Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media
Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media
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DOI：
10.1128/iai.00318-07
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发表时间：
2007-08-01
影响因子：
3.1
通讯作者：
Goldstein, Richard
中科院分区：
文献类型：
--
作者：
Leroy, Magali;Cabral, Howard;Goldstein, Richard
典型的回收量的非分型流感嗜血杆菌（NTHi）细菌在离体中耳（ME）吸出物从灰鼠模型的实验性中耳炎是不足以直接分析基因表达的微阵列或脂多糖糖型的质谱。这促使我们研究多个连续灌洗取样的策略以增加离体细菌回收。由于多个连续灌洗液样本显著增加了鼻咽定殖或ME感染期间收集的细菌CFU总数，这使我们评估从连续灌洗液中顺序获得的细菌是否相似。通过显微镜对完整的离体样品系列的比较观察最初显示ME炎性流体仅由反张相NTHi组成。相比之下，随后的灌洗采样相同的感染耳朵揭示了存在的细菌在两个额外的生长状态，丝状和生物膜包裹。这种离体样品的基因表达分析与连续灌洗样品中不同细菌生长阶段的基因表达分析雅阁。存在形态上不同的NTHi亚群与不同水平的基因表达表明，样本的汇集需要谨慎，直到他们的分离方法的开发。基于多次连续灌洗的这项研究与先前的报道一致，即NTHi在体内形成生物膜，描述了在不牺牲动物的情况下直接获取离体生物膜样品的方法，并且对于粘膜感染的研究具有广泛的适用性。此外，这种方法揭示了实验性中耳炎中细菌的实际负担显著大于先前报道的。这些发现可能对针对NTHi的抗生素治疗和疫苗开发具有直接影响。
The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.