Muneki Hotomi

Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci

ABSTRACT We demonstrated that during colonization with Streptococcus pneumoniae the nasal mucosal tissues of mice support two populations of pneumococci. Transparent-phase pneumococci can be readily washed from the outer surface, while a second population composed of primarily opaque-phase pneumococci is released only by homogenization of the nasal tissue. The fact that the opaque phase has previously been associated with invasion and the fact that opaque-phase pneumococci were released by homogenization of previously washed nasal tissue suggest that the opaque-phase pneumococci may have invaded the nasal tissue. Consistent with this hypothesis was our observation that there was inflammation in portions of the nasal mucosa of the colonized mice but not in the mucosa of noncolonized mice, but this observation did not prove the hypothesis. If the opaque-phase pneumococci released from the nasal tissue were from within the tissue and/or if resistance of the opaque-phase subpopulation to antibody, complement, and phagocytes is essential for long-term carriage, it seems likely that the virulence factors of S. pneumoniae that are necessary for killing humans exist to facilitate carriage. Although this speculation is unproven, the observation that there are separate populations of pneumococci during colonization may help guide future attempts to understand the biology of nasal colonization by this pathogen.

Clinical bacteriology and immunology in acute otitis media in children

Acute otitis media (AOM) is the most common disease seen in childhood. Streptococcus pneumoniae, non-typeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis are the most frequent pathogens of all AOM episodes. The high prevalence of drug-resistant pathogens such as penicillin-resistant S. pneumoniae (PRSP) and betalactamase producing or nonproducing ampicillin-resistant H. influenzae (BLPAR or BLNAR) is causing serious clinical problems worldwide. PRSP and BLNAR have become important risk factors for intractable clinical outcome of AOM. PRSP causes a three times higher incidence of intractable AOM than susceptible strains. BLNAR strains show penicillin-binding protein gene mutation and are not only resistant to ampicillin, but also have reduced susceptibility to cephalosporin. The resistant H. influenzae pathogen has shown clonal dissemination in Japan in ways different from those of penicillin-resistant S. pneumoniae. Protection against AOM due to these pathogens may depend on pathogen-specific antibodies. Pneumococcal capsular polysaccharides (PCPs) are type specific and poorly immunogenic in children younger than 2 years old. Approximately 50% of otitis-prone children showed subnormal levels of anti-PCP IgG2 antibody. In our immunological study in children with otitis media, however, otitis-prone children were not unusually vulnerable to infections except those resulting in otitis media. This fact seems to refute the presence of a broad immunological deficit in these children. Some pathogen-specific antibodies may be directed against protein immunogens such as pneumococcal surface protein A (PspA) of S. pneumoniae, P6 of NTHi, and UspA of M. catarrhalis. The levels of antibody to P6 of NTHi in healthy children were significantly higher than those in the otitis-prone children after the age of 18 months. In general, individual antibody levels in otitis-prone individuals did not have an age-dependent rise. The failure to develop a good antibody response to common antigens such as PspA and P6 may enable the pathogen to cause persistent or recurrent disease.

Genetic Characteristics and Clonal Dissemination of β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae Strains Isolated from the Upper Respiratory Tract of Patients in Japan

ABSTRACT We evaluated the recent prevalence of antimicrobial-resistant Haemophilus influenzae isolated from the upper respiratory tracts (URT) of patients in Japan. Mutations in the ftsI gene, which encodes penicillin binding protein 3 (PBP3), and the clonal dissemination of the resistant strains were also investigated. A total of 264 H. influenzae isolates were collected from patients with URT infections. According to the criteria of the Clinical and Laboratory Standards Institute for the susceptibility of H. influenzae to ampicillin (AMP), the isolates were distributed as follows: 161 (61.0%) susceptible strains (MIC ≤ 1 μg/ml), 37 (14.0%) intermediately resistant strains (MIC = 2 μg/ml), and 66 (25.0%) resistant strains (MIC ≥ 4 μg/ml). According to PCR-based genotyping, 172 (65.1%) of the isolates had mutations in the ftsI gene and were negative for the β-lactamase (bla) gene. These 172 isolates were thus defined as genetically β-lactamase-negative ampicillin-resistant (gBLNAR) strains. The ftsI mutant group included 98 (37.1%) strains with group I/II mutations in the variable mutated region (group I/II gBLNAR) and 74 (28.0%) strains with group III mutations in the highly mutated region (group III gBLNAR). Eighty-seven (33.0%) of the isolates were genetically β-lactamase-negative ampicillin-susceptible (gBLNAS) strains. The group III gBLNAR strains showed resistance to β-lactams. Only five strains (1.9%) were positive for a bla gene encoding TEM-type β-lactamase. The three clusters consisting of 16 strains found among the 61 BLNAR strains (MIC ≥ 4 μg/ml and without the bla gene) showed identical or closely related DNA restriction fragment patterns. Those isolates were frequently identified among strains with a MIC to AMP of 16 μg/ml. The current study demonstrates the apparent dissemination and spread of a resistant clone of H. influenzae among medical centers in Japan. The gBLNAR strains show a remarkable prevalence among H. influenzae isolates, with the prevalence increasing with time. This fact should be taken into account when treating URT infections.

Specific mucosal immunity and enhanced nasopharyngeal clearance of nontypeable Haemophilus influenzae after intranasal immunization with outer membrane protein P6 and cholera toxin

Nontypeable Haemophilus influenzae (NTHi) is one of the leading pathogens in otitis media. Studies of vaccines against NTHi have focused on outer membrane proteins (OMPs). One outer membrane protein P6 is highly conserved among strains and is an attractive candidate for a subunit bacterial vaccine. In this study, mucosal immunity induced by intranasal immunization with P6 and cholera toxin (CT) was investigated in a mouse model. Intranasal immunization with P6 and CT evoked a good mucosal IgA as well as a systemic IgG response against P6. On the other hand, intranasal immunization with P6 alone induced a weak mucosal IgA response. Enzyme linked immunospot assay detected anti-P6 specific antibody producing cells in the nasopharyngeal mucosa of immunized mice. The protective response of intranasal immunization was demonstrated by enhancement of nasopharyngeal clearance of NTHi and inhibition of adherence of NTHi to cultured human epithelial cells. Based on these results, intranasal immunization with P6 and CT may be an effective approach to protect human from H. influenzae infections in the upper respiratory tract.

Detection of Haemophilus influenzae in middle ear of otitis media with effusion by polymerase chain reaction

Otitis media with effusion (OME) is one of the major causes of hearing loss in childhood. The pathogenesis still remains unclear, though it is closely related to acute otitis media with bacterial infections. It is known that Haemophilus influenzae is one of the most common bacteria isolated from middle ear effusions (MEEs). Recently, in vitro DNA amplification by polymerase chain reaction (PCR) is a new technology that has considerable implication for diagnosis of viral and bacterial infections because of its potentially precise specificity and sensitivity. In the present experiment polymerase chain reaction (PCR) was applied to the detection of DNA genome of H. influenzae contained in middle ear effusions. By Southern blot hybridization, two characteristic bands for H. influenzae DNA were detected at 273 b.p. and 550 b.p. position in 15 of 27 MEEs. However, no organism was cultured by conventional methods. Our results indicate the PCR technique is more specific and sensitive in detection of bacteria in middle ear effusion of OME, compared with conventional methods. It strongly suggests more involvement of the bacteria, especially H. influenzae, in OME onset.

Treatment and outcome of severe and non-severe acute otitis media

To determine outcomes in acute otitis media (AOM) according to severity of disease and to assess different initial treatment regimens, 308 with AOM were enrolled and divided into severe ( n =277; 89.9%) and non-severe ( n =31; 10.1%) groups based on symptoms and tympanic membrane changes. Children in the severe group were initially managed with amoxicillin (AMPC) whereas children in the non-severe group were initially managed without antibiotics. Children were monitored on days 1, 5, 10, 14 and 28. Five outcome measures were assessed: disappearance of symptoms at day 5, resolution of tympanic membrane changes by day 28, disappearance of middle ear effusions by day 28, recurrence of acute symptoms prior to day 28, and need to change treatment regimens. Children with severe disease were more often male (57% versus 36%, P <0.05) and more often colonized with pathogens (77% versus 55%, P <0.05 than children with non-severe disease. The two groups were similar with respect to age and day care attendance. Despite differences in initial treatment regimens between the two groups, symptoms improved at the same rate for severe and non-severe disease, 94% by day 5. In contrast, tympanic membranes returned to normal in 69% of the severe and 81% of the non-severe groups by day 28; however, as early as day 5, 10% of the severe and 55% of the non-severe groups demonstrated normal tympanic membranes. Middle ear effusions similarly disappeared more slowly in the severe group, 52% versus 74% by day 14 and 76% versus 84% by day 28. Recurrence rates of acute symptoms occurred with equal frequency in the severe, 15%, and non-severe groups, 10%. Failure of the symptoms or the tympanic membranes to improve led to antibiotic changes in 59.9% of the severe group and to the addition of antibiotics in 51.6% of the non-severe group. Children in the severe group who failed to improve with an initial course of amoxicillin were younger (40.2 months versus 45.8 months, P <0.05), had higher tympanic membrane scores (4.5 versus 4.1, P <0.05), and were more often colonized with penicillin-resistant Streptococcus pneumoniae (33.8% versus 6.5%, P <0.01) than children who responded to AMPC. In a similar manner, children with non-severe disease who failed to improve without antibiotics were younger (40.7 months versus 54.8 months, P <0.05) and more often colonized with pathogens (75.0% versus 33.4%, P <0.05). Conclusion:Severe disease occurred more often among males and among children colonized with pathogens. Response to treatment was impaired in younger children and in children colonized with pathogens, especially penicillin-resistant Streptococcus pneumoniae.

Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media

OBJECTIVES The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease. METHODS Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI). RESULTS 84.3% clinical isolates of NTHi were biofilm forming strains (BFI> or =0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC. CONCLUSION We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.

Antibody Responses to the Outer Membrane Protein P6 of Non-typeable Haemophilus influenzae and Pneumococcal Capsular Polysaccharides in Otitis-prone Children

Acute otitis media (AOM) is a common infectious disease in children. Some children experience recurrent episodes of AOM. Recent investigations demonstrate antigen-specific immunological deficiencies in children prone to AOM. In the present study, the immune responses to non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (S. pneumoniae) were further investigated in otitis-prone children and normal children. Forty-eight percent of otitis-prone children exhibited reduced IgG2 levels to S. pneumoniae and 55% exhibited reduced IgG levels to NTHi. These data suggest that otitis proneness appears to be related to numerous immunological derangements. Pathogen-specific antibodies are a reliable measure of otitis proneness.

Brain Regions Responsible for Tinnitus Distress and Loudness: A Resting-State fMRI Study

Subjective tinnitus is characterized by the perception of phantom sound without an external auditory stimulus. We hypothesized that abnormal functionally connected regions in the central nervous system might underlie the pathophysiology of chronic subjective tinnitus. Statistical significance of functional connectivity (FC) strength is affected by the regional autocorrelation coefficient (AC). In this study, we used resting-state functional MRI (fMRI) and measured regional mean FC strength (mean cross-correlation coefficient between a region and all other regions without taking into account the effect of AC (rGC) and with taking into account the effect of AC (rGCa) to elucidate brain regions related to tinnitus symptoms such as distress, depression and loudness. Consistent with previous studies, tinnitus loudness was not related to tinnitus-related distress and depressive state. Although both rGC and rGCa revealed similar brain regions where the values showed a statistically significant relationship with tinnitus-related symptoms, the regions for rGCa were more localized and more clearly delineated the regions related specifically to each symptom. The rGCa values in the bilateral rectus gyri were positively correlated and those in the bilateral anterior and middle cingulate gyri were negatively correlated with distress and depressive state. The rGCa values in the bilateral thalamus, the bilateral hippocampus, and the left caudate were positively correlated and those in the left medial superior frontal gyrus and the left posterior cingulate gyrus were negatively correlated with tinnitus loudness. These results suggest that distinct brain regions are responsible for tinnitus symptoms. The regions for distress and depressive state are known to be related to depression, while the regions for tinnitus loudness are known to be related to the default mode network and integration of multi-sensory information.

Factors associated with clinical outcomes in acute otitis media.

Acute otitis media (AOM) is a common disease in childhood. If predictors of outcome in AOM were known, it would be possible to individualize therapy. Our aim was to identify factors that predict the outcome in AOM. We enrolled 368 children with AOM (ages, 10 to 86 months). The severity of symptoms and the severity of tympanic membrane changes were graded with a scoring system. Nasopharyngeal colonization with middle ear pathogens was determined on day 1. Three outcomes were assessed: persistence of symptoms at day 5, persistence of tympanic membrane changes at day 28, and recurrence of acute symptoms prior to day 28. Persistence of symptoms at day 5 was associated with younger age (35 versus 44 months; p < .001), higher symptom score on day 1 (3.5 versus 2.9; p < .05), and colonization with Streptococcus pneumoniae (61% versus 41%; p < .05). Persistence of tympanic membrane changes at day 28 was associated with younger age (39 versus 45 months; p < .01), higher tympanic membrane score on day 1 (4.1 versus 3.6; p < .01), and nasopharyngeal colonization with S pneumoniae, especially drug-resistant S pneumoniae (33% versus 13%; p < .05). Recurrence of acute symptoms prior to day 28 occurred in 14% of the children. Streptococcus pneumoniae was the only pathogen associated with an increased recurrence rate (23%) as compared to the group without pathogens (7%; p < .05). Age, severity of disease at presentation, and nasopharyngeal colonization patterns were proven to be important determinants of outcome in AOM.