Department of Earth and Planetary Sciences, University of California Berkeley, Berkeley, CA 94720, USA.
Microbiome. 2014 Jan 28;2(1):1. doi: 10.1186/2049-2618-2-1.
The source inoculum of gastrointestinal tract (GIT) microbes is largely influenced by delivery mode in full-term infants, but these influences may be decoupled in very low birth weight (VLBW, <1,500 g) neonates via conventional broad-spectrum antibiotic treatment. We hypothesize the built environment (BE), specifically room surfaces frequently touched by humans, is a predominant source of colonizing microbes in the gut of premature VLBW infants. Here, we present the first matched fecal-BE time series analysis of two preterm VLBW neonates housed in a neonatal intensive care unit (NICU) over the first month of life.
Fresh fecal samples were collected every 3 days and metagenomes sequenced on an Illumina HiSeq2000 device. For each fecal sample, approximately 33 swabs were collected from each NICU room from 6 specified areas: sink, feeding and intubation tubing, hands of healthcare providers and parents, general surfaces, and nurse station electronics (keyboard, mouse, and cell phone). Swabs were processed using a recently developed 'expectation maximization iterative reconstruction of genes from the environment' (EMIRGE) amplicon pipeline in which full-length 16S rRNA amplicons were sheared and sequenced using an Illumina platform, and short reads reassembled into full-length genes. Over 24,000 full-length 16S rRNA sequences were produced, generating an average of approximately 12,000 operational taxonomic units (OTUs) (clustered at 97% nucleotide identity) per room-infant pair. Dominant gut taxa, including Staphylococcus epidermidis, Klebsiella pneumoniae, Bacteroides fragilis, and Escherichia coli, were widely distributed throughout the room environment with many gut colonizers detected in more than half of samples. Reconstructed genomes from infant gut colonizers revealed a suite of genes that confer resistance to antibiotics (for example, tetracycline, fluoroquinolone, and aminoglycoside) and sterilizing agents, which likely offer a competitive advantage in the NICU environment.
We have developed a high-throughput culture-independent approach that integrates room surveys based on full-length 16S rRNA gene sequences with metagenomic analysis of fecal samples collected from infants in the room. The approach enabled identification of discrete ICU reservoirs of microbes that also colonized the infant gut and provided evidence for the presence of certain organisms in the room prior to their detection in the gut.
在足月婴儿中，胃肠道（GIT）微生物的源接种物在很大程度上受分娩方式的影响，但通过常规广谱抗生素治疗，这些影响可能在极低出生体重（VLBW，<1500g）新生儿中被分离。我们假设，建筑环境（BE），特别是人类经常接触的房间表面，是早产儿肠道中定植微生物的主要来源。在这里，我们展示了首例在生命的第一个月内对住在新生儿重症监护病房（NICU）中的两名早产 VLBW 新生儿的粪便 - 环境时间序列进行的匹配分析。
每 3 天收集一次新鲜粪便样本，并在 Illumina HiSeq2000 设备上对宏基因组进行测序。对于每个粪便样本，从 NICU 房间的 6 个指定区域采集大约 33 个拭子：水槽、喂养和插管管、医护人员和父母的手、一般表面以及护士站电子设备（键盘、鼠标和手机）。使用最近开发的“从环境中最大化期望重建基因”（EMIRGE）扩增子管道处理拭子，其中全长 16S rRNA 扩增子被剪切并使用 Illumina 平台进行测序，并将短读取重新组装成全长基因。产生了超过 24000 个全长 16S rRNA 序列，每个房间 - 婴儿对平均产生大约 12000 个操作分类单位（OTUs）（在 97%核苷酸同一性下聚类）。包括表皮葡萄球菌、肺炎克雷伯菌、脆弱拟杆菌和大肠杆菌在内的主要肠道分类群广泛分布于整个房间环境中，有许多肠道定植菌在超过一半的样本中被检测到。从婴儿肠道定植菌中重建的基因组揭示了一系列赋予抗生素（例如四环素、氟喹诺酮和氨基糖苷类）和杀菌剂抗性的基因，这可能在 NICU 环境中提供竞争优势。
我们开发了一种高通量的、无需培养的方法，该方法将基于全长 16S rRNA 基因序列的房间调查与从房间内婴儿收集的粪便样本的宏基因组分析相结合。该方法能够识别出离散的 ICU 微生物库，这些微生物也定植在婴儿肠道中，并提供了在肠道中检测到某些微生物之前，它们在房间中存在的证据。
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