Complete genome sequence of Streptococcus pneumoniae strain ST556, a multidrug-resistant isolate from an otitis media patient.
ABSTRACT: Streptococcus pneumoniae is a major pathogen causing bacterial infection in the middle ear of humans. We previously used S. pneumoniae strain ST556, a low-passage 19F isolate from an otitis media patient, to perform a whole-genome screen for ear infection-associated genes in a chinchilla model. This report presents the complete genome sequence of ST556. The genome sequence will provide information complementary to the experimental data from our genetic study of this strain.
Project description:Ear infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.STM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.These findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.
Project description:Among 34 Streptococcus pneumoniae (Spn) sequential isolates from middle ear fluid, we found a case of a nontypeable S. pneumoniae (NT-Spn) in a child with acute otitis media (AOM). The strain was pneumolysin PCR positive and capsule gene PCR negative. Virulence of the NT-Spn was confirmed in a chinchilla model of AOM.
Project description:Non-vaccine Streptococcus pneumoniae serotypes are increasingly associated with disease. We evaluated isolates of the same sequence type (ST199) but different serotypes (15B/C, 19A) for growth in vitro, and pathogenic potential in a chinchilla otitis media model. We also developed a quantitative PCR (qPCR) assay to quantitatively assess each isolate, circumventing the need for selectable markers. In vitro studies showed faster growth of serotype 19A over 15B/C. Both were equally capable of colonization and middle ear infection in this model. Serotype 19A is included in new conjugate vaccine formulations while serotype 15B/C is not. Non-capsular vaccine targets will be important in disease prevention efforts.
Project description: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.
Project description:OTO-201 can provide sustained release to the middle ear and effectively treat otitis media, when compared with FDA-approved ciprofloxacin otic drop formulations.There is an unmet medical need for antibiotic therapy that can provide a full course of treatment from a single administration by an otolaryngologist at the time of tympanostomy tube placement, obviating the need for twice daily multiday treatment with short-acting otic drops.Studies in guinea pigs and chinchillas were conducted. OTO-201 was administered as a single intratympanic injection and compared with the twice daily multi-day treatment with Ciprodex or Cetraxal otic drops.OTO-201 demonstrated sustained release of ciprofloxacin in the middle ear compartment for days to approximately 2 weeks depending on the dose. The substantial C(max) values and steady drug exposure yielded by OTO-201 were in contrast to the pulsatile short lasting exposure seen with Ciprodex and Cetraxal. OTO-201 was also effective in a preclinical chinchilla model of Streptococcus pneumoniae-induced otitis media. The degree of cure was comparable to that afforded by Ciprodex and Cetraxal. There was no evidence of middle or inner ear pathology in guinea pigs treated with OTO-201, unlike Ciprodex and Cetraxal, which both demonstrated mild cochlear ototoxicity. No adverse effects of the poloxamer 407 vehicle were noted.Intratympanic injection of OTO-201 constitutes an attractive treatment option to twice daily multiday dosing with ciprofloxacin ear drops for the treatment of otitis media, as evidenced by superior middle ear drug exposure, efficacy in an acute otitis media model, safety of administration, and convenience of a single dose regimen.
Project description:Nontypeable Haemophilus influenzae (NTHi) is a leading causative agent of otitis media. Much of the inflammation occurring during NTHi disease is initiated by lipooligosaccharides (LOS) on the bacterial surface. Phosphorylcholine (PCho) is added to some LOS forms in a phase-variable manner, and these PCho(+) variants predominate in vivo. Thus, we asked whether this modification confers some advantage during infection. Virulence of an otitis media isolate (NTHi strain 86-028NP) was compared with that of an isogenic PCho transferase (licD) mutant using a chinchilla (Chinchilla lanigera) model of otitis media. Animals infected with NTHi 86-028NP licD demonstrated increased early inflammation and a delayed increase in bacterial counts compared to animals infected with NTHi 86-028NP. LOS purified from chinchilla-passed NTHi 86-028NP had increased PCho content compared to LOS purified from the inoculum. Both strains were recovered from middle ear fluids as long as 14 days postinfection. Biofilms were macroscopically visible in the middle ears of euthanized animals infected with NTHi 86-028NP 7 days and 14 days postchallenge. Conversely, less dense biofilms were observed in animals infected with NTHi 86-028NP licD 7 days postinfection, and none of the animals infected with NTHi 86-028NP licD had a visible biofilm by 14 days. Fluorescent antibody staining revealed PCho(+) variants within biofilms, similar to our prior results with tissue culture cells in vitro (S. L. West-Barnette, A. Rockel, and W. E. Swords, Infect. Immun. 74:1828-1836, 2006). Animals coinfected with equal proportions of both strains had equal persistence of each strain and somewhat greater severity of disease. We thus conclude that PCho promotes NTHi infection and persistence by reducing the host inflammatory response and by promoting formation of stable biofilm communities.
Project description:The arginine deiminase system (ADS) is associated with arginine catabolism and plays a role in virulence of several pathogenic bacteria. In Streptococcus pneumoniae, the ADS genes exist as a locus consisting of arcABCDT. A recent genome-wide mutagenesis approach revealed that both arcD and arcT are potentially essential in a chinchilla otitis media (OM) model. In the present study, we generated ΔarcD, ΔarcT, and ΔarcDT mutants by homologous recombination and evaluated their infectivity. Our results showed that only arcD, and not arcT, of an OM isolate is required during chinchilla middle ear infection. Additionally, D39 ΔarcD exhibited enhanced nasopharyngeal colonization and was attenuated in both mouse pneumonia and bacteremia models. In vitro, D39 ΔarcD displayed enhanced adherence to A549 epithelial cells and increased phagocytosis by J774A.1 macrophages compared to those with the parental strain. This mutant also exhibited an impaired capsule, as detected using electron microscopy, immunofluorescence, and a capsule assay. We demonstrated that the capsule defect in the D39 ΔarcD mutant may not be associated with a deficiency in arginine but rather is likely caused by a loss of interaction between the capsule and the transmembrane protein ArcD.
Project description:Otitis media, a common and often recurrent bacterial infection of childhood, is a major reason for physician visits and the prescription of antimicrobials. Haemophilus influenzae is the cause of approximately 20% of episodes of bacterial otitis media, but most strains lack the capsule, a factor known to play a critical role in the virulence of strains causing invasive H. influenzae disease. Here we show that in capsule-deficient (nontypeable) strains, sialic acid, a terminal residue of the core sugars of H. influenzae lipopolysaccharide (LPS), is a critical virulence factor in the pathogenesis of experimental otitis media in chinchillas. We used five epidemiologically distinct H. influenzae isolates, representative of the genetic diversity of strains causing otitis media, to inoculate the middle ear of chinchillas. All animals developed acute bacterial otitis media that persisted for up to 3 wk, whereas isogenic sialic acid-deficient mutants (disrupted sialyltransferase or CMP-acetylneuraminic acid synthetase genes) were profoundly attenuated. MS analysis indicated that WT bacteria used to inoculate animals lacked any sialylated LPS glycoforms. In contrast, LPS of ex vivo organisms recovered from chinchilla middle ear exudates was sialylated. We conclude that sialylated LPS glycoforms play a key role in pathogenicity of nontypeable H. influenzae and depend on scavenging the essential precursors from the host during the infection.
Project description:Cationic antimicrobial peptides (AMPs), a component of the innate immune system, play a major role in defense of mucosal surfaces against a wide spectrum of microorganisms such as viral and bacterial co-pathogens of the polymicrobial disease otitis media (OM). To further understand the role of AMPs in OM, we cloned a cDNA encoding a cathelicidin homolog (cCRAMP) from upper respiratory tract (URT) mucosae of the chinchilla, the predominant host used to model experimental OM. Recombinant cCRAMP exhibited alpha-helical secondary structure and killed the three main bacterial pathogens of OM. In situ hybridization showed cCRAMP mRNA production in epithelium of the chinchilla Eustachian tube and RT-PCR was used to amplify cCRAMP mRNA from several other tissues of the chinchilla URT. Quantitative RT-PCR analysis of chinchilla middle ear epithelial cells (CMEEs) incubated with either viral (influenza A virus, adenovirus, or RSV) or bacterial (nontypeable H. influenzae, M. catarrhalis, or S. pneumoniae) pathogens associated with OM demonstrated distinct microbe-specific patterns of altered expression. Collectively, these data showed that viruses and bacteria modulate AMP messages in the URT, which likely contributes to the disease course of OM.
Project description:Acute otitis media (AOM) is one of the most common infectious diseases primarily caused by Streptococcus pneumoniae (S.pn) among children. Progranulin (PGRN) is a multifunctional growth factor widely expressed in various tissues and cells. Studies have confirmed that PGRN is involved in the development of a variety of inflammatory diseases. We found that the expression of PGRN increased significantly in the middle ear of wild mice with AOM. However, its physiological functions in AOM still remain unknown. To examine the role of PGRN during AOM, we established an acute otitis media model in both C57BL/6 wild mice and PGRN-deficient (PGRN-/-) mice via transbullar injection with S.pn clinical strain serotype 19F. Interestingly, we observed dual results: on one hand, macrophage recruitment notably increased in PGRN-/- mice compared with WT mice; on the other hand, the overall bacterial clearance was surprisingly dampened in PGRN-/- mice. The enhanced recruitment of macrophages was associated with increased production of chemokine (C-C motif) ligand 2 (CCL2), while the decreased bacterial clearance was associated with impaired endocytosis capacity of macrophages. The scavenging ability of bacteria in PGRN-/- mice was recovered with administration of recombinant PGRN. These results suggested a novel dual role of PGRN in affecting the activities of macrophages.