The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

ABSTRACT Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1–35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa. By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1–35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli. The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

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Synergistic Efficacy of Aedes aegypti Antimicrobial Peptide Cecropin A2 and Tetracycline against Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00686-17 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 31

Author(s):

Zhaojun Zheng ◽

Nagendran Tharmalingam ◽

Qingzhong Liu ◽

Elamparithi Jayamani ◽

Wooseong Kim ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Aedes Aegypti ◽

Mammalian Cells ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Synergistic Activity ◽

Content Type

ABSTRACT The increasing prevalence of antibiotic resistance has created an urgent need for alternative drugs with new mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates that could address the spread of multidrug-resistant bacteria, either alone or in combination with conventional antibiotics. We studied the antimicrobial efficacy and bactericidal mechanism of cecropin A2, a 36-residue α-helical cationic peptide derived from Aedes aegypti cecropin A, focusing on the common pathogen Pseudomonas aeruginosa. The peptide showed little hemolytic activity and toxicity toward mammalian cells, and the MICs against most clinical P. aeruginosa isolates were 32 to 64 μg/ml, and its MICs versus other Gram-negative bacteria were 2 to 32 μg/ml. Importantly, cecropin A2 demonstrated synergistic activity against P. aeruginosa when combined with tetracycline, reducing the MICs of both agents by 8-fold. The combination was also effective in vivo in the P. aeruginosa/Galleria mellonella model (P < 0.001). We found that cecropin A2 bound to P. aeruginosa lipopolysaccharides, permeabilized the membrane, and interacted with the bacterial genomic DNA, thus facilitating the translocation of tetracycline into the cytoplasm. In summary, the combination of cecropin A2 and tetracycline demonstrated synergistic antibacterial activity against P. aeruginosa in vitro and in vivo, offering an alternative approach for the treatment of P. aeruginosa infections.

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Engineered Endolysin-Based “Artilysins” To Combat Multidrug-Resistant Gram-Negative Pathogens

mBio ◽

10.1128/mbio.01379-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 144

Author(s):

Yves Briers ◽

Maarten Walmagh ◽

Victor Van Puyenbroeck ◽

Anneleen Cornelissen ◽

William Cenens ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Wall ◽

Outer Membrane ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Gram Negative ◽

Content Type ◽

Bacterial Viruses ◽

Cell Wall Hydrolysis

ABSTRACTThe global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, includingPseudomonas aeruginosaandAcinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strainsin vitrowith a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effectivein vitro(human keratinocytes) andin vivo(Caenorhabditis elegans).IMPORTANCEBacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active againstPseudomonas aeruginosaandAcinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.

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Rhamnolipids Nano-Micelles as a Potential Hand Sanitizer

Antibiotics ◽

10.3390/antibiotics10070751 ◽

2021 ◽

Vol 10 (7) ◽

pp. 751

Author(s):

Marwa Reda Bakkar ◽

Ahmed Hassan Ibrahim Faraag ◽

Elham R. S. Soliman ◽

Manar S. Fouda ◽

Amir Mahfouz Mokhtar Sarguos ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Active Sites ◽

Specific Interaction ◽

Virus Transmission ◽

Multidrug Resistant ◽

Future Perspective ◽

Resistant Bacteria ◽

In Silico Studies

COVID-19 is a pandemic disease caused by the SARS-CoV-2, which continues to cause global health and economic problems since emerging in China in late 2019. Until now, there are no standard antiviral treatments. Thus, several strategies were adopted to minimize virus transmission, such as social distancing, face covering protection and hand hygiene. Rhamnolipids are glycolipids produced formally by Pseudomonas aeruginosa and as biosurfactants, they were shown to have broad antimicrobial activity. In this study, we investigated the antimicrobial activity of rhamnolipids against selected multidrug resistant bacteria and SARS-CoV-2. Rhamnolipids were produced by growing Pseudomonas aeruginosa strain LeS3 in a new medium formulated from chicken carcass soup. The isolated rhamnolipids were characterized for their molecular composition, formulated into nano-micelles, and the antibacterial activity of the nano-micelles was demonstrated in vitro against both Gram-negative and Gram-positive drug resistant bacteria. In silico studies docking rhamnolipids to structural and non-structural proteins of SARS-CoV-2 was also performed. We demonstrated the efficient and specific interaction of rhamnolipids with the active sites of these proteins. Additionally, the computational studies suggested that rhamnolipids have membrane permeability activity. Thus, the obtained results indicate that SARS-CoV-2 could be another target of rhamnolipids and could find utility in the fight against COVID-19, a future perspective to be considered.

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Pseudomonas aeruginosa PAO 1 In Vitro Time–Kill Kinetics Using Single Phages and Phage Formulations—Modulating Death, Adaptation, and Resistance

Antibiotics ◽

10.3390/antibiotics10070877 ◽

2021 ◽

Vol 10 (7) ◽

pp. 877

Author(s):

Ana Mafalda Pinto ◽

Alberta Faustino ◽

Lorenzo M. Pastrana ◽

Manuel Bañobre-López ◽

Sanna Sillankorva

Keyword(s):

Pseudomonas Aeruginosa ◽

Phage Therapy ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Chronic Infections ◽

Swarming Motility ◽

Resistance Development ◽

Healthcare Settings ◽

Time Kill

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

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Antimicrobial Activity of Ceftazidime-Avibactam Tested against Multidrug-Resistant Enterobacteriaceae and Pseudomonas aeruginosa Isolates from U.S. Medical Centers, 2013 to 2016

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01045-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 52

Author(s):

Helio S. Sader ◽

Mariana Castanheira ◽

Dee Shortridge ◽

Rodrigo E. Mendes ◽

Robert K. Flamm

Keyword(s):

Pseudomonas Aeruginosa ◽

Multidrug Resistant ◽

Drug Resistant ◽

Large Collection ◽

Content Type ◽

Negative Results ◽

Medical Centers ◽

Extensively Drug Resistant ◽

Genes Encoding

ABSTRACT The in vitro activity of ceftazidime-avibactam and many comparator agents was determined against various resistant subsets of organisms selected among 36,380 Enterobacteriaceae and 7,868 Pseudomonas aeruginosa isolates. The isolates were consecutively collected from 94 U.S. hospitals, and all isolates were tested for susceptibility by reference broth microdilution methods in a central monitoring laboratory (JMI Laboratories). Enterobacteriaceae isolates resistant to carbapenems (CRE) and/or ceftazidime-avibactam (MIC ≥ 16 μg/ml) were evaluated for the presence of genes encoding extended-spectrum β-lactamases and carbapenemases. Ceftazidime-avibactam inhibited >99.9% of all Enterobacteriaceae at the susceptible breakpoint of ≤8 μg/ml and was active against multidrug-resistant (MDR; n = 2,953; MIC50/90, 0.25/1 μg/ml; 99.2% susceptible), extensively drug-resistant (XDR; n = 448; MIC50/90, 0.5/2 μg/ml; 97.8% susceptible), and CRE (n = 513; MIC50/90, 0.5/2 μg/ml; 97.5% susceptible) isolates. Only 82.2% of MDR Enterobacteriaceae (n = 2,953) and 64.2% of ceftriaxone-nonsusceptible Klebsiella pneumoniae (n = 1,063) isolates were meropenem susceptible. Among Enterobacter cloacae (22.2% ceftazidime nonsusceptible), 99.8% of the isolates, including 99.3% of the ceftazidime-nonsusceptible isolates, were ceftazidime-avibactam susceptible. Only 23 of 36,380 Enterobacteriaceae (0.06%) isolates were ceftazidime-avibactam nonsusceptible, including 9 metallo-β-lactamase producers and 2 KPC-producing strains with porin alteration; the remaining 12 strains showed negative results for all β-lactamases tested. Ceftazidime-avibactam showed potent activity against P. aeruginosa (MIC50/90, 2/4 μg/ml; 97.1% susceptible), including MDR (MIC50/90, 4/16 μg/ml; 86.5% susceptible) isolates, and inhibited 71.8% of isolates nonsusceptible to meropenem, piperacillin-tazobactam, and ceftazidime (n = 628). In summary, ceftazidime-avibactam demonstrated potent activity against a large collection (n = 44,248) of contemporary Gram-negative bacilli isolated from U.S. patients, including organisms resistant to most currently available agents, such as CRE and meropenem-nonsusceptible P. aeruginosa.

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In VitroActivity of RX-P873 against Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04840-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2280-2285 ◽

Cited By ~ 5

Author(s):

Robert K. Flamm ◽

Paul R. Rhomberg ◽

Ronald N. Jones ◽

David J. Farrell

Keyword(s):

Pseudomonas Aeruginosa ◽

Acinetobacter Baumannii ◽

Active Agent ◽

Multidrug Resistant ◽

Surveillance Program ◽

Gram Negative ◽

Content Type ◽

Highly Active ◽

Bacterial Ribosome

ABSTRACTRX-P873 is a novel antibiotic from the pyrrolocytosine series which exhibits high binding affinity for the bacterial ribosome and broad-spectrum antibiotic properties. The pyrrolocytosines have shownin vitroactivity against multidrug-resistant Gram-negative and Gram-positive strains of bacteria known to cause complicated urinary tract, skin, and lung infections, as well as sepsis.Enterobacteriaceae(657),Pseudomonas aeruginosa(200), andAcinetobacter baumannii(202) isolates from North America and Europe collected in 2012 as part of a worldwide surveillance program were testedin vitroby broth microdilution using Clinical and Laboratory Standards Institute (CLSI) methodology. RX-P873 (MIC90, 0.5 μg/ml) was >32-fold more active than ceftazidime and inhibited 97.1% and 99.5% ofEnterobacteriaceaeisolates at MIC values of ≤1 and ≤4 μg/ml, respectively. There were only three isolates with an MIC value of >4 μg/ml (all were indole-positiveProtea). RX-P873 (MIC50/90, 2/4 μg/ml) was highly active againstPseudomonas aeruginosaisolates, including isolates which were nonsusceptible to ceftazidime or meropenem. RX-P873 was 2-fold less active againstP. aeruginosathan tobramycin (MIC90, 2 μg/ml; 91.0% susceptible) and colistin (MIC90, 2 μg/ml; 99.5% susceptible) and 2-fold more potent than amikacin (MIC90, 8 μg/ml; 93.5% susceptible) and meropenem (MIC90, 8 μg/ml; 76.0% susceptible). RX-P873, the most active agent againstAcinetobacter baumannii(MIC90, 1 μg/ml), was 2-fold more active than colistin (MIC90, 2 μg/ml; 97.0% susceptible) and 4-fold more active than tigecycline (MIC90, 4 μg/ml). This novel agent merits further exploration of its potential against multidrug-resistant Gram-negative bacteria.

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Imipenem/Cilastatin/Relebactam Alone and in Combination against Pseudomonas aeruginosa in the In Vitro Pharmacodynamic Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01764-20 ◽

2020 ◽

Vol 64 (12) ◽

Author(s):

Iris H. Chen ◽

David P. Nicolau ◽

Joseph L. Kuti

Keyword(s):

Pseudomonas Aeruginosa ◽

Plasma Concentrations ◽

Multidrug Resistant ◽

Pharmacodynamic Model ◽

Resistance Development ◽

Content Type ◽

Combination Studies ◽

The Mean ◽

Combined Drugs

ABSTRACT Combination therapy may enhance imipenem/cilastatin/relebactam’s (I/R) activity against Pseudomonas aeruginosa and suppress resistance development. Human-simulated unbound plasma concentrations of I/R at 1.25 g every 6 h (h), colistin at 360 mg daily, and amikacin at 25 mg/kg daily were reproduced alone and in combination against six imipenem-nonsusceptible P. aeruginosa isolates in an in vitro pharmacodynamic model over 24 h. For I/R alone, the mean reductions in CFU ± the standard errors by 24 h were −2.52 ± 0.49, −1.49 ± 0.49, −1.15 ± 0.67, and −0.61 ± 0.10 log10 CFU/ml against isolates with MICs of 1/4, 2/4, 4/4, and 8/4 μg/ml, respectively. Amikacin alone also resulted in 24 h CFU reductions consistent with its MIC, while colistin CFU reductions did not differ. Resistant subpopulations were observed after 24 h in 1, 4, and 3 I/R-, colistin-, and amikacin-exposed isolates, respectively. The combination of I/R and colistin resulted in synergistic (n = 1) or additive (n = 2) interactions against three isolates with 24-h CFU reductions ranging from −2.62 to −4.67 log10 CFU/ml. The combination of I/R and amikacin exhibited indifferent interactions against all isolates, with combined drugs achieving −0.51- to −3.33-log10 CFU/ml reductions. No resistant subpopulations were observed during I/R and colistin combination studies, and when added to amikacin, I/R prevented the emergence of amikacin resistance. Against these six multidrug-resistant P. aeruginosa, I/R alone achieved significant CFU reductions against I/R-susceptible isolates. Combinations of I/R plus colistin resulted in additivity or synergy against some P. aeruginosa, whereas the addition of amikacin did not provide further antibacterial efficacy against these isolates.

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In VitroEvaluation of Novel Compounds against Selected Resistant Pseudomonas aeruginosa Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05944-11 ◽

2011 ◽

Vol 56 (3) ◽

pp. 1646-1649 ◽

Cited By ~ 2

Author(s):

Seth T. Housman ◽

Christina Sutherland ◽

David P. Nicolau

Keyword(s):

Pseudomonas Aeruginosa ◽

Broad Spectrum ◽

Large Subunit ◽

Multidrug Resistant ◽

Content Type ◽

Bacterial Ribosome ◽

Multidrug Resistant Pathogens

ABSTRACTWe describe the activities of RX-P763, RX-P766, RX-P770, RX-P792, RX-P793, and RX-P808 against strains of resistantPseudomonas aeruginosa. These compounds target the large subunit of the bacterial ribosome and have broad-spectrum activities against multidrug-resistant pathogens. All compounds demonstratedin vitroactivity againstP. aeruginosa, with MIC90values of 4 to 8 μg/ml (range, 0.5 to 64). These novel compounds had narrow MIC distributions and maintained activity despite resistance phenotypes to other commonly utilized agents.

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Evolution of Pseudomonas aeruginosa Virulence as a Result of Phage Predation

Applied and Environmental Microbiology ◽

10.1128/aem.01421-13 ◽

2013 ◽

Vol 79 (19) ◽

pp. 6110-6116 ◽

Cited By ~ 36

Author(s):

Zeinab Hosseinidoust ◽

Theo G. M. van de Ven ◽

Nathalie Tufenkji

Keyword(s):

Pseudomonas Aeruginosa ◽

Mammalian Cells ◽

Bacterial Infections ◽

Membrane Damage ◽

Human Keratinocytes ◽

Host Population ◽

Resistant Bacteria ◽

Content Type ◽

Metabolic Action

ABSTRACTThe rapid increase in the emergence of antibiotic-resistant bacteria has attracted attention to bacteriophages for treating and preventing bacterial infections. Bacteriophages can drive the diversification ofPseudomonas aeruginosa, giving rise to phage-resistant variants with different phenotypes from their ancestral hosts. In this study, we sought to investigate the effect of phage resistance on cytotoxicity of host populations toward cultured mammalian cells. The library of phage-resistantP. aeruginosaPAO1 variants used was developed previously via experimental evolution of an isogenic host population using phages PP7 and E79. Our results presented herein indicate that the phage-resistant variants developed in a heterogeneous phage environment exhibit a greater ability to impede metabolic action of cultured human keratinocytes and have a greater tendency to cause membrane damage even though they cannot invade the cells in large numbers. They also show a heightened resistance to phagocytosis by model murine macrophages. Furthermore, all isolates produced higher levels of at least one of the secreted virulence factors, namely, total proteases, elastase, phospholipase C, and hemolysins. Reverse transcription-quantitative PCR (RT-qPCR) revealed upregulation in the transcription of a number of genes associated with virulence ofP. aeruginosafor the phage-resistant variants. The results of this study indicate a significant change in thein vitrovirulence ofP. aeruginosafollowing phage predation and highlight the need for caution in the selection and design of phages and phage cocktails for therapeutic use.

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The Concerted Action of Two B3-Like Prophage Genes Excludes Superinfecting Bacteriophages by Blocking DNA Entry into Pseudomonas aeruginosa

Journal of Virology ◽

10.1128/jvi.00953-20 ◽

2020 ◽

Vol 94 (15) ◽

Author(s):

Marco Antonio Carballo-Ontiveros ◽

Adrián Cazares ◽

Pablo Vinuesa ◽

Luis Kameyama ◽

Gabriel Guarneros

Keyword(s):

Pseudomonas Aeruginosa ◽

Pathogenic Bacteria ◽

Phage Therapy ◽

Alternative Therapy ◽

Multidrug Resistant ◽

Open Reading Frames ◽

Resistant Bacteria ◽

Content Type ◽

Secondary Infections ◽

Genes Encoding

ABSTRACT In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa. Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host’s cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.

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