Exploring bacterial diversity in hospital environments by GS-FLX Titanium pyrosequencing

Abstract

Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive care units (ICU). In this work we present an exploration of bacterial diversity at inanimate surfaces of the ICU wards of the University Hospital A Coruña (Spain), as an example of confined hospital environment subjected to selective pressure, taking the entrance hall of the hospital, an open and crowded environment, as reference. Surface swab samples were collected from both locations and recovered DNA used as template to amplify a hypervariable region of the bacterial 16S rRNA gene. Sequencing of the amplicons was performed at the Roche 454 Sequencing Center using GS-FLX Titanium procedures. Reads were pre-processed and clustered into OTUs (operational taxonomic units), which were further classified. A total of 16 canonical bacterial phyla were detected in both locations. Members of the phyla Firmicutes (mainly Staphylococcus and Streptococcus) and Actinobacteria (mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae) were over-represented in the ICU with respect to the Hall. The phyllum Proteobacteria was also well represented in the ICU, mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae. In the Hall sample, the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria were over-represented with respect to the ICU. Over-representation of Proteobacteria was mainly due to the high abundance of Enterobacteriaceae members. The presented results demonstrate that bacterial diversity differs at the ICU and entrance hall locations. Reduced diversity detected at ICU, relative to the entrance hall, can be explained by its confined character and by the existence of antimicrobial selective pressure. This is the first study using deep sequencing techniques made in hospital wards showing substantial hospital microbial diversity.

Figure 1. Venn diagram representation of shared richness at a distance of 0.03.

Figure 2. Rarefaction curves for the ICU and Hall samples at distances of 0.03, 0.05 and 0.1.

Figure 3. Normalized abundance of reads associated with taxonomic nodes at the order level, expressed as the number of occurrences in 100000 reads.

Abundances are shown in logarithmic (left panel) and decimal (right panel) scales.

Figure 4. Schematic representation of the relative abundance of families described in Hall and ICU samples.

Figure 5. Hierarchical pie chart representing bacterial diversity assessed by pyrosequencing.

Bacteria recovered from (a) Hall and (b) ICU samples during the experimental period.

Figure 6. Hierarchical pie chart, representing bacterial diversity in clinical samples from ICU-hospitalized patients during the experimental period.