ◽

Kerin L. Tyrrell ◽

Ellie J. C. Goldstein

Keyword(s):

Bactericidal Activity ◽

Test Method ◽

In Vitro Test ◽

Gram Negative ◽

Methicillin Resistant ◽

E Coli ◽

Minimum Inhibitory Concentrations ◽

Test Conditions ◽

Vitro Test

Ceftaroline is a new cephalosporin with bactericidal activity against methicillin-resistant S. aureus (MRSA) as well as gram-negative pathogens. Variations of in vitro test conditions were found to affect ceftaroline activity, with 5% NaCl inhibiting growth and/or reducing the minimum inhibitory concentrations (MICs) for E. coli, K. pneumoniae, M. catarrhalis, H. influenzae, and streptococci, while an inoculum of 106 CFU/mL raised MICs of some E. coli, K. pneumoniae, and M. catarrhalis strains.

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Investigating In Vitro Antibacterial Activities of Medicinal Plants Having Folkloric Repute in Ethiopian Traditional Medicine

Journal of Evidence-Based Integrative Medicine ◽

10.1177/2515690x19886276 ◽

2019 ◽

Vol 24 ◽

pp. 2515690X1988627 ◽

Cited By ~ 1

Author(s):

Mekonnen Sisay ◽

Negussie Bussa ◽

Tigist Gashaw ◽

Getnet Mengistu

Keyword(s):

Antibacterial Activity ◽

Medicinal Plants ◽

Antimicrobial Agents ◽

Bacterial Isolates ◽

Zone Of Inhibition ◽

Traditional Use ◽

Gram Negative ◽

E Coli ◽

Slow Development

Medicinal plants are targeted in the search for new antimicrobial agents. Nowadays, there is an alarmingly increasing antimicrobial resistance to available agents with a very slow development of new antimicrobials. It is, therefore, necessary to extensively search for new agents based on the traditional use of herbal medicines as potential source. The antibacterial activity of 80% methanol extracts of the leaves of Verbena officinalis (Vo-80ME), Myrtus communis (Mc-80ME), and Melilotus elegans (Me-80ME) was tested against 6 bacterial isolates using agar well diffusion technique. In each extract, 3 concentrations of 10, 20, and 40 mg/well were tested for each bacterium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were also determined. Vo-80ME and Mc-80ME exhibited promising antibacterial activity against Staphylococcus aureus with the highest zone of inhibition being 18.67 and 26.16 mm, respectively at concentration of 40 mg/well. Regarding gram-negative bacteria, Vo-80ME exhibited an appreciable activity against Escherichia coli and Salmonella typhi. Mc-80ME displayed remarkable activity against all isolates including Pseudomonas aeruginosa with the maximum zone of inhibition being 22.83 mm. Me-80ME exhibited better antibacterial activity against E coli, but its secondary metabolites had little or no activity against other gram-negative isolates. The MIC values of Vo-80ME ranged from 0.16 to 4.00 mg/mL. The lowest MIC was observed in Mc-80ME, with the value being 0.032 mg/mL. Mc-80ME had bactericidal activity against all tested bacterial isolates. Mc-80ME showed remarkable zone of inhibitions in all tested bacterial isolates. Besides, Vo-80ME showed good antibacterial activity against S aureus, E coli, and S typhi. Conversely, Me-80ME has shown good activity against E coli only. Generally, M communis L and V officinalis have good MIC and MBC results.

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Structural basis for transcription inhibition by E. coli SspA

Nucleic Acids Research ◽

10.1093/nar/gkaa672 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9931-9942 ◽

Cited By ~ 1

Author(s):

Fulin Wang ◽

Jing Shi ◽

Dingwei He ◽

Bei Tong ◽

Chao Zhang ◽

...

Keyword(s):

Structural Information ◽

Protein A ◽

Acid Tolerance ◽

In Vitro Transcription ◽

Nucleotide Metabolism ◽

Structural Basis ◽

E Coli ◽

Promoter Escape ◽

Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

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Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains

Scientific Reports ◽

10.1038/s41598-019-50110-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Minh Tan Nguyen ◽

Musharrat Jahan Prima ◽

Jung-A. Song ◽

Julee Kim ◽

Bich Hang Do ◽

...

Keyword(s):

Metabolic Diseases ◽

Protein A ◽

Genetically Engineered ◽

Oncostatin M ◽

Glutathione S Transferase ◽

Skeletal Tissue ◽

E Coli ◽

Th17 Cell Differentiation ◽

Tissue Alteration

Abstract Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b‘a’ domain of PDI (PDIb‘a’), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

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In Vitro Activity of Newer and Conventional Antimicrobial Agents, Including Fosfomycin and Colistin, against Selected Gram-Negative Bacilli in Kuwait

Pathogens ◽

10.3390/pathogens7030075 ◽

2018 ◽

Vol 7 (3) ◽

pp. 75 ◽

Cited By ~ 5

Author(s):

Wadha Alfouzan ◽

Rita Dhar ◽

David Nicolau

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

The Other ◽

In Vitro Activity ◽

Limited Data ◽

Gram Negative ◽

E Coli ◽

Microbroth Dilution

Limited data are available on susceptibilities of these organisms to some of the recently made accessible antimicrobial agents. The in vitro activities of newer antibiotics, such as, ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA) along with some “older” antibiotics, for example fosfomycin (FOS) and colistin (CL) were determined against selected strains (resistant to ≥ 3 antimicrobial agents) of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Minimum inhibitory concentrations (MIC) were determined by Clinical and Laboratory Standards Institute microbroth dilution. 133 isolates: 46 E. coli, 39 K. pneumoniae, and 48 P. aeruginosa were tested. Results showed that E. coli isolates with MIC50/90, 0.5/1 μ g / mL for CL; 4/32 μ g / mL for FOS; 0.25/32 μ g / mL for C/T; 0.25/8 μ g / mL for CZA, exhibited susceptibility rates of 95.7%, 97.8%, 76.1%, and 89.1%, respectively. On the other hand, K. pneumoniae strains with MIC50/90, 0.5/1 μ g / mL for CL; 256/512 μ g / mL for FOS; 2/128 μ g / mL for C/T; 0.5/128 μ g / mL for CZA showed susceptibility rates of 92.3%, 7.7%, 51.3%, and 64.1%, respectively. P. aeruginosa isolates with MIC50/90, 1/1 μ g / mL for CL; 128/128 μ g / mL for C/T; 32/64 μ g / mL for CZA presented susceptibility rates of 97.9%, 33.3%, and 39.6%, respectively. Higher MICs were demonstrated against most of the antibiotics. However, CL retained efficacy at low MICs against most of the isolates tested.

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Volatile Oils ofNepeta tenuifolia(Jing Jie) as an Alternative Medicine against Multidrug-Resistant Pathogenic Microbes

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2018/8347403 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Yi-Hsuan Lee ◽

Chao-Min Wang ◽

Po-Yu Liu ◽

Ching-Chang Cheng ◽

Zong-Yen Wu ◽

...

Keyword(s):

Essential Oils ◽

In Vitro Study ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Gram Negative ◽

E Coli ◽

Wide Range ◽

Main Component ◽

Reference Strains

Essential oils from the dried spikes ofNepeta tenuifolia(Benth) are obtained by steam distillation. Pulegone was identified as the main component in the spikes ofN. tenuifoliathrough analysis, with greater than 85% purity obtained in this study. The essential oils are extremely active against all Gram-positive and some Gram-negative reference bacteria, particularlySalmonella enterica,Citrobacter freundii, andEscherichia coli. The minimum inhibitory concentration was found to be between 0.08 and 0.78% (againstS. enterica), 0.39 and 0.78% (againstC. freundii), and 0.097 and 0.39% (againstE. coli), whereas the minimum bactericidal concentration varied in range from 0.097% to 1.04%. In general, the essential oils show a strong inhibitory action against all tested reference strains and clinical isolates. However, the antibacterial activity of EOs against bothPseudomonas aeruginosareference strains and clinical isolates was relatively lower than other Gram-negative pathogens. The essential oils ofN. tenuifoliaalso displayed bactericidal activities (MBC/MIC < 4) in this study. These findings reflect the bactericidal activity of the essential oils against a wide range of multidrug-resistant clinical pathogens in an in vitro study. In addition, we propose the fragmentation pathways of pulegone and its derivatives by LC-ESI-MS/MS in this study.

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