Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease

Chronic respiratory infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients, and are characterized by the development of multidrug resistance (MDR) phenotype and biofilm formation, generally recalcitrant to treatment with conventional antibiotics. Hence, novel effective strategies are urgently needed. Antimicrobial peptides represent new promising therapeutic agents. Here, we analyze for the first time the efficacy of three versions of a cryptide identified in human apolipoprotein B (ApoB, residues 887-922) towards bacterial strains clinically isolated from CF patients. Antimicrobial and anti-biofilm properties of ApoB-derived cryptides have been analyzed by broth microdilution assays, crystal violet assays, confocal laser scanning microscopy and scanning electron microscopy. Cell proliferation assays have been performed to test cryptide effects on human host cells. ApoB-derived cryptides have been found to be endowed with significant antimicrobial and anti-biofilm properties towards Pseudomonas and Burkholderia strains clinically isolated from CF patients. Peptides have been also found to be able to act in combination with the antibiotic ciprofloxacin, and they are harmless when tested on human bronchial epithelial mesothelial cells. These findings open interesting perspectives to cryptide applicability in the treatment of chronic lung infections associated with CF disease.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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Polymeric Nanoparticles Active against Dual-Species Bacterial Biofilms

Molecules ◽

10.3390/molecules26164958 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4958

Author(s):

Jessa Marie V. Makabenta ◽

Jungmi Park ◽

Cheng-Hsuan Li ◽

Aritra Nath Chattopadhyay ◽

Ahmed Nabawy ◽

...

Keyword(s):

Antimicrobial Activity ◽

Laser Scanning ◽

Polymeric Nanoparticles ◽

Bacterial Species ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Bacterial Biofilms ◽

Global Public Health ◽

Bacterial Strains

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.

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The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation

Cellular Physiology and Biochemistry ◽

10.1159/000487566 ◽

2018 ◽

Vol 45 (4) ◽

pp. 1399-1409 ◽

Cited By ~ 7

Author(s):

Supeng Yin ◽

Bei Jiang ◽

Guangtao Huang ◽

Yulong Zhang ◽

Bo You ◽

...

Keyword(s):

Human Serum ◽

Biofilm Formation ◽

Laser Scanning ◽

Serum Proteins ◽

Venous Catheter ◽

Bacterial Biofilm ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Bacterial Strains

Background/Aims: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. Methods: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. Results: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. Conclusions: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

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Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections

Cells ◽

10.3390/cells9010194 ◽

2020 ◽

Vol 9 (1) ◽

pp. 194 ◽

Cited By ~ 4

Author(s):

Adriely Goes ◽

Philipp Lapuhs ◽

Thomas Kuhn ◽

Eilien Schulz ◽

Robert Richter ◽

...

Keyword(s):

Outer Membrane ◽

Mammalian Cells ◽

Laser Scanning ◽

Respiratory Infections ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Potential Applicability ◽

Intracellular Infections

In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.

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Organo-Selenium-Containing Polyester Bandage Inhibits Bacterial Biofilm Growth on the Bandage and in the Wound

Biomedicines ◽

10.3390/biomedicines8030062 ◽

2020 ◽

Vol 8 (3) ◽

pp. 62

Author(s):

Phat Tran ◽

Tyler Enos ◽

Keaton Luth ◽

Abdul Hamood ◽

Coby Ray ◽

...

Keyword(s):

Wound Infection ◽

Wound Dressing ◽

Laser Scanning ◽

Bacterial Biofilm ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Bacterial Strains ◽

Biofilm Growth ◽

Biofilm Model

The dressing material of a wound plays a key role since bacteria can live in the bandage and keep re-infecting the wound, thus a bandage is needed that blocks biofilm in the bandage. Using an in vivo wound biofilm model, we examined the effectiveness of an organo-selenium (OS)-coated polyester dressing to inhibit the growth of bacteria in a wound. Staphylococcus aureus (as well as MRSA, Methicillin resistant Staph aureus), Stenotrophomonas maltophilia, Enterococcus faecalis, Staphylococcus epidermidis, and Pseudomonas aeruginosa were chosen for the wound infection study. All the bacteria were enumerated in the wound dressing and in the wound tissue under the dressing. Using colony-forming unit (CFU) assays, over 7 logs of inhibition (100%) was found for all the bacterial strains on the material of the OS-coated wound dressing and in the tissue under that dressing. Confocal laser scanning microscopy along with IVIS spectrum in vivo imaging confirmed the CFU results. Thus, the dressing acts as a reservoir for a biofilm, which causes wound infection. The same results were obtained after soaking the dressing in PBS at 37 °C for three months before use. These results suggest that an OS coating on polyester dressing is both effective and durable in blocking wound infection.

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A Novel Hybrid Peptide Composed of LfcinB6 and KR-12-a4 With Enhanced Antimicrobial, Anti-Inflammatory and Anti-Biofilm Activities

10.21203/rs.3.rs-1109468/v1 ◽

2021 ◽

Author(s):

Chelladurai Ajish ◽

Sungtae Yang ◽

S. Dinesh Kumar ◽

Eun Young Kim ◽

Hye Jung Min ◽

...

Keyword(s):

Antimicrobial Activity ◽

Laser Scanning ◽

Antimicrobial Agents ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Bacterial Cells ◽

Bacterial Strains ◽

Hybrid Peptide ◽

Cell Selectivity ◽

Anti Inflammatory

Abstract Hybridizing two known antimicrobial peptides (AMPs) is a simple and effective strategy for designing antimicrobial agents with enhanced cell selectivity against bacterial cells. Here, we generated a hybrid peptide Lf-KR in which LfcinB6 and KR-12-a4 were linked with a Pro hinge to obtain a novel AMP with potent antimicrobial, anti-inflammatory, and anti-biofilm activities. Lf-KR exerted superior cell selectivity for bacterial cells over sheep red blood cells. Lf-KR showed broad-spectrum antimicrobial activities (MIC: 4–8 mM) against tested 12 bacterial strains and retained its antimicrobial activity in the presence of salts at physiological concentrations. Membrane depolarization and dye leakage assays showed that the enhanced antimicrobial activity of Lf-KR was due to increased permeabilization and depolarization of microbial membranes. Lf-KR significantly inhibited the expression and production of pro-inflammatory cytokines (NO and TNF-a) in LPS-stimulated mouse macrophage RAW264.7 cells. In addition, Lf-KR showed a powerful eradication effect on preformed multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms. We confirmed using confocal laser scanning microscopy that a large portion of the preformed MDRPA biofilm structure was perturbed by the addition of Lf-KR. Collectively, our results suggest that Lf-KR can be an antimicrobial, anti-inflammatory, and anti-biofilm candidate as a pharmaceutical agent.

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Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

Applied and Environmental Microbiology ◽

10.1128/aem.00039-11 ◽

2011 ◽

Vol 77 (10) ◽

pp. 3413-3421 ◽

Cited By ~ 21

Author(s):

Rebecca Peyyala ◽

Sreenatha S. Kirakodu ◽

Jeffrey L. Ebersole ◽

Karen F. Novak

Keyword(s):

Laser Scanning ◽

Bacterial Species ◽

Disease Process ◽

Laser Scanning Microscopy ◽

Host Cells ◽

Confocal Laser ◽

Content Type ◽

Oral Biofilms ◽

Multispecies Biofilms

ABSTRACTOral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novelin vitromodel system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprisingStreptococcus gordonii,Streptococcus oralis, andStreptococcus sanguinis;S. gordonii,Actinomyces naeslundii, andFusobacterium nucleatum; orS. gordonii,F. nucleatum, andPorphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

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Salmonella spvC Gene Inhibits Autophagy of Host Cells and Suppresses NLRP3 as Well as NLRC4

Frontiers in Immunology ◽

10.3389/fimmu.2021.639019 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liting Zhou ◽

Yuanyuan Li ◽

Song Gao ◽

Haibo Yuan ◽

Lingli Zuo ◽

...

Keyword(s):

Hela Cells ◽

Laser Scanning ◽

Systemic Infection ◽

Laser Scanning Microscopy ◽

Host Cells ◽

Confocal Laser ◽

Autophagic Flux ◽

Virulence Determinants ◽

Pyrin Domain ◽

Lyase Activity

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents.

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Antimicrobial and Anticorrosion Activity of a Novel Composite Biocide against Mixed Bacterial Strains in Taiwanese Marine Environments

Materials ◽

10.3390/ma14206156 ◽

2021 ◽

Vol 14 (20) ◽

pp. 6156

Author(s):

Soul-Yi Chang ◽

Shih-Yen Huang ◽

Yu-Ren Chu ◽

Shun-Yi Jian ◽

Kai-Yin Lo ◽

...

Keyword(s):

Stainless Steel ◽

Laser Scanning ◽

Electrochemical Impedance ◽

Laser Scanning Microscopy ◽

304 Stainless Steel ◽

Single Type ◽

Confocal Laser ◽

Subtropical Climate ◽

Marine Environments ◽

Bacterial Strains

Taiwan is an island with a humid subtropical climate. The relatively warm seawater results in biofouling of the surfaces of marine facilities. Biocide application is a common practice for combating and eliminating adhesive fouling. However, a single type of biocide may have limited antimicrobial effects due to the relatively high microbial diversity in marine environments. Therefore, applying a mixture of various biocides may be necessary. In this study, the antimicrobial and anticorrosion properties of a newly designed composite biocide, namely a combination of thymol and benzyldimethyldodecylammonium chloride, were investigated by applying the biocide to 304 stainless steel substrates immersed in inocula containing bacterial strains from Tamsui and Zuoying harbors. The ability of 3TB and 5TB treatments to prevent sessile cells and biofilm formation on the 304 stainless steel coupon surface was determined through scanning electron microscopy investigation. In addition, confocal laser scanning microscopy indicated that the 5TB treatment achieved a greater bactericidal effect in both the Tamsui and Zuoying inocula. Moreover, electrochemical impedance spectroscopy revealed that the diameter of the Nyquist semicircle was almost completely unaffected by Tamsui or Zuoying under the 5TB treatment. Through these assessments of antimicrobial activity and corrosion resistance, 5TB treatment was demonstrated to have superior bactericidal activity against mixed strains in both southern and northern Taiwanese marine environments.

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Evaluation of a novel, multi-functional inhibitor compound for prevention of biofilm formation on carbon steel in marine environments

Scientific Reports ◽

10.1038/s41598-021-94827-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benjamin Tuck ◽

Elizabeth Watkin ◽

Maria Forsyth ◽

Anthony Somers ◽

Mahdi Ghorbani ◽

...

Keyword(s):

Carbon Steel ◽

Laser Scanning ◽

Surface Condition ◽

Cell Number ◽

Laser Scanning Microscopy ◽

Microbiologically Influenced Corrosion ◽

Confocal Laser ◽

Viable Cell Number ◽

Initial Surface ◽

Bacterial Strains

AbstractChemical biocides remain the most effective mitigation strategy against microbiologically influenced corrosion (MIC), one of the costliest and most pervasive forms of corrosion in industry. However, toxicity and environmental concerns associated with these compounds are encouraging the development of more environmentally friendly MIC inhibitors. In this study, we evaluated the antimicrobial effect of a novel, multi-functional organic corrosion inhibitor (OCI) compound, cetrimonium trans-4-hydroxy-cinnamate (CTA-4OHcinn). Attachment of three bacterial strains, Shewanella chilikensis, Pseudomonas balearica and Klebsiella pneumoniae was evaluated on wet-ground (120 grit finish) and pre-oxidised carbon steel surfaces (AISI 1030), in the presence and absence of the new OCI compound. Our study revealed that all strains preferentially attached to pre-oxidised surfaces as indicated by confocal laser scanning microscopy, scanning electron microscopy and standard colony forming unit (CFU) quantification assays. The inhibitor compound at 10 mM demonstrated 100% reduction in S. chilikensis attachment independent of initial surface condition, while the other two strains were reduced by at least 99.7% of the original viable cell number. Our results demonstrate that CTA-4OHcinn is biocidal active and has promise as a multifunctional, environmentally sound MIC inhibitor for industrial applications.

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