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菌。近75.8%的菌株有青霉素结合蛋白1a、2b和2x(PBP)突变，通过对pbp1a、pbp2x和pbp2b基因异常的聚合酶链式反应(PCR)鉴定，可将其分为7类：(1)无异常pBP基因的青霉素敏感肺炎链球菌(PSSP)(24.2%)；(2)只有pbp2x基因异常的青霉素中间链球菌(GPisp)(26%)；(Iii)只有pbp1a基因异常[gPisp(/a)](0.1%)(Iv)只有pbp2b基因异常[gPisp(2b)](2.2%)(V)pbp1a和pbp2x基因异常的gPISP群(2.8%)；(Vi)pbp2x和pbp2b基因异常的gPISP群(2.2%)；以及(Vii)PBP基因3个异常的耐青霉素肺炎链球菌(GPRSP)(38.5%)。流感嗜血杆菌分为61.0%的敏感株(MIC值小于或等于1pg/ml)，37株(14.0%)为中等…耐药株较多(MIC=2μg/ml)，耐药株(MIC>4 pg/ml)66株(25.0%)。5株产TEM型β-内酰胺酶。分成3株(1.2%)有fts1基因突变的菌株(gBLPACR：耐药的β-内酰胺酶)和2株(0.8%)未突变的感染1基因的菌株(耐药的β-内酰胺酶)。根据聚合酶链式反应的基因分型，172株(65.1%)菌株有fts1基因突变而不产生β-内酰胺酶(gBLNAR：遗传的β-内酰胺酶不产生氨苄西林耐药)。其中变异区I/II群突变98株(37.1%)，高变区III群突变74株(28.0%)。其余87株(33.0%)为非产β内酰胺酶敏感菌株，未发现fts1基因和blA基因突变。第III组gBLNAR菌株对青霉素和头孢菌素均耐药。PBP基因突变不仅使流感嗜血杆菌对氨苄西林耐药，而且降低了对头孢菌素的敏感性。在治疗上呼吸道感染性疾病时，应考虑到流感嗜血杆菌gBLNAR株的高流行率，在2岁以下儿童中诱导有效的保护性免疫是非常重要的。在本研究中，我们评估了流感嗜血杆菌P6的母体免疫，以激发针对P6的特异性抗体并将其传递给后代。用P6经鼻腔免疫母鼠，观察其对母鼠血清和母乳中特异性抗体的诱导作用。根据母亲的哺乳情况，对免疫母亲所产后代的特异性抗体进行调查，以评价免疫母亲母乳喂养的重要性。我们的发现有力地表明，P6母体鼻腔免疫将是预防儿童早期NTHi感染的一种有吸引力的策略。它可以在怀孕期间通过胎盘和出生后通过母乳提供保护性抗体。较少

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