scholarly journals Effect of Sodium Hypochlorite and Benzalkonium Chloride on the Structural Parameters of the Biofilms Formed by Ten Salmonella enterica Serotypes

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 154 ◽  
Author(s):  
Rosa Capita ◽  
Silvia Fernández-Pérez ◽  
Laura Buzón-Durán ◽  
Carlos Alonso-Calleja
Keyword(s):  
Sodium Hypochlorite ◽  
Salmonella Enterica ◽  
Benzalkonium Chloride ◽  
Laser Scanning ◽  
Surface Coverage ◽  
Structural Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Low Concentrations ◽  
Practical Field

The influence of the strain on the ability of Salmonella enterica to form biofilms on polystyrene was investigated by confocal laser scanning microscopy. The effects of sodium hypochlorite with 10% active chlorine (SHY; 25,000, 50,000, or 100,000 ppm), and benzalkonium chloride (BZK; 1000, 5000, or 10,000 ppm) on twenty-four-hour-old biofilms was also determined. The biofilms of ten Salmonella enterica isolates from poultry (S. Agona, S. Anatum, S. Enteritidis, S. Hadar, S. Infantis, S. Kentucky, S. Thompson, S. Typhimurium, monophasic variant of S. Typhimurium 1,4,(5),12:i:-, and S. Virchow) were studied. Biofilms produced by S. Anatum, S. Hadar, S. Kentucky, and S. Typhimurium showed a trend to have the largest biovolume and the greatest surface coverage and thickness. The smallest biofilms (P < 0.01) in the observation field (14.2 × 103 µm2) were produced by S. Enteritidis and S. 1,4,(5),12:i:- (average 12.9 × 103 ± 9.3 × 103 µm3) compared to the rest of the serotypes (44.4 × 103 ± 24.7 × 103 µm3). Biovolume and surface coverage decreased after exposure for ten minutes to SHY at 50,000 or 100,000 ppm and to BZK at 5000 or 10,000 ppm. However, the lowest concentrations of disinfectants increased biovolume and surface coverage in biofilms of several strains (markedly so in the case of BZK). The results from this study suggest that the use of biocides at low concentrations could represent a public health risk. Further research studies under practical field conditions should be appropriate to confirm these findings.

Effects of Bacteriophage P100 at Different Concentrations on the Structural Parameters of Listeria monocytogenes Biofilms

2018 ◽  
Vol 81 (12) ◽  
pp. 2040-2044 ◽  
Author(s):  
CRISTINA RODRÍGUEZ-MELCÓN ◽  
ROSA CAPITA ◽  
CAMINO GARCÍA-FERNÁNDEZ ◽  
CARLOS ALONSO-CALLEJA
Keyword(s):  
Image Analysis ◽  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
Digital Image Analysis ◽  
Surface Coverage ◽  
Structural Parameters ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Foodborne Diseases ◽  
Average Thickness

ABSTRACT Because listeriosis is one of the deadliest foodborne diseases, controlling and eradicating Listeria monocytogenes biofilms is a serious challenge for food safety. Biofilms (24 h old) formed on polystyrene by a L. monocytogenes strain of food origin were exposed for a further 24 h to 12 different concentrations (from 100 to 1011 PFU/mL) of the bacteriophage P100 (Listex P100). The structural parameters of biofilms were studied by using confocal laser scanning microscopy and digital image analysis. The biovolume in the observation field (14,121 μm2) of control (untreated) biofilms was 237,333.1 ± 2,692.6 μm3. The biomass of treated biofilms ranged from 164.7 ± 89.0 μm3 (biofilms exposed to 1010 PFU/mL) to 231,170.5 ± 15,142.0 μm3 (100 PFU/mL). The lowest biomass was achieved after treatment with 108 PFU/mL, with no further decrease in biovolume when higher phage concentrations were used. A strong (P &lt; 0.001) correlation was found between phage concentration (log units) and biovolume (−0.965), surface coverage (−0.939), roughness (0.976), maximum thickness (−0.853), and average thickness (−0.965). Findings from this research suggest that bacteriophage P100 at concentrations equal to or greater than 8 log PFU/mL successfully removes L. monocytogenes biofilms from polystyrene surfaces.

scholarly journals Er,Cr:YSGG Laser-Activation Enhances Antimicrobial and Antibiofilm Action of Low Concentrations of Sodium Hypochlorite in Root Canals

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 232
Author(s):  
Pablo Betancourt ◽  
Josep María Sierra ◽  
Octavi Camps-Font ◽  
Josep Arnabat-Domínguez ◽  
Miguel Viñas
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Sodium Hypochlorite ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Root Canals ◽  
Low Concentrations ◽  
Scanning Electron

The onset and persistence of endodontic infections due to residual biofilm after chemical disinfection promotes secondary bacterial infection. Alternative methods to disinfect operated root canals are a matter of great interest. The aim was to evaluate the antibacterial effectiveness of sodium hypochlorite (NaOCl) at low concentrations activated by the Er,Cr:YSGG laser-activated irrigation (LAI) against 10-day-old intracanal Enterococcus faecalis biofilm. Biofilms were formed inside the root canals and divided into 7 groups (n13): 0.5% NaOCl + Er,Cr:YSGG; Saline + Er,Cr:YSGG; 0.5% NaOCl + syringe irrigation(SI); 2.5% NaOCl + SI; 5% NaOCl + SI; positive and negative controls. Bacterial survivors were counted and specimens visualized under scanning electron and confocal laser scanning microscopy. Treatments with 0.5% NaOCl + Er,Cr:YSGG and 2.5% NaOCl + SI gave a significant reduction in the number of CFU/mm2. Moreover, scanning electron microscopy and confocal laser scanning microscopy imaging confirmed and reinforced bacteriological data. Thus, Er,Cr:YSGG LAI proved to be able to improve the intracanal distribution of 0.5% NaOCl after 60 s of activation, reaching the same level of effectiveness than 2.5% NaOCl. This is regarded as of clinical interest, since working with lower concentrations may contribute to reduce undesired effects.

Morphological and biochemical changes inPseudomonas fluorescensbiofilms induced by sub-inhibitory exposure to antimicrobial agents

2009 ◽  
Vol 55 (2) ◽  
pp. 163-178 ◽  
Author(s):  
James J. Dynes ◽  
John R. Lawrence ◽  
Darren R. Korber ◽  
George D.W. Swerhone ◽  
Gary G. Leppard ◽  
...  
Keyword(s):  
Antimicrobial Agent ◽  
Pseudomonas Fluorescens ◽  
Benzalkonium Chloride ◽  
Laser Scanning ◽  
Antimicrobial Agents ◽  
Membrane Integrity ◽  
Biochemical Changes ◽  
Laser Scanning Microscopy ◽  
Spectral Signature ◽  
Confocal Laser

Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM) were used to examine the morphological and biochemical changes in Pseudomonas fluorescens biofilms grown in the presence of subinhibitory concentrations of 4 antimicrobial agents: triclosan, benzalkonium chloride, chlorhexidine dihydrochloride, and trisodium phosphate. CLSM analyses using the stains SYTO9 and propidium iodide indicated that the antimicrobial agents affected cell membrane integrity and cellular density to differing degrees. However, fluorescein diacetate assays and plate counts demonstrated that the cells remained metabolically active. Fluorescent lectin binding assays showed that changes in the arrangement and composition of the exopolymer matrix of the biofilms also occurred and that these changes depended on the antimicrobial agent. Detailed single cell analyses using STXM provided evidence that the cell morphology, and the spatial distribution and relative amounts of protein, lipids and polysaccharides in the biofilms and within the cells were different for each antimicrobial. The distribution of chlorhexidine in the biofilm, determined from its distinct spectral signature, was localized mainly inside the bacterial cells. Each antimicrobial agent elicited a unique response; P. fluorescens cells and biofilms changed their morphology and architecture, as well as the distribution and abundance of biomacromolecules, in particular the exopolymer matrix. Pseudomonas fluorescens also exhibited adaptation to benzalkonium chloride at 10 µg/mL. Our observations point to the importance of changes in the quantity and chemistry of the exopolymeric matrix in the response to antimicrobial agents and suggest their importance as targets for control.

Interspecies Interactions in Dual-Species Biofilms Formed by Psychrotrophic Bacteria and the Tolerance of Sessile Communities to Disinfectants

2020 ◽  
Vol 83 (6) ◽  
pp. 951-958 ◽  
Author(s):  
LEI YUAN ◽  
NI WANG ◽  
FAIZAN A. SADIQ ◽  
GUOQING HE
Keyword(s):  
Hydrogen Peroxide ◽  
Sodium Hypochlorite ◽  
Peracetic Acid ◽  
Laser Scanning ◽  
Structural Changes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interspecies Interactions ◽  
Mixed Species ◽  
Psychrotrophic Bacteria

ABSTRACT Biofilms on the surface of food processing equipment act as potential reservoirs of microbial contamination. Bacterial interactions are believed to play key roles in both biofilm formation and antimicrobial tolerance. In this study, Aeromonas hydrophila, Chryseobacterium oncorhynchi, and Pseudomonas libanensis, which were previously isolated from Chinese raw milk samples, were selected to establish two dual-species biofilm models (P. libanensis plus A. hydrophila and P. libanensis plus C. oncorhynchi) on stainless steel at 7°C. Subsequently, three disinfectants, hydrogen peroxide (100 ppm), peracetic acid (100 ppm), and sodium hypochlorite (100 ppm), were used to treat the developed sessile communities for 10 min. Structural changes after exposure to disinfectants were analyzed with confocal laser scanning microscopy. The cell numbers of both A. hydrophila and C. oncorhynchi recovered from surfaces increased when grown as dual species biofilms with P. libanensis. Dual-species biofilms were more tolerant of disinfectants than were each single-species biofilm. Peracetic acid was the most effective disinfectant for removing biofilms, followed by hydrogen peroxide and sodium hypochlorite. The results expand the knowledge of mixed-species biofilms formed by psychrotrophic bacteria and will be helpful for developing effective strategies to eliminate bacterial mixed-species biofilms. HIGHLIGHTS

scholarly journals Combined Effect of Melittin and DNase on Enterococcus faecalis Biofilms and Its Susceptibility to Sodium Hypochlorite

Materials ◽  
2020 ◽  
Vol 13 (17) ◽  
pp. 3740
Author(s):  
Sujitha Ramaraj ◽  
Mi-Ah Kim ◽  
Vinicius Rosa ◽  
Prasanna Neelakantan ◽  
Won-Jun Shon ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Sodium Hypochlorite ◽  
Laser Scanning ◽  
Formation Rate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
P Value ◽  
Water Control ◽  
Sem Images ◽  
Root Canals

Biofilm communities are tolerant to antimicrobials and difficult to eradicate. This study aimed to investigate the effect of melittin, an antimicrobial peptide, either alone or in combination with deoxyribonuclease (DNase), an inhibitor of extracellular deoxyribonucleic acid (eDNA), against Enterococcus faecalis (E. faecalis) biofilms, and biofilm susceptibility to sodium hypochlorite (NaOCl). Biofilms of E. faecalis were developed in root canals of bovine teeth. The biofilms were treated with distilled water (control), melittin, DNase, or DNase+melittin. The antibiofilm effects of the treatments were analyzed using colony forming unit (CFU) assay, crystal violet staining, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscope (FE-SEM). The susceptibility of DNase+melittin-treated biofilms to NaOCl (0%, 2.5% and 5%) was investigated by the CFU assay. The data were statistically analyzed using one-way analysis of variance, followed by Tukey’s test. A p-value of <0.05 was considered significant. Specimens treated with DNase+melittin showed a more significant decrease in the CFUs, eDNA level, and biofilm formation rate than those treated only with melittin or DNase (p < 0.05). CLSM analysis showed DNase+melittin treatment significantly reduced the volume of biofilms and extracellular polymeric substance compared to either treatment alone (p < 0.05). FE-SEM images showed a high degree of biofilm disruption in specimens that received DNase+melittin. 2.5% NaOCl in specimens pretreated with DNase+melittin showed higher antibacterial activity than those treated only with 5% NaOCl (p < 0.05). This study highlighted that DNase improved the antibiofilm effects of melittin. Moreover, DNase+melittin treatment increased the susceptibility of biofilms to NaOCl. Thus, the complex could be a clinical strategy for safer use of NaOCl by reducing the concentration.

scholarly journals Fitness of Salmonella enterica serovar Thompson in the Cilantro Phyllosphere

2002 ◽  
Vol 68 (7) ◽  
pp. 3614-3621 ◽  
Author(s):  
Maria T. Brandl ◽  
Robert E. Mandrell
Keyword(s):  
Salmonella Enterica ◽  
Laser Scanning ◽  
Fruits And Vegetables ◽  
Fluorescent Protein ◽  
Bacterial Flora ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Pathogen ◽  
Dry Conditions ◽  
Green Fluorescent

ABSTRACT The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.

scholarly journals Hydrosol of Thymbra capitata Is a Highly Efficient Biocide against Salmonella enterica Serovar Typhimurium Biofilms

2016 ◽  
Vol 82 (17) ◽  
pp. 5309-5319 ◽  
Author(s):  
Foteini Karampoula ◽  
Efstathios Giaouris ◽  
Julien Deschamps ◽  
Agapi I. Doulgeraki ◽  
George-John E. Nychas ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Laser Scanning ◽  
High Efficiency ◽  
3D Structure ◽  
Time Lapse ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Cells ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTSalmonellais recognized as one of the most significant enteric foodborne bacterial pathogens. In recent years, the resistance of pathogens to biocides and other environmental stresses, especially when they are embedded in biofilm structures, has led to the search for and development of novel antimicrobial strategies capable of displaying both high efficiency and safety. In this direction, the aims of the present work were to evaluate the antimicrobial activity of hydrosol of the Mediterranean spiceThymbracapitataagainst both planktonic and biofilm cells ofSalmonella entericaserovar Typhimurium and to compare its action with that of benzalkonium chloride (BC), a commonly used industrial biocide. In order to achieve this, the disinfectant activity following 6-min treatments was comparatively evaluated for both disinfectants by calculating the concentrations needed to achieve the same log reductions against both types of cells. Their bactericidal effect against biofilm cells was also comparatively determined byin situand real-time visualization of cell inactivation through the use of time-lapse confocal laser scanning microscopy (CLSM). Interestingly, results revealed that hydrosol was almost equally effective against biofilms and planktonic cells, whereas a 200-times-higher concentration of BC was needed to achieve the same effect against biofilm compared to planktonic cells. Similarly, time-lapse CLSM revealed the significant advantage of the hydrosol to easily penetrate within the biofilm structure and quickly kill the cells, despite the three-dimensional (3D) structure ofSalmonellabiofilm.IMPORTANCEThe results of this paper highlight the significant antimicrobial action of a natural compound, hydrosol ofThymbra capitata, against both planktonic and biofilm cells of a common foodborne pathogen. Hydrosol has numerous advantages as a disinfectant of food-contact surfaces. It is an aqueous solution which can easily be rinsed out from surfaces, it does not have the strong smell of the essential oil (EO) and it is a byproduct of the EO distillation procedure without any industrial application until now. Consequently, hydrosol obviously could be of great value to combat biofilms and thus to improve product safety not only for the food industries but probably also for many other industries which experience biofilm-related problems.

scholarly journals Growth-Inhibitory Effect of Heparin on Babesia Parasites

2004 ◽  
Vol 48 (1) ◽  
pp. 236-241 ◽  
Author(s):  
Sabine Bork ◽  
Naoaki Yokoyama ◽  
Yuzuru Ikehara ◽  
Sanjay Kumar ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescein Isothiocyanate ◽  
Inhibitory Effect ◽  
Laser Scanning Microscopy ◽  
Babesia Bovis ◽  
Confocal Laser ◽  
Intracellular Parasites ◽  
Low Concentrations

ABSTRACT We examined the inhibitory effects of three heparins on the growth of Babesia parasites. The multiplication of Babesia bovis, B. bigemina, B. equi, and B. caballi in in vitro cultures and that of B. microti in vivo were significantly inhibited in the presence of heparins, as determined by light microscopy. Treatment with various concentrations of heparin showed complete clearance of the intracellular parasites. Interestingly, a higher percentage of abnormally multidividing B. bovis parasites was observed in the presence of low concentrations of heparin. Furthermore, fluorescein isothiocyanate-labeled heparin was preferably found on the surfaces of extracellular merozoites, as detected by confocal laser scanning microscopy. These findings indicate that the heparin covers the surfaces of babesial merozoites and inhibits their subsequent invasion of erythrocytes.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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