Immunological and molecular-biological studies on pathogenesis and treatments of intractable obits media

肺炎链球菌和流感嗜血杆菌是儿童急性中耳炎的主要病原体。75.8%的菌株青霉素结合蛋白la、2b和2x （PBP）发生突变，通过PCR检测异常pbp1a、pbp2x和pbp2b基因，将其分为7个基因型类：(1)无pbp基因异常的青霉素敏感肺炎链球菌（PSSP）分离株（24.2%），(2)仅含异常pbp2x基因[gPISP (2x)]的基因型肺炎链球菌（gPISP）分离株（26%），(3)仅含异常pbp1a基因[gPISP (/a)](0.1%),(4)仅含异常pbp2b基因[gPISP (2b)](2.2%),(5)含异常pbp1a和pbp2x基因的gPISP分离株（2.8%），(6)含异常pbp2x和pbp2b基因的gPISP分离株（2.2%），（vii） pbp基因3个异常的基因型耐青霉素肺炎链球菌（gPRSP）分离株（38.5%）。其中敏感株（MIC≤1 pg/ml）占61.0%，中等耐药株（MIC =2 μg/ml）占37株（14.0%），耐药株（MIC≥4 pg/ml）占66株（25.0%）。5株菌株产生TEM型β-内酰胺酶。将其分为3株（1.2%）fts1基因突变株（gBLPACR：产生阿莫西林-克拉维酸的基因突变株）和2株（0.8%）fts1基因突变株（gBLPAR：产生氨苄西林的基因突变株）。根据pcr基因分型，172株（65.1%）分离株存在不产生β-内酰胺酶的fts1基因突变（gBLNAR：基因上不产生氨苄西林的β-内酰胺酶）。在可变突变区（1/II组）出现I/II组突变98株（37.1%），在高突变区（III组）出现III组突变74株（28.0%）。其余87株（33.0%）为gBLNAS（遗传β-内酰胺酶不产氨苄西林敏感）菌株，fts1基因和bla基因均未突变。III组gBLNAR菌株对青霉素和头孢菌素均有耐药性。PBP基因突变后的流感嗜血杆菌不仅对氨苄西林耐药，而且对头孢菌素的敏感性降低。在治疗上呼吸道传染病时，应考虑到流感嗜血杆菌gBLNAR菌株的高流行率。在2岁以下儿童中诱导有效的保护性免疫非常重要。在本研究中，我们评估了母体免疫流感嗜血杆菌P6以产生针对P6的特异性抗体并将其转移给后代。我们用P6经鼻免疫母鼠，研究了P6在血清和乳汁中的诱导作用。根据母乳喂养情况，对免疫母亲所产子代进行特异性抗体检测，评价免疫母亲母乳喂养的重要性。我们的研究结果强烈表明，母亲鼻内接种P6将是一种有吸引力的策略，可以在儿童早期预防NTHi感染。它可以在怀孕期间通过胎盘移植和出生后通过母乳提供保护性抗体。少

S.pneumoniae and H.influenzae have been major pathogens in acute otitis media of children. Almost 75.8% strains had mutation in their penicillin binding protein la, 2b and 2x (PBP) and they were classified into seven genotypic classes after PCR identification of abnormal pbp1a, pbp2x, and pbp2b genes: (i) penicillin-susceptible S. pneumoniae (PSSP) isolates with no abnormal pbp genes (24.2%), (ii) genotypic penicillin-intermediate S. pneumoniae (gPISP) isolates with only an abnormal pbp2x gene [gPISP (2x)] (26%), (iii) with only an abnormal pbp1a gene [gPISP (/a)](0.1%) (iv) with only an abnormal pbp2b gene [gPISP (2b)](2.2%) (V) gPISP isolates with abnormal pbp1a and pbp2x genes (2.8%), (vi) gPISP isolates with abnormal pbp2x and pbp2b genes (2.2%), and (vii) genotypic penicillin-resistant S. pneumoniae (gPRSP) isolates with three abnormal pbp genes (38.5%). H. influenzae isolates were divided into 61.0% susceptible strains (MIC less than or equal 1 pg/ml), 37 (14.0%) intermediately r … More esistant strains (MIC =2 μg/ml) and 66 (25.0%) resistant strains (MIC greater than or equal 4 pg/ml). Five strains produced TEM type β-lactamase. They were divided into 3 (1.2%) strains with mutations in fts1 gene (gBLPACR: genetically β-lactamase producing amoxicillin-clavulanate resistant) and 2 (0.8%) strains without mutations infts1 gene gBLPAR (genetically β-lactamase producing ampicillin resistant). According to PCR-based genotyping, 172 (65.1%) isolates had mutations in fts1 gene without producing β-lactamase (gBLNAR: genetically β-lactamase nonproducing ampicillin resistant). They were 98 (37.1%) strains with group I/II mutations in variable mutated region (Group 1/II gBLNAR) and 74 (28.0%) strains with group III mutations in highly mutated region (Group III gBLNAR). The rest of 87 (33.0%) isolate were gBLNAS (genetically β-lactamase non-producing ampicillin susceptible) strains with neither mutations in fts1 gene nor bla gene. The Group III gBLNAR strains showed resistance to both penicillin and cephalosporin. PBP gene mutated H. influenzae not only resistance to ampicillin but also had reduce susceptibility to cephalosporin. The high prevalence of gBLNAR strains of H. influenzae should be taken into account when treating the upper respiratory tract infectious diseases.It is very important to induce effective protective immunity among children younger than 2 years of age. In this study, we evaluated the maternal immunization of P6 of H.influenzae to evoke specific antibody to P6 and to transfer it to offspring. We intranasally immunized mother mice with P6 and investigated the induction of specific antibody in sera and breast milk. The specific antibody among offspring delivered from immunized mother was also investigated according to the nursing status to evaluate the importance of breast feedings by immunized mothers. Our findings strongly suggest that maternal intranasal immunization with P6 would be an attractive strategy against NTHi infections during early childhood. It can supply protective antibodies via transplacental during pregnancy and via breast milk after birth. Less