scholarly journals Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production

2016 ◽  
Vol 62 (12) ◽  
pp. 1013-1020
Author(s):  
Najla A. Obaid ◽  
Stephen Tristram ◽  
Christian K. Narkowicz ◽  
Glenn A. Jacobson
Keyword(s):  
Haemophilus Influenzae ◽  
Biofilm Formation ◽  
Frozen Storage ◽  
Major Effect ◽  
Nontypeable Haemophilus Influenzae ◽  
Microtitre Plate ◽  
Biofilm Production ◽  
Solid Media ◽  
Inoculum Preparation

Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18–20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at –80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

scholarly journals Role of Sialic Acid and Complex Carbohydrate Biosynthesis in Biofilm Formation by Nontypeable Haemophilus influenzae in the Chinchilla Middle Ear

2005 ◽  
Vol 73 (6) ◽  
pp. 3210-3218 ◽  
Author(s):  
Joseph Jurcisek ◽  
Laura Greiner ◽  
Hiroshi Watanabe ◽  
Anthony Zaleski ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Mammalian Host ◽  
Nontypeable Haemophilus Influenzae ◽  
Biofilm Production ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an important pathogen in respiratory tract infections, including otitis media (OM). NTHI forms biofilms in vitro as well as in the chinchilla middle ear, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of OM. We've previously shown that SiaA, SiaB, and WecA are involved in biofilm production by NTHI in vitro. To investigate whether these gene products were also involved in biofilm production in vivo, NTHI strain 2019 and five isogenic mutants with deletions in genes involved in carbohydrate biosynthesis were inoculated into the middle ears of chinchillas. The wild-type strain formed a large, well-organized, and viable biofilm; however, the wecA, lsgB, siaA, pgm, and siaB mutants were either unable to form biofilms or formed biofilms of markedly reduced mass, organization, and viability. Despite their compromised ability to form a biofilm in vivo, wecA, lsgB, and siaA mutants survived in the chinchilla, inducing culture-positive middle ear effusions, whereas pgm and siaB mutants were extremely sensitive to the bactericidal activity of chinchilla serum and thus did not survive. Lectin analysis indicated that sialic acid was an important component of the NTHI 2019 biofilm produced in vivo. Our data suggested that genes involved in carbohydrate biosynthesis and assembly play an important role in the ability of NTHI to form a biofilm in vivo. Collectively, we found that when modeled in a mammalian host, whereas biofilm formation was not essential for survivability of NTHI in vivo, lipooligosaccharide sialylation was indispensable.

Nontypeable Haemophilus influenzae infection impedes Pseudomonas aeruginosa colonization and persistence in mouse respiratory tract

2021 ◽  
Author(s):  
Natalie Lindgren ◽  
Lea Novak ◽  
Benjamin C. Hunt ◽  
Melissa S. McDaniel ◽  
W. Edward Swords
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Respiratory Tract ◽  
Haemophilus Influenzae ◽  
Respiratory Infections ◽  
Bacterial Species ◽  
Young Patients ◽  
Nontypeable Haemophilus Influenzae ◽  
Potential Significance ◽  
And Diffusion

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa ( Pa ). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract. Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

scholarly journals LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface

2009 ◽  
Vol 77 (9) ◽  
pp. 4081-4091 ◽  
Author(s):  
Chelsie E. Armbruster ◽  
Wenzhou Hong ◽  
Bing Pang ◽  
Kristin E. Dew ◽  
Richard A. Juneau ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Opportunistic Infections ◽  
Bacterial Species ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nontypeable Haemophilus Influenzae ◽  
Bacterial Persistence ◽  
Soluble Mediator

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

scholarly journals Association between Biofilm-Production and Antibiotic Resistance in Uropathogenic Escherichia coli (UPEC): An In Vitro Study

Diseases ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 17 ◽  
Author(s):  
Payam Behzadi ◽  
Edit Urbán ◽  
Márió Gajdács
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
In Vitro Study ◽  
Point Of View ◽  
Diffusion Method ◽  
Medical Attention ◽  
Disk Diffusion Method ◽  
Biofilm Production ◽  
Tract Infections

Urinary tract infections (UTIs) are among the most common infections requiring medical attention worldwide. The production of biofilms is an important step in UTIs, not only from a mechanistic point of view, but this may also confer additional resistance, distinct from other aspects of multidrug resistance (MDR). A total of two hundred and fifty (n = 250) Escherichia coli isolates, originating from clean-catch urine samples, were included in this study. The isolates were classified into five groups: wild-type, ciprofloxacin-resistant, fosfomycin-resistant, trimethoprim-sulfamethoxazole-resistant and extended spectrum β-lactamase (ESBL)-producing strains. The bacterial specimens were cultured using eosine methylene blue agar and the colony morphology of isolates were recorded. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests. Biofilm-formation of the isolates was carried out with the crystal violet tube-adherence method. n = 76 isolates (30.4%) produced large colonies (>3 mm), mucoid variant colonies were produced in n = 135 cases (54.0%), and n = 119 (47.6%) were positive for biofilm formation. The agreement (i.e., predictive value) of mucoid variant colonies in regard to biofilm production in the tube-adherence assay was 0.881 overall. Significant variation was seen in the case of the group of ESBL-producers in the ratio of biofilm-producing isolates. The relationship between biofilm-production and other resistance determinants has been extensively studied. However, no definite conclusion can be reached from the currently available data.

scholarly journals In-Vitro Biofilm Formation and Antimicrobial Resistance of Escherichia coli in Diabetic and Nondiabetic Patients

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Sunayana Raya ◽  
Ankit Belbase ◽  
Laxmi Dhakal ◽  
Krishna Govinda Prajapati ◽  
Reena Baidya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Diabetic Patients ◽  
Microtitre Plate ◽  
Plate Method ◽  
E Coli ◽  
Biofilm Production

Background. Diabetic patients are more susceptible to urinary tract infection compared to nondiabetic patients, Escherichia coli being the most common uropathogen causing UTI. Unreasonable and incorrect antibiotic prescription for UTI in these patients may induce the development of antibiotic-resistant urinary pathogens resulting in delayed recovery and longer hospitalization. In addition to these, biofilm forming capacity of the pathogen may worsen the problem. The main aim of this cross-sectional study (conducted from March to September 2015) is to detect the biofilm forming capacity of UTI causing micro-organisms and compare the antibiotic resistance pattern of Escherichia coli, the most common cause of UTI, which will help the physician in choosing the best antibiotic. Method. Total of 1,099 clean-catch mid stream urine (CCMSU) was processed by standard microbiological technique; 182 were from the diabetic group and 917 nondiabetic. Following identification, all isolates were subjected to antibiotic susceptibility testing using modified Kirby-Bauer disc diffusion method. In-vitro biofilm forming capacity of the isolates were detected by Microtitre plate method. The data were analyzed using SPSS software 16. Result. Urinary tract infection was found to be significantly higher in diabetic patients (42.9%) compared to nondiabetic patients (17.4%) with Escherichia coli as the most common uropathogen in both diabetic and nondiabetic groups. Similarly, UTI was more common in elderly population (29.5%). Imipenem, nitrofurantoin and amikacin were found to be the most effective drug for uropathogenic E. coli in both diabetic and nondiabetic patients, whereas amoxicillin, ciprofloxacin, and cotrimoxazole were least effective. Of the total bacterial isolates, 43.3% showed positive results for in-vitro biofilm production by the Microtitre plate method. A significantly higher resistance rate was observed among biofilm producing E. coli for quinolones, cotrimoxazole, and third generation cephalosporin ceftriaxone. Most of the biofilm producers (79.5%) were found to be MDR (p-value 0.015). Conclusion. Elderly populations with diabetes are at a higher risk of UTI. Higher biofilm production and resistance to in-use antimicrobial agents in this study render its inefficacy for empirical treatment and point out the importance of biofilm screening to ensure the effective management of infection.

scholarly journals Conversion of Staphylococcus epidermidis Strains from Commensal to Invasive by Expression of the ica Locus Encoding Production of Biofilm Exopolysaccharide

2005 ◽  
Vol 73 (5) ◽  
pp. 3188-3191 ◽  
Author(s):  
Hualin Li ◽  
Lin Xu ◽  
Jianping Wang ◽  
Yumei Wen ◽  
Cuong Vuong ◽  
...  
Keyword(s):  
Central Venous Catheter ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Venous Catheter ◽  
Infection Model ◽  
Polysaccharide Intercellular Adhesin ◽  
Central Venous ◽  
Biofilm Production ◽  
Central Venous Catheter Infection

ABSTRACT To test if biofilm formation in Staphylococcus epidermidis is dependent on the polysaccharide intercellular adhesin, whose biosynthesis is driven by the ica locus, a plasmid containing the ica locus was transferred to three ica-negative strains. Using in vitro biofilm assays and a rat central venous catheter infection model, we confirmed the importance of the ica locus for biofilm production and pathogenesis of S. epidermidis.

scholarly journals In vitro antifungal susceptibility of candidemia agents and detection of their biofilm production by two different methods

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Yasemin Oz ◽  
Iman Qoraan ◽  
Egemen Gokbolat
Keyword(s):  
Production Rate ◽  
Biofilm Formation ◽  
Colorimetric Assay ◽  
Formation Rate ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Antifungal Resistance ◽  
Detection Methods ◽  
Biofilm Production

Candida bloodstream infections are a significant cause of morbidity and mortality in hospitalized patients. The most important contribution of biofilm is the higher antifungal resistance than planktonic cells. We aimed to investigate the biofilm formation rate and antifungal susceptibility characteristics of our bloodstream isolates, and evaluate two different biofilm detection methods. A total of 200 bloodstream Candida isolates were included. The biofilms were formed on 96-well microtiter plates and measured by spectrophotometric percent transmittance and 2,3-bis(2- methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium- 5-carboxanilide colorimetric assay. In addition antifungal susceptibilities of these isolates were evaluated against caspofungin, anidulafungin and amphotericin B by reference method. Biofilm production rate was considerably high among our bloodstream isolates. The most important biofilm producer species was C. tropicalis; C. glabrata had the lowest biofilm production rate. The consistency rate between biofilm detection methods was 66%. Remarkable antifungal resistance was not observed among our isolates in general. In conclusion, biofilm production in Candida species is an important virulence factor, and its rate is considerably high in bloodstream isolates. At present, a standardized method has not been established to detect the biofilm formation.

scholarly journals Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

2014 ◽  
Vol 83 (3) ◽  
pp. 950-957 ◽  
Author(s):  
Christine Cho ◽  
Aroon Chande ◽  
Lokesh Gakhar ◽  
Lauren O. Bakaletz ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Microscopic Analysis ◽  
Human Infection ◽  
Extracellular Dna ◽  
Wild Type ◽  
Nontypeable Haemophilus Influenzae ◽  
Dispersal Pattern ◽  
Content Type ◽  
Biofilm Dispersal

NontypeableHaemophilus influenzae(NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease ofStaphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P< 0.042). Microscopic analysis of biofilms grownin vitroshowed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.

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