Contribution of DNA metabarcoding to the environmental fungal assessments in hospitals

Background: Hospitals are particularly sensitive environments where immunosuppressed patients might acquire invasive fungal infections. Therefore, it is necessary to carry out periodical environmental microbiological assessments that evaluate the fungal bioburden in air and surfaces from different hospital zones. Current microbiological monitoring protocols at healthcare settings are mostly based on cultivation, while environmental DNA (eDNA) assessments are still scarce and should be further evaluated. To fill this gap, this study combines a large sampling scheme, comprising > 200 samples (air, surface, dust and soil) collected from four zones at three Spanish hospitals in two campaigns (winter and autumn), with two eDNA approaches (DNA metabarcoding and quantitative PCR) to characterize the hospital mycobiomes (diversity, community composition and airborne load), compared to a parallel culture-dependent study. Results: Fungal richness was significantly higher in soil and air samples compared to indoor surface samples (vents and high-touch surfaces), as well as in samples collected in winter compared those taken in autumn. Intensive care units showed lower fungal richness compared to regular patient rooms, waiting rooms and entrance halls. The most important explanatory factors for the variance in community composition were the hospital and zone where samples were collected, the type of sample, and the sampling campaign. Hospital mycobiomes, represented by 1,900 operational taxonomic units, were affiliated to 4 phyla (mostly Ascomycota - 53 % and Basidiomycota - 41.3 %), 35 classes, 114 orders, 305 families, 643 genera, and 535 species. The dominant genera, in both air and surfaces samples from the three hospitals, were Cladosporium, Alternaria, Aureobasidium, Penicillium, Neodidymelliopsis, Aspergillus, Pseudopithomyces and Stemphylium. The yeasts Candida and Clavispora were particularly abundant in high-touch surfaces indoors. Conclusions: DNA metabarcoding revealed a much more comprehensive inventory of hospital fungi compared to culturing, however, both approaches found similar dominant taxa including a variety of potentially opportunistic human pathogens. DNA metabarcoding can assist hospital managers under certain demanding situations, e.g. construction works or reported microbial outbreaks, providing an in-depth characterization of hospital mycobiomes. In addition, qPCR proved to be a reliable method to quantify the fungal load in air samples, which can complement colony forming units and particle counts in environmental assessments.

研究背景：医院属于高度敏感的特殊环境，免疫功能低下患者在此类环境中可能罹患侵袭性真菌感染。因此，对不同医院区域的空气与表面的真菌生物负荷开展周期性环境微生物评估十分必要。当前医疗场所的微生物监测方案大多基于培养法，而环境DNA（environmental DNA, eDNA）相关评估仍较为匮乏，有待进一步研究验证。为填补这一研究空白，本研究开展了大规模采样方案：在两次采样季（冬季与秋季）中，从3所西班牙医院的4个区域采集了超过200份样本（涵盖空气、表面、灰尘与土壤），同时结合DNA宏条形码（DNA metabarcoding）与定量PCR（quantitative PCR, qPCR）两种eDNA分析方法，对医院真菌组（mycobiome）的多样性、群落组成及空气传播负荷进行表征，并与平行开展的培养依赖性研究进行对比。
研究结果：土壤与空气样本中的真菌丰富度显著高于室内表面样本（通风口与高频接触表面）；冬季采集的样本真菌丰富度也显著高于秋季样本。重症监护病房的真菌丰富度显著低于普通病房、候诊室与门诊大厅。影响群落组成变异的最重要解释因子包括样本采集所在的医院与区域、样本类型以及采样季。本次研究涉及的医院真菌组涵盖1900个操作分类单元（operational taxonomic unit, OTU），隶属于4个门（以子囊菌门Ascomycota占比53%、担子菌门Basidiomycota占比41.3%为主）、35个纲、114个目、305个科、643个属以及535个物种。三家医院的空气与表面样本中的优势菌属包括Cladosporium、Alternaria、Aureobasidium、Penicillium、Neodidymelliopsis、Aspergillus、Pseudopithomyces以及Stemphylium。在室内高频接触表面中，念珠菌属Candida与Clavispora的丰度尤为突出。
研究结论：相较于传统培养法，DNA宏条形码可更全面地覆盖医院真菌的物种清单；但两种方法均检测到了相似的优势类群，其中包含多种潜在的人类机会致病性真菌。在部分特殊场景（如医院基建施工或已报道的微生物暴发事件）中，DNA宏条形码可协助医院管理人员对医院真菌组进行深度表征。此外，定量PCR（qPCR）被证实是量化空气样本中真菌负荷的可靠方法，可在环境评估中弥补菌落形成单位（colony forming unit, CFU）计数与颗粒物计数的不足。