A new protocol for multispecies bacterial infections in zebrafish and their monitoring through automated image analysis.

The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogen-host interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish.

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media.

BACKGROUND: Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation. METHODS: Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2. RESULTS: Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs. CONCLUSIONS: Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear.

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.

Pseudomonas aeruginosa infection in the hypoventilated middle ear: an experimental model.

CONCLUSION: This is a suitable model for studying different aspects of the pathophysiology of chronic suppurative otitis media. OBJECTIVE: To analyze the methodological features of an animal model of chronic suppurative otitis media induced by intratympanic inoculation of Pseudomonas aeruginosa. MATERIAL AND METHODS: Otitis media was induced by inoculation of P. aeruginosa through the inferior aspect of the rat bulla and cauterization of the Eustachian tube via a transpalatal approach. Inspection of the tympanic membrane, culturing of middle ear effusion and processing of the temporal bones for light microscopy were performed. RESULTS: Abnormal otomicroscopic findings and persistence of infection were correlated with the histopathological changes found in middle ear tissues.

Modified titration intratympanic gentamicin injection for unilateral intractable Ménière's disease.

This study looked into the efficacy of a modified titration protocol of intratympanic gentamicin injection (ITG) in the patients with unilateral intractable Ménière's disease (MD). Modified titration protocol of ITG at a low dose (20 mg/mL) was administered to 10 patients with definite unilateral intractable MD. After initial first two fixed ITGs on weekly basis, the patients might or might not be given any more injections, depending on the appearance of unilateral vestibular loss (UVL). ITG was terminated if the patients satisfied the criteria of UVL. All patients were followed-up for at least two years. The effects of ITG on the vertigo attack, functional level scores and postural balance were evaluated. Of the 10 cases, 8 showed the sign of UVL after receiving initial two ITGs and were not given any more intratympanic injections, and the other 2 patients were administered three ITGs. A two-year follow-up revealed that complete and substantial vertigo control was achieved in 9 cases, and limited vertigo control in 1 patient. Hearing level was lowered in 2 patients. The posture stability and functional level scores were improved. Our study showed that the modified titration protocol of ITG at a low dose could effectively control vertigo in patients with unilateral intractable MD.

Interactions between the middle ear and the inner ear: bacterial products.

The round-window membrane (RWM) is extremely thin and is the only soft-tissue barrier between the middle ear and the inner ear. Under inflammatory conditions of the middle ear the various layers of the triple-layered RWM undergo characteristic changes parallel to the changes of the middle-ear mucosa. Several studies report that bacterial products, exo- and endotoxins, from bacteria invading the middle ear may result in profound inflammatory changes in the inner ear, followed by severe damage to the inner-ear function. The present review, in which we summarized experimental and clinical observations, on bacterial products in interactions between the middle and inner ear, focused on: 1. Bacteria and bacterial products in an inflamed middle ear that may influence inner-ear function. 2. RWM structure and RWM permeability under the influence of bacteria and bacterial products. 3. Morphological and functional inner-ear effects of bacterial infection of the middle ear, and the possible mechanisms involved. 4. Future studies to be directed in this field.

A clinical pathologic study of hearing loss in congenital cytomegalovirus infection.

Cytomegalovirus (CMV) infection is currently reported as the most common cause of congenital viral induced deafness. However, few systematic studies of the audiovestibular sequelae of this infection are present in the literature. A clinical pathologic study was conducted from 1976 to 1982 to evaluate this. Fifty-two pairs of infant and children's temporal bone studied demonstrated no evidence of CMV endolabyrinthitis even in the single case with evidence of extensive congenital CMV infection. Over 2,000 umbilical cord sera were screened to detect asymptomatic CMV infection with an incidence of 0.38% (and slightly greater than 1% when extrapolated to correct for the sensitivity of the method of detection) in a central Pennsylvania study population. No sensorineural abnormalities were detected in five asymptomatic children and 30 control children. However, three out of six (50%) infants, symptomatic at birth and followed to a mean age of 5.5 years, showed significant and progressive sensorineural loss and vestibular deficits.

Cytomegalovirus endolabyrinthitis.

A premature male infant, who died 22 days after birth with hyaline membrane disease, was found to have had cytomegalic inclusion disease at autopsy. Histopathologic examination of the temporal bones showed cytomegalovirus (CMV) infection of the entire endolabyrinth without involvement of the neural and sensory structures. These findings support the thesis that late gestational or perinatal fetal CMV infection results in an endolymphatic labyrinthitis. We hypothesize that blood-borne virus passes from the stria vascularis into the endolymphatic spaces and infects the nonneurosensory epithelium. This pattern of infection differs from the perilabyrinthitis of human varicellazoster and experimentally produced mouse CMV.

Auditory brain stem response changes after application of endotoxin to the round window membrane in experimental otitis media.

The effects of endotoxin (purified Escherichia coli lipopolysaccharide 0111:B4) on cochlear function in normal and otitis media animals were evaluated. Two types of experimental otitis media models were developed in guinea pigs: eustachian tube obstruction and intratympanic injection of endotoxin. In normal animals, three different concentrations (0.01, 0.1, or 1.0 mg/ml) of endotoxin were applied onto the round window membrane, and auditory brain stem responses were recorded at 1, 3, 6, and 12 hours and 1, 2, 3, and 14 days after the application of endotoxin. Concentrations of 0.01 and 0.1 mg/ml of endotoxin did not affect the auditory brain stem response thresholds, whereas a concentration of 1.0 mg/ml resulted in elevation of the auditory brain stem response thresholds. Alteration of the auditory brain stem response threshold began at 3 hours, reached a peak at 24 or 48 hours, and returned to a normal level 2 weeks after the application of endotoxin. However, when the same concentration (1.0 mg/ml) of endotoxin was applied to the round window membranes of animals that underwent eustachian tube obstruction or intratympanic injection of endotoxin, the endotoxin did not cause any alteration of the auditory brain stem response threshold compared with normal animals.

Outbreak of otitis media caused by Burkholderia gladioli infection in immunocompromised mice.

An athymic nude mouse with severe head tilt due to otitis media was identified. Within weeks of identification of this first case, immune-deficient mice of various genotypes from the same facility were similarly affected, and cases from other facilities were found within two months. Culture of ear exudate specimens from affected mice yielded bacteria that were initially identified as Burkholderia cepacia, a plant pathogen considered an important opportunistic pathogen in persons with cystic fibrosis or chronic granulomatous disease. Several of these isolates, however, were subsequently identified as B. gladioli on the basis of results of biochemical analysis and a species-specific polymerase chain reaction (PCR) assay. Genotyping analysis revealed clonality among the isolates, indicating a shared strain among affected mice. A 16S rDNA-based PCR assay specific for the genera Burkholderia and Ralstonia, and a selective culture medium were used in efforts to characterize the epidemiology of this outbreak. In addition to culture of specimens from the oropharyngeal cavity of affected mice, samples were obtained from the environment, feces, sipper tubes, drinking water, and soiled bedding from cages of affected individuals. Burkholderia gladioli was most consistently detected in oropharyngeal swab specimens from affected mice. The PCR assay was equivalent to selective culture in identifying mice in the carrier state that did not have clinical signs of infection. However, neither detection method had sufficient sensitivity to reliably identify all carrier mice, causing the organism to persist at low levels unless entire colonies of immune-deficient mice were removed. The organism was highly resistant to antibiotic therapy. The source and epidemiology of this organism remain unknown. This epizootic serves as an important reminder that immunocompromised rodent colonies may harbor important human opportunistic pathogens.

Cytomegalovirus isolation from a human inner ear.

Cytomegalovirus (CMV) infection of the fetus has been associated with congenital deafness or hearing loss. This association has previously been based on clinical or pathological studies. We report an infant who died with the congenital CMV syndrome in which CMV was isolated from the perilymph of the inner ear providing additional evidence that this virus can infect the labyrinth.

Viral labyrinthitis -- an experimental study.

Summary--An attempt was made to produce viral labyrinthitis in the rhesus monkey. Rhesus monkeys are susceptible to the mumps virus. Nine animals were used. After removal of the stapes, the left oval windows were plugged with Gelfoam soaked in a culture of live mumps virus; right oval windows were plugged with Gelfoam soaked in killed cultures. Animals were sacrificed at different time intervals, postinfection. In eight of the nine animals there was seroconversion from negative to positive; however, none of the animals developed the histologic changes of viral labyrinthitis.

Spiral modiolar vein: its importance in viral load of the inner ear.

Guinea pig-specific cytomegalovirus and Sendai virus were inoculated into the cochleas of seronegative guinea pigs to study the route of entry of cells participating in inner ear inflammation. Inflammatory cells accumulated around the spiral modiolar vein and appeared to be streaming from this vein into the scala tympani via a collecting venule. Inactivated virus inoculated into the cochlea and normal control cochlea failed to show inflammatory cell infiltrates. The spiral modiolar vein appears to play an important role in the movement of cells from the systemic circulation into the inner ear as part of the host's normal defense against invading pathogens such as viruses.

Acute labyrinthitis associated with systemic Candida albicans infection in ageing mice.

The yeast Candida albicans is an important opportunistic pathogen that has been associated with disease of the inner ear. This study describes the histopathology of acute labyrinthitis caused by systemic infection with C. albicans in aging inbred mice. Within four days after infection, yeast and hyphal forms of C.albicans were found in the membranous labyrinth. The utricle and the adjacent parts of the ampullary regions of the semicircular canals were most severely affected, but damage was also seen in the scala media, the scala tympani, the saccule, and the scala vestibuli. In the utricle, the lining epithelium of the membranous labyrinth was disrupted, and the lining cells of the vestibular membrane showed foci in which the membrane was disrupted. The data suggest that age may represent a risk factor for fungal labyrinthitis.

Interrelations between the middle and inner ear in otitis media.

This article reviews the importance of the round-window membrane in exposing the labyrinth to or protecting it from the toxic effects of otitis media. Characteristics of the immune system in the human middle ear and middle-ear mechanisms against bacteria are explained. The role of bacteria and bacterial products in inner-ear damage is detailed, and related pathological events are described. The hypothetical role of inflammatory mediators in bacteria-induced inner-ear toxicity is particularly emphasized. Clinical conditions causing these events are detailed, and the most frequently involved microorganisms are mentioned. Finally, round-window membrane macroscopic and microscopic anatomy is discussed, and considerations about the exact role of membrane inflammation--protection versus damage of the inner ear--are expressed.

Bacterial invasion of the inner ear in association with pneumococcal meningitis.

OBJECTIVE: To examine the pathways of bacterial invasion and subsequent spreading in the inner ear during pneumococcal meningitis. STUDY DESIGN: A well-established adult rat model of Streptococcus pneumoniae meningitis was used. METHODS: Thirty rats were inoculated intrathecally with S. pneumoniae serotype 1, 3 or 9 V and received no additional treatment. The rats were sacrificed when reaching terminal illness or on Day 7 and then prepared for serial sectioning and PAS-Alcian blue staining for light microscopy. RESULTS: During the first few days after inoculation, bacteria invade the inner ear through the cochlear aqueduct, into the scala tympani of the cochlea (perilymphatic space). From here, bacteria spreads apically toward the helicotrema and subsequently basally through the scala vestibuli, toward the vestibule and the vestibular system. When the bacteria after 5 to 6 days had reached scala vestibuli of the basal turn of the cochlea, hematogenous spreading occurred to the spiral ligament and into the cochlear endolymph, subsequently to the vestibular endolymph. We found no evidence of alternative routes for bacterial invasion in the inner ear. Several internal barriers to bacterial spreading were found within the inner ear. Bacterial elimination was evidenced by engulfment by macrophages within the inner ear. CONCLUSION: From the meninges, pneumococci invade the inner ear through the cochlear aqueduct during the first days of infection, whereas hematogenous invasion via the spiral ligament capillary bed occur at later stages. Although internal barriers exist within the inner ear, the spreading of bacteria occurs via the natural pathways of the fluid compartments. Bacterial elimination occurs by local macrophage engulfment.

Microbial flora of cochlear implants by gene pyrosequencing.

OBJECTIVE: To describe the microbial flora associated with cochlear implants (CIs) removed for infectious and noninfectious indications. STUDY DESIGN: Prospective, controlled. SETTING: Academic, tertiary medical center. PATIENTS: All patients undergoing CI removal. INTERVENTION: CIs were removed with aseptic technique and processed for microbial identification. MAIN OUTCOME MEASURE: CI microbes were identified with routine culture and speciation and 16s deoxyribonucleic acid 454-pyrosequencing. RESULTS: All CIs had evidence of microbes. Propionibacterium acnes and Acidovorax facilis were more common on noninfected CIs (p = 0.005, 0.031). Staphylococcus aureus was more common on infected CIs (p = 0.003). The microbial profiles associated with CI infection were different from, but overlapped with those of noninfected CIs. Microbial culture with selective media identified pathogens not identified on pyrosequencing. CONCLUSION: Bacteria are present on all CIs, both with and without evidence of clinical infection, but species differ with clinical status. Empiric therapy for CI infections should include coverage for S. aureus. Gene pyrosequencing and selective culture techniques may yield complementary results that may impact the management of CI infections.

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears.

OBJECTIVE: The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear. RESULTS: Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10(2)CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group. CONCLUSIONS: LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.