scholarly journals Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

2014 ◽  
Vol 80 (22) ◽  
pp. 7088-7095 ◽  
Author(s):  
Carmen Puig ◽  
Arnau Domenech ◽  
Junkal Garmendia ◽  
Jeroen D. Langereis ◽  
Pascal Mayer ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Respiratory Disease ◽  
Otitis Media ◽  
Crystal Violet ◽  
Solid Surface ◽  
Biofilm Formation ◽  
Invasive Disease ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Initial Adhesion

ABSTRACTBiofilm formation by nontypeable (NT)Haemophilus influenzaeremains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NTH. influenzaeby comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NTH. influenzaeisolation, with significantly increased biofilm formation for NTH. influenzaeisolates collected from patients with invasive disease and OM compared with NTH. influenzaeisolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.

scholarly journals Epigenetic Regulation Alters Biofilm Architecture and Composition in Multiple Clinical Isolates of Nontypeable Haemophilus influenzae

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Kenneth L. Brockman ◽  
Patrick N. Azzari ◽  
M. Taylor Branstool ◽  
John M. Atack ◽  
Benjamin L. Schulz ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Biofilm Formation ◽  
Human Pathogens ◽  
Content Type ◽  
Alkaline Conditions ◽  
Tract Infections ◽  
Biofilm Structure

ABSTRACT Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases. IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phase variable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Letícia Roloff Stallbaum ◽  
Beatriz Bohns Pruski ◽  
Suelen Cavalheiro Amaral ◽  
Stella Buchhorn de Freitas ◽  
Daniela Rodriguero Wozeak ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Multidrug Resistant ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

scholarly journals Green Tea Polyphenols and Padma Hepaten Inhibit Candida albicans Biofilm Formation

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Yosi Farkash ◽  
Mark Feldman ◽  
Isaac Ginsburg ◽  
Doron Steinberg ◽  
Miriam Shalish
Keyword(s):  
Candida Albicans ◽  
Green Tea ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Host Cells ◽  
Green Tea Polyphenols ◽  
Crystal Violet Staining ◽  
Biofilm Inhibition ◽  
Therapeutic Concept ◽  
Biofilm Development

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

scholarly journals First Complete Genome Sequence of Haemophilus influenzae Serotype a

2017 ◽  
Vol 5 (3) ◽  
Author(s):  
Mariam Iskander ◽  
Kristy Hayden ◽  
Gary Van Domselaar ◽  
Raymond Tsang
Keyword(s):  
Haemophilus Influenzae ◽  
Genome Sequence ◽  
Invasive Disease ◽  
Indigenous Populations ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Human Pathogen ◽  
Small Children ◽  
Content Type ◽  
Serotype A

ABSTRACT Haemophilus influenzae is an important human pathogen that primarily infects small children. In recent years, H. influenzae serotype a has emerged as a significant cause of invasive disease among indigenous populations. Here, we present the first complete whole-genome sequence of H. influenzae serotype a.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

scholarly journals Haemophilus influenzae biofilm formation in chronic otitis media with effusion

2016 ◽  
Vol 273 (11) ◽  
pp. 3553-3560 ◽  
Author(s):  
Helen Van Hoecke ◽  
Ann-Sophie De Paepe ◽  
Edward Lambert ◽  
Jonas D Van Belleghem ◽  
Piet Cools ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Biofilm Formation ◽  
Otitis Media With Effusion ◽  
Chronic Otitis Media

scholarly journals First Whole-Genome Sequence of a Haemophilus influenzae Type e Strain Isolated from a Patient with Invasive Disease in Italy

2017 ◽  
Vol 5 (13) ◽  
Author(s):  
Maria Giufrè ◽  
Rita Cardines ◽  
Marina Cerquetti
Keyword(s):  
Haemophilus Influenzae ◽  
Genome Sequence ◽  
Invasive Disease ◽  
Whole Genome Sequence ◽  
Haemophilus Influenzae Type ◽  
Whole Genome ◽  
Type B ◽  
Genomic Information ◽  
Conjugate Vaccines ◽  
Content Type

ABSTRACT In the present era of conjugate vaccines against Haemophilus influenzae type b, non-vaccine-preventable strains are of concern. Here, we report the first whole-genome sequence of an invasive H. influenzae type e strain. This genomic information will enable further investigations on encapsulated non-type b H. influenzae strains.

scholarly journals Closed Complete Annotated Genome Sequences of Five Haemophilus influenzae Biogroup aegyptius Strains

2019 ◽  
Vol 8 (45) ◽  
Author(s):  
Zachary N. Phillips ◽  
Greg Tram ◽  
Michael P. Jennings ◽  
John M. Atack
Keyword(s):  
Haemophilus Influenzae ◽  
Complete Genome ◽  
Invasive Disease ◽  
Genome Sequences ◽  
Content Type ◽  
Brazilian Purpuric Fever

Haemophilus influenzae biogroup aegyptius is a cause of conjunctivitis in children. Biogroup aegyptius strains also caused fatal outbreaks of invasive disease, known as Brazilian purpuric fever (BPF), in the 1980s. BPF is fatal if untreated. Here, we report the complete genome sequences of five strains of Haemophilus influenzae biogroup aegyptius.

scholarly journals Bacterial Lysis through Interference with Peptidoglycan Synthesis Increases Biofilm Formation by Nontypeable Haemophilus influenzae

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Sara Marti ◽  
Carmen Puig ◽  
Alexandra Merlos ◽  
Miguel Viñas ◽  
Marien I. de Jonge ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Host Immune System ◽  
Content Type ◽  
Resistance To Antibiotics ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Subinhibitory Concentrations

ABSTRACT Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds. Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of β-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with β-lactam antibiotics, especially for β-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds.

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