scholarly journals The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Luke V. Blakeway ◽  
Aimee Tan ◽  
Joseph A. Jurcisek ◽  
Lauren O. Bakaletz ◽  
John M. Atack ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Moraxella Catarrhalis ◽  
Nitrosative Stress ◽  
Bacterial Load ◽  
Phase Variation ◽  
In Vivo Model ◽  
Phase Variable ◽  
Variable Expression

Abstract Background Moraxella catarrhalis is a leading cause of otitis media (OM) and chronic obstructive pulmonary disease (COPD). M. catarrhalis contains a Type III DNA adenine methyltransferase (ModM) that is phase-variably expressed (i.e., its expression is subject to random, reversible ON/OFF switching). ModM has six target recognition domain alleles (modM1–6), and we have previously shown that modM2 is the predominant allele, while modM3 is associated with OM. Phase-variable DNA methyltransferases mediate epigenetic regulation and modulate pathogenesis in several bacteria. ModM2 of M. catarrhalis regulates the expression of a phasevarion containing genes important for colonization and infection. Here we describe the phase-variable expression of modM3, the ModM3 methylation site and the suite of genes regulated within the ModM3 phasevarion. Results Phase-variable expression of modM3, mediated by variation in length of a 5′-(CAAC)n-3′ tetranucleotide repeat tract in the open reading frame was demonstrated in M. catarrhalis strain CCRI-195ME with GeneScan fragment length analysis and western immunoblot. We determined that ModM3 is an active N6-adenine methyltransferase that methylates the sequence 5′-ACm6ATC-3′. Methylation was detected at all 4446 5′-ACATC-3′ sites in the genome when ModM3 is expressed. RNASeq analysis identified 31 genes that are differentially expressed between modM3 ON and OFF variants, including five genes that are involved in the response to oxidative and nitrosative stress, with potential roles in biofilm formation and survival in anaerobic environments. An in vivo chinchilla (Chinchilla lanigera) model of otitis media demonstrated that transbullar challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to a modM3 ON variant. In addition, co-infection experiments with NTHi and M. catarrhalis modM3 ON or modM3 OFF variants revealed that phase variation of modM3 altered survival of NTHi in the middle ear during early and late stage infection. Conclusions Phase variation of ModM3 epigenetically regulates the expression of a phasevarion containing multiple genes that are potentially important in the progression of otitis media.

Experimental Otitis Media Evaluated by Magnetic Resonance Imaging: An in Vivo Model

1992 ◽  
Vol 101 (3) ◽  
pp. 248-254 ◽  
Author(s):  
Kenny H. Chan ◽  
William J. Doyle ◽  
J. Douglas Swarts ◽  
David Kardatzke ◽  
Yoshie Hashida ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Magnetic Resonance ◽  
Contrast Agent ◽  
Otitis Media ◽  
Middle Ear ◽  
Washout Rate ◽  
In Vivo Model ◽  
Resonance Imaging ◽  
Middle Ear Mucosa

The use of magnetic resonance imaging in otitis media research is being explored in our laboratory. In this study, we present a new method for studying changes in the middle ear cleft due to an episode of induced otitis media in the chinchilla model. It uses gadolinium-diethylenetriamine pentaacetic acid, a magnetic resonance imaging contrast agent, to examine the uptake and washout characteristics of middle ear mucosa during an inflammatory episode. Parameters such as the time to maximum intensity of the mucosa and the washout rate of the contrast agent from the mucosa were significantly correlated to the duration of the infection.

scholarly journals Evaluation of Phase Variation of NontypeableHaemophilus influenzae Lipooligosaccharide during Nasopharyngeal Colonization and Development of Otitis Media in the Chinchilla Model

2000 ◽  
Vol 68 (8) ◽  
pp. 4593-4597 ◽  
Author(s):  
H. H. Tong ◽  
L. E. Blue ◽  
M. A. James ◽  
Y. P. Chen ◽  
T. F. DeMaria
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Phase Variation ◽  
Lavage Fluid ◽  
Nasal Lavage ◽  
Phase Variable ◽  
Nasopharyngeal Colonization ◽  
Nthi Strain ◽  
Nasal Lavage Fluid ◽  
Culture Positive

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures.lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 tolic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP− to the ChoP+ phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP+ phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP− variant (P < 0.05). The concentration of cells with the ChoP+ phenotype in the middle ear was 3 log units higher than that of cells with the ChoP− variant (P < 0.01). There was a statistically significant association between ChoP+ expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP+phenotype promotes enhanced nasopharyngeal colonization and development of OM.

In Vivo Sensitivity Test in Otitis Media: Efficacy of Antibiotics

PEDIATRICS ◽  
1985 ◽  
Vol 75 (1) ◽  
pp. 8-13 ◽  
Author(s):  
Virgil M. Howie ◽  
Ruth Dillard ◽  
Barbara Lawrence
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Penicillin G ◽  
Treatment Results ◽  
Double Blind ◽  
Phenoxymethyl Penicillin ◽  
Procaine Penicillin ◽  
The Poor

During a 10-year period, antibiotics were assigned in random, double-blind fashion in six combinations to treat 948 episodes of otitis media in children. Exudate from the middle ear of all patients was cultured before treatment. Three follow-up visits were conducted; the first follow-up visit was three to five days after the start of therapy, and the second and third visits were 14 and 31 days after onset of treatment. Exudates were recultured for 75% of the patients on the first follow-up visit. Comparison of treatment results showed that triple sulfonamide combined with either phenoxymethyl penicillin, or benzathine and procaine penicillin G given intramuscularly (IM) was as effective as was ampicillin or amoxicillin. Phenoxymethyl penicillin and cyclacillin alone were usually effective against pneumococci but relatively ineffective against Haemophilus influenzae. Cefaclor and trimethoprim-sulfamethoxazole produced unsatisfactory results in about half the cases caused by pneumococci or H influenzae. Although production of β-lactamase by some otitis-causing Haemophilus and Staphylococcus species may explain the ineffectiveness of some treatments, the percentage of organisms positive for β-lactamase was too small to be responsible for the poor results with certain drugs.

In vivo optical characterization of middle ear effusions associated with otitis media

2021 ◽  
Author(s):  
Jungeun Won ◽  
Guillermo L. Monroy ◽  
Ryan G. Porter ◽  
Michael A. Novak ◽  
Ronit Barkalifa ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Optical Characterization

scholarly journals Nontypeable Haemophilus influenzae Gene Expression Induced In Vivo in a Chinchilla Model of Otitis Media

2003 ◽  
Vol 71 (6) ◽  
pp. 3454-3462 ◽  
Author(s):  
Kevin M. Mason ◽  
Robert S. Munson ◽  
Lauren O. Bakaletz
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Middle Ear ◽  
Fluorescent Protein ◽  
Otitis Media With Effusion ◽  
Acute Otitis Media ◽  
Bacterial Gene ◽  
Nthi Strain

ABSTRACT The gram-negative bacterium nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic otitis media with effusion and, with Streptococcus pneumoniae and Moraxella catarrhalis, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of H. influenzae strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.

scholarly journals Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

2005 ◽  
Vol 73 (4) ◽  
pp. 2213-2221 ◽  
Author(s):  
Ruth Griffin ◽  
Andrew D. Cox ◽  
Katherine Makepeace ◽  
James C. Richards ◽  
E. Richard Moxon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Phase Variation ◽  
Phase Variable ◽  
Type B ◽  
Variable Expression ◽  
Virulent Type ◽  
Unknown Structure

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

scholarly journals Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions

2003 ◽  
Vol 71 (7) ◽  
pp. 3821-3830 ◽  
Author(s):  
Ravenna Flitman-Tene ◽  
Sigalit Mudahi-Orenstein ◽  
Sharon Levisohn ◽  
David Yogev
Keyword(s):  
Dna Recombination ◽  
Small Ruminants ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Phase Variable ◽  
Multiple Sequence ◽  
Site Specific ◽  
Mycoplasma Agalactiae ◽  
Variable Expression

ABSTRACT Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.

scholarly journals Moraxella catarrhalis Might Be More Common than Expected in Acute Otitis Media in Young Finnish Children

2016 ◽  
Vol 54 (9) ◽  
pp. 2373-2379 ◽  
Author(s):  
Saara Sillanpää ◽  
Sami Oikarinen ◽  
Markku Sipilä ◽  
Lenka Kramna ◽  
Markus Rautiainen ◽  
...  
Keyword(s):  
Otitis Media ◽  
Middle Ear ◽  
Moraxella Catarrhalis ◽  
Acute Otitis Media ◽  
Pcr Analysis ◽  
Considerable Proportion ◽  
Bacterial Dna ◽  
Content Type ◽  
Conventional Culture

According to studies based on bacterial cultures of middle ear fluids,Streptococcus pneumoniae,Haemophilus influenzae, andMoraxella catarrhalishave been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence ofS. pneumoniae,H. influenzae,M. catarrhalis,Alloiococcusotitidis,Staphylococcus aureus, andPseudomonas aeruginosa. One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis,M. catarrhalisresults were positive in 42 (47%) samples,H. influenzaein 30 (33%),S. pneumoniaein 27 (30%),A. otitidisin 6 (6.7%),S. aureusin 5 (5.6%), andP. aeruginosain 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, andM. catarrhaliswas detected in most (85%) of those cases. Fifteen signals forM. catarrhaliswere strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even thoughM. catarrhalisis often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.

scholarly journals Pulmonary Damage and Bacterial Load in Assessment of the Efficacy of Simulated Human Treatment-Like Amoxicillin (2,000 Milligrams) Therapy of Experimental Pneumococcal Pneumonia Caused by Strains for Which Amoxicillin MICs Differ

2005 ◽  
Vol 49 (3) ◽  
pp. 996-1001 ◽  
Author(s):  
Matilde Gracia ◽  
Carmina Martínez-Marín ◽  
Lorena Huelves ◽  
Maria J. Giménez ◽  
Lorenzo Aguilar ◽  
...  
Keyword(s):  
Bacterial Load ◽  
In Vitro Study ◽  
In Vivo Model ◽  
Lung Weight ◽  
End Points ◽  
Dosing Interval ◽  
Pulmonary Damage ◽  
Amoxicillin Clavulanate

ABSTRACT An experimental rat pneumonia model using two amoxicillin-susceptible (MICs, ≤0.015 and 2 μg/ml) and two non-amoxicillin-susceptible (MIC, 4 μg/ml) Streptococcus pneumoniae strains was developed for testing the efficacy of amoxicillin administered to simulate human serum kinetics after treatment with amoxicillin-clavulanate (2,000 and 125 mg, respectively, twice a day, for 2.5 days). The end points for efficacy were reductions in bacterial loads in the lungs and reductions in levels of pulmonary damage. For the amoxicillin-susceptible strains (serotypes 23F and 14), a decrease greater than 4.5 log10 CFU/pair of lungs was obtained, and the time for which the serum antibiotic concentration (SAC) was higher than the MIC (T S A C > MIC) was greater than 60% of the dosing interval. For non-amoxicillin-susceptible strains, the decrease in bacterial load was 1.34 to 1.75 log10 CFU/pair of lungs, with a T S A C > MIC of 46.7% of the dosing interval. An in vitro study showed that serotype 9V non-amoxicillin-susceptible strains behaved as tolerant-like to concentrations similar to those in the in vivo model. The high and maintained SACs (T S A C > MIC, >46% for all strains) significantly diminished lung injury (affected area of the lung and lung weight), compared to that in controls, by all strains, regardless of the MIC, bactericidal behavior in in vitro killing curves, or the serotype of the infecting strain. These results show the importance of host therapeutic end points in the evaluation of antibiotic efficacy. The antibiotic was more efficacious, for one nonsusceptible strain tested, when the treatment was started early (1 h postinoculation [p.i.]) than when treatment was delayed (24 h p.i.).

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