The Wound Microbiome: Modern Approaches to Examining the Role of Microorganisms in Impaired Chronic Wound Healing

Abstract

Significance: Bacterial burden is believed to play a significant role in impaired wound healing of chronic wounds and the development of infection-related complications. The standard of care in the clinic relies upon cultivation-dependent methods to identify microorganisms. These assays are biased toward microorganisms that thrive in isolation under laboratory conditions. Recent Advances: Significant advances in genomic technologies have enabled less-biased, culture-independent approaches to characterize microbial communities, or microbiomes. The aggregate sequencing and analysis of 16S ribosomal RNA genes has demonstrated that cultures under-represent true microbial diversity and load. Critical Issues: Despite recent advances that enable culture-independent analyses of microbiomes, those organisms that are important in impaired healing remain ambiguous. Inconsistent findings across various studies highlight the need to characterize microbiomes of chronic wounds with homogenous etiology to determine differences in microbiomes that may be driven by the wound environment and that may affect wound outcomes. Rigorous analyses of wound microbiomes in light of the three dimensions of bioburden (microbial diversity, microbial load, and pathogenic organisms), clinical metadata, and wound outcomes will be a significant step forward in our quest to understand the role of microorganisms in impaired healing. Future Directions: Longitudinal studies employing serial sampling are needed to appreciate the role of the dynamic microbial community in chronic wound healing. The value of clinical metadata needs to be examined as potential biomarkers of problematic microbiota and wound outcomes. Lastly, better characterization and understanding of wound microbiomes will open avenues for improved diagnostic and therapeutic tools for the nonhealing wound.

Figures

Elizabeth A. Grice, PhD

Figure 1.

The workflow of a 16S ribosomal RNA (16S rRNA) gene targeted microbiome next-generation sequencing project. A heterogeneous mixture of genomic DNA is extracted from samples taken from a wound. Primers specific for the desired regions of the 16S rRNA gene are used to amplify bacterial DNA. Each sample has a unique identifying sequence on the primer known as a barcode to facilitate the multiplexing of samples on the sequencer. The resulting polymerase chain reaction (PCR) products are pooled and sequenced using platforms, such as Roche 454, Illumina MiSeq, or Life Technologies Ion Torrent. After filtering out low-quality sequences, various analyses are performed, including assignment to taxonomy, analysis of shared phylogeny, and analysis of microbial community membership, structure, and diversity. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 2.

Model of the impact of three dimensions of bioburden and nonmicrobial factors on wound outcomes. The three dimensions of bioburden are covarying components of the wound microbiome. We propose that the microbiome is one of several factors that lead to ultimate wound outcomes, such as healing or the development of infection-related complications.

Figure 3.

16S rRNA gene sequencing-based methods provide a more comprehensive view of bacterial diversity compared to culture-based methods. A comparison of bacterial diversity assessed by culture-based and 16S rRNA gene sequencing results. The symbols represent the mean bacterial taxa count per sample included in each study. In Price et al., the number of cultured genera was compared to the number of sequenced genera. In Han et al., the number of cultured species was compared to the number of sequenced genera. In Gardner et al., the number of cultured species was compared to the number of sequenced species. The horizontal line indicates the mean of the three studies surveyed. References to each study are denoted in brackets.

Figure 4.

The 10 most abundant bacterial genera observed in select chronic wound microbiome publications. Each large circle represents the top 10 most abundant genera reported from the publication indicated. Only the top 10 most abundant genera are included. Staphylococcus, Corynebacterium, and Anaerococcus were common to the 10 most abundant genera in all three studies. “Unc.” denotes unclassified bacteria. *Denotes strictly anaerobic or obligately anaerobic bacteria. References to each study are denoted in brackets. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 5.

Bacterial families reported to colonize chronic wounds in culture-independent studies. Twenty-one bacterial families account for the majority of microbiota colonizing chronic wounds in five studies utilizing culture-independent methods. The bacterial phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria are represented. Here, we show the results of these publications as relative abundance charts, with the etiology of the wounds, and the method of collection noted. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound