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.

scholarly journals Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae

2004 ◽  
Vol 72 (1) ◽  
pp. 106-113 ◽  
Author(s):  
W. Edward Swords ◽  
Miranda L. Moore ◽  
Luciana Godzicki ◽  
Gail Bukofzer ◽  
Michael J. Mitten ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Biofilm Formation ◽  
Model System ◽  
Model Systems ◽  
Bacterial Persistence ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.

scholarly journals Nontypeable Haemophilus influenzae: Pathogenesis and Prevention

1998 ◽  
Vol 62 (2) ◽  
pp. 294-308 ◽  
Author(s):  
A. Ruth Foxwell ◽  
Jennelle M. Kyd ◽  
Allan W. Cripps
Keyword(s):  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Phenotypic Diversity ◽  
Outer Membrane Proteins ◽  
Mucosal Vaccine ◽  
Mammalian Host ◽  
Nontypeable Haemophilus Influenzae ◽  
Vaccine Strategy ◽  
Tract Infections

SUMMARY In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.

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 Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

2006 ◽  
Vol 74 (8) ◽  
pp. 4590-4597 ◽  
Author(s):  
Jennifer D. Boddicker ◽  
Rebecca A. Anderson ◽  
Jennifer Jagnow ◽  
Steven Clegg
Keyword(s):  
Extracellular Matrix ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Matrix Material ◽  
Infection Process ◽  
Bladder Epithelium ◽  
Tract Infections ◽  
Type 3

ABSTRACT Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.

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 VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

2007 ◽  
Vol 189 (14) ◽  
pp. 5041-5048 ◽  
Author(s):  
Dayle A. Daines ◽  
Mack H. Wu ◽  
Sarah Y. Yuan
Keyword(s):  
Haemophilus Influenzae ◽  
Growth Inhibition ◽  
Gene Pair ◽  
Gene Families ◽  
Upper Respiratory Tract ◽  
Nontypeable Haemophilus Influenzae ◽  
E Coli ◽  
Conserved Gene

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

scholarly journals Comparison of transcription of the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear

BMC Genomics ◽  
2013 ◽  
Vol 14 (1) ◽  
pp. 925 ◽  
Author(s):  
Paul W Whitby ◽  
Timothy M VanWagoner ◽  
Thomas W Seale ◽  
Daniel J Morton ◽  
Terrence L Stull
Keyword(s):  
Haemophilus Influenzae ◽  
Middle Ear ◽  
Iron Heme

scholarly journals An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth

2007 ◽  
Vol 56 (12) ◽  
pp. 1600-1607 ◽  
Author(s):  
Analía Lima ◽  
Pablo Zunino ◽  
Bruno D'Alessandro ◽  
Claudia Piccini
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Outer Membrane ◽  
Proteus Mirabilis ◽  
Outer Membrane Proteins ◽  
Mammalian Host ◽  
Tract Infections

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.

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