Vishakha Sabharwal

Hepatocyte-specific mutation of both NF-κB RelA and STAT3 abrogates the acute phase response in mice

The acute phase response is an evolutionarily conserved reaction in which physiological stress triggers the liver to remodel the blood proteome. Although thought to be involved in immune defense, the net biological effect of the acute phase response remains unknown. As the acute phase response is stimulated by diverse cytokines that activate either NF-κB or STAT3, we hypothesized that it could be eliminated by hepatocyte-specific interruption of both transcription factors. Here, we report that the elimination in mice of both NF-κB p65 (RelA) and STAT3, but neither alone, abrogated all acute phase responses measured. The failure to respond was consistent across multiple different infectious, inflammatory, and noxious stimuli, including pneumococcal pneumonia. When the effects of infection were analyzed in detail, pneumococcal pneumonia was found to alter the expression of over a thousand transcripts in the liver. This outcome was inhibited by the combined loss of RelA and STAT3. Moreover, this interruption of the acute phase response increased mortality and exacerbated bacterial dissemination during pneumonia, possibly as a result of acute humoral enhancement of macrophage opsonophagocytosis, which was impaired in the mutant mice. Thus, we conclude that RelA and STAT3 are essential for stress-induced transcriptional remodeling in the liver and the subsequent activation of the acute phase response, whose functional role includes compartmentalization of local infection.

Role of Complement in Host Defense against Pneumococcal Otitis Media

ABSTRACT Strategies to limit complement deposition on Streptococcus pneumoniae are established as virulence features for invasive disease, but their role in respiratory tract infection requires further analysis. We evaluated complement C3 protein deposition on discordant S. pneumoniae isolates of the same serotype (6A) and their capacity to cause nasopharyngeal (NP) colonization and experimental otitis media (EOM) in an animal model. We compared C3 binding to five 6A isolates from asymptomatic NP carriers with five 6A strains that caused invasive disease, and we observed less C3 (∼10-fold less fluorescence) binding to invasive isolates. We selected two high-level C3-binding carriage and two low-level C3-binding invasive 6A isolates for further study. In the EOM model, 11/12 (92%) ears challenged with a low-level C3-binding 6A strain became infected. Only 2/8 (25%) ears challenged with the discordant high-level C3-binding 6A isolate developed disease (P = 0.005). Results with the second discordant 6A isolate pair were comparable. Cobra venom factor (CoVF) treatment, which depletes C3 and consumes complement, restored virulence of the high-level C3-binding strain; 8/8 (100%) ears in CoVF-treated animals developed EOM compared to only 25% of ears in naïve animals (P = 0.007). These studies demonstrate the critical role for complement evasion in pneumococcal EOM. Colonization with carriage isolates that bound high levels of C3 caused EOM in fewer animals compared to low-level C3-binding invasive strains. Thus, limiting C3 deposition on the surface of S. pneumoniae correlates with increased incidence of EOM following NP colonization and barotrauma in the animal model.

Nontypeable Streptococcus pneumoniae as an Otopathogen

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.

Formulations for trans-tympanic antibiotic delivery.

We have developed a drug delivery system for prolonged trans-tympanic antibiotic delivery from a single dose administration. Increased permeability to ciprofloxacin of the intact tympanic membrane (TM) was achieved by chemical permeation enhancers (CPEs--bupivacaine, limonene, sodium dodecyl sulfate); this was also seen by CPEs contained within a hydrogel (poloxamer 407) to maintain the formulation at the TM. The CPE-hydrogel formulation had minimal effects on auditory thresholds and tissue response in vivo. CPE-hydrogel formulations have potential for ototopical delivery of ciprofloxacin for the treatment of acute otitis media (AOM) and other middle ear diseases.

How we prevent and manage infection in sickle cell disease

Sickle cell disease (SCD) affects approximately 100 000 people in the US, 12 500 in the UK, and millions worldwide. SCD is typified by painful vaso‐occlusive episodes, haemolytic anaemia and organ damage. A secondary complication is infection, which can be bacterial, fungal or viral. Universal newborn screening, routine use of penicillin prophylaxis, availability of conjugated vaccines against S. pneumoniae and comprehensive care programmes instituted during the past few decades in industrialized countries have dramatically reduced childhood mortality and improved life expectancy. Yet patients with SCD remain at increased risk of infection. Unfortunately, the treatment of most bacterial infections that are common in SCD is not based on the results of randomized controlled clinical trials. In their absence, treatment decisions are based on consensus guidelines, clinical experience or adapting treatment applied in other diseases. This leads to wide variation in treatment among institutions and even between treating physicians in a single institution. Prevention of infection, when possible, is most important and we focus on prevention through targeted prophylaxis and vaccination. We will share our management strategies for managing the more common infections in SCD and provide the rationale for our recommendations.

Variation of Pneumococcal Pilus-1 Expression Results in Vaccine Escape during Experimental Otitis Media [EOM]

The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease. Methods Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry. Results Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid. Conclusion Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.

Streptococcus pneumoniae Serotype 6C: an Intra- and Interclonal Complex Comparison

We report the annotated draft genome sequences of four serotype 6C Streptococcus pneumoniae isolates of differing genetic backgrounds. Serotype 6C isolates are increasing in prevalence and becoming progressively more resistant to antibiotics. As a result, these strains are likely to become more important in the near future.

Perinatal Transmission of Hepatitis C Virus: Defining the Cascade of Care

Objectives The US National Viral Hepatitis Action Plan calls for major efforts to expand hepatitis C virus (HCV) diagnosis and treatment; prenatal care settings are potential venues for expanding HCV testing. We aimed to characterize the HCV diagnostic cascade for women and infants and investigate factors associated with linkage and follow‐up. Study design We used electronic health records for a 10‐year cohort of 879 women with opioid use disorder from an obstetric clinic serving women with substance use disorders. Results Altogether, 744 women (85%) were screened for HCV; 510 (68%) were seropositive, of whom 369 (72%) had nucleic acid testing performed and of these 261 (71%) were viremic. Of 404 infants born to HCV‐seropositive women, 273 (68%) were tested at least once for HCV, 180 (45%) completed the American Academy of Pediatrics‐recommended perinatal HCV screening, and 5 (2.8%) were diagnosed with HCV infection and linked to care. More recent delivery date (2014‐2015) was associated with maternal linkage to care (aOR, 2.5; 95% CI, 1.4‐4.7). Maternal coinfection with HIV (aOR, 9.0; 95% CI, 1.1‐72.8) and methadone maintenance therapy, compared with buprenorphine (aOR, 1.5; 95% CI, 0.9‐2.5), were associated with higher rates of infant HCV testing. Conclusions HCV prevalence among pregnant women with opioid use is high and infant HCV screening is imperfect. Programmatic changes to improve both mother and infant follow‐up may help to bridge identified gaps in the cascade to cure.

Treatment of Streptococcus pneumoniae otitis media in a chinchilla model by transtympanic delivery of antibiotics

Otits media (OM) is the most frequent indication for antimicrobial prescription to US children. Streptococcus pneumoniae (S. pneumoniae) remains one of the most common pathogens causing OM. Successful eradication of S. pneumoniae in the middle ear can be achieved by adhering to a 7-10 day regimen of oral antibiotics. However, oral drug administration is challenging for parents. Lack of adherence has been associated with treatment failure or early relapse. To overcome this challenge, we used a noninvasive formulation to achieve high transtympanic antibiotic flux and cured S. pneumoniae OM in chinchillas. The formulation consists of a thermosensitive in situ gelling hydrogel, chemical permeation enhancers, and an antibiotic. The direct transport of drugs into the middle ear produced high concentrations of ciprofloxacin (in the range of hundreds of micrograms per milliliter) within the first 24 hours of administration. Drug concentrations above the minimum inhibitory concentration (MIC) for S. pneumoniae were sustained throughout the 7-day treatment. S. pneumoniae OM in a chinchilla model was successfully eradicated, without causing tissue toxicity. Transtympanic delivery minimized systemic drug exposure, as evidenced by undetectable levels in blood, measured by high-performance liquid chromatography.

76Lack of Middle Ear Virulence of Streptococcus pneumoniae 33F Isolates in Chinchillas is Associated with Absence of the PsrP Pathogenicity Island

Background. The identification of site specific genes and functions essential for virulence may provide further understanding of the pathogenesis of pneumococcal disease and potentially reveal novel targets for treatment and prevention. Methods. We screened more than 10 selected serotypes of Streptococus pneumoniae in our chinchilla model of experimental otitis media (EOM), initially using nasopharyngeal inoculation followed by barotrauma and subsequently with direct intrabullar challenge. Genomic DNA of these isolates was extracted by standard methods and sequenced using the HiSeq Illumina platform. De novo assembled sequences were annotated with RAST (Rapid Annotation by Subsystem Technology). Functional comparative genomic analysis was performed in SEED viewer; genes of subsystems identified as uniquely absent in 33F strains underwent detailed analysis in SEED, BRIG and BLAST. Results. The serotype 33F isolates colonized the nasopharynx comparable to all other serotypes but failed to produce either clinically apparent or culture-positive middle ear disease. The lack of virulence was confirmed by the failure to develop middle ear disease following direct intrabullar challenge as well as with 2 additional 33F isolates. Functional genomic comparison against OM producing strains as well as ∼50 invasive strains revealed that all three 33F strains lack the pneumococcal serine-rich repeat protein (PsrP) pathogenicity island. The PsrP island appeared to be present in the sequenced IPD strains. Conclusion. Serotype 33F failed to produce EOM using either NP colonization and barotrauma or direct inoculation. Comparative genomic analysis revealed the absence of the PsrP pathogenicity island in these strains. PsrP is a representative of serine-rich repeat proteins found in many pathogenic streptococci and Staphylococcus aureus. Although non-essential for survival, it plays an important role in the formation of biofilms and adhesion to host cells. Further characterization of the function of this island in S. pneumoniae is necessary to define its role in virulence both for middle ear infection as well as invasive disease and pneumonia. Disclosures. All authors: No reported disclosures.