Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples.

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.

Examining Different Analysis Protocols Targeting Hospital Sanitary Facility Microbiomes.

Indoor spaces exhibit microbial compositions that are distinctly dissimilar from one another and from outdoor spaces. Unique in this regard, and a topic that has only recently come into focus, is the microbiome of hospitals. While the benefits of knowing exactly which microorganisms propagate how and where in hospitals are undoubtedly beneficial for preventing hospital-acquired infections, there are, to date, no standardized procedures on how to best study the hospital microbiome. Our study aimed to investigate the microbiome of hospital sanitary facilities, outlining the extent to which hospital microbiome analyses differ according to sample-preparation protocol. For this purpose, fifty samples were collected from two separate hospitals-from three wards and one hospital laboratory-using two different storage media from which DNA was extracted using two different extraction kits and sequenced with two different primer pairs (V1-V2 and V3-V4). There were no observable differences between the sample-preservation media, small differences in detected taxa between the DNA extraction kits (mainly concerning Propionibacteriaceae), and large differences in detected taxa between the two primer pairs V1-V2 and V3-V4. This analysis also showed that microbial occurrences and compositions can vary greatly from toilets to sinks to showers and across wards and hospitals. In surgical wards, patient toilets appeared to be characterized by lower species richness and diversity than staff toilets. Which sampling sites are the best for which assessments should be analyzed in more depth. The fact that the sample processing methods we investigated (apart from the choice of primers) seem to have changed the results only slightly suggests that comparing hospital microbiome studies is a realistic option. The observed differences in species richness and diversity between patient and staff toilets should be further investigated, as these, if confirmed, could be a result of excreted antimicrobials.

Analysis of the cervical microbiome in women from the German national cervical cancer screening program.

PURPOSE: Cervical cancer (CC) is caused by a persistent high-risk human papillomavirus (hrHPV) infection. The cervico-vaginal microbiome may influence the development of (pre)cancer lesions. Aim of the study was (i) to evaluate the new CC screening program in Germany for the detection of high-grade CC precursor lesions, and (ii) to elucidate the role of the cervico-vaginal microbiome and its potential impact on cervical dysplasia. METHODS: The microbiome of 310 patients referred to colposcopy was determined by amplicon sequencing and correlated with clinicopathological parameters. RESULTS: Most patients were referred for colposcopy due to a positive hrHPV result in two consecutive years combined with a normal PAP smear. In 2.1% of these cases, a CIN III lesion was detected. There was a significant positive association between the PAP stage and Lactobacillus vaginalis colonization and between the severity of CC precursor lesions and Ureaplasma parvum. CONCLUSION: In our cohort, the new cervical cancer screening program resulted in a low rate of additional CIN III detected. It is questionable whether these cases were only identified earlier with additional HPV testing before the appearance of cytological abnormalities, or the new screening program will truly increase the detection rate of CIN III in the long run. Colonization with U. parvum was associated with histological dysplastic lesions. Whether targeted therapy of this pathogen or optimization of the microbiome prevents dysplasia remains speculative.

Tropical leg lymphedema caused by podoconiosis is associated with increased colonisation by anaerobic bacteria.

The non-filarial and non-communicable disease podoconiosis affects around 4 million people and is characterized by severe leg lymphedema accompanied with painful intermittent acute inflammatory episodes, called acute dermatolymphangioadenitis (ADLA) attacks. Risk factors have been associated with the disease but the mechanisms of pathophysiology remain uncertain. Lymphedema can lead to skin lesions, which can serve as entry points for bacteria that may cause ADLA attacks leading to progression of the lymphedema. However, the microbiome of the skin of affected legs from podoconiosis individuals remains unclear. Thus, we analysed the skin microbiome of podoconiosis legs using next generation sequencing. We revealed a positive correlation between increasing lymphedema severity and non-commensal anaerobic bacteria, especially Anaerococcus provencensis, as well as a negative correlation with the presence of Corynebacterium, a constituent of normal skin flora. Disease symptoms were generally linked to higher microbial diversity and richness, which deviated from the normal composition of the skin. These findings show an association of distinct bacterial taxa with lymphedema stages, highlighting the important role of bacteria for the pathogenesis of podoconiosis and might enable a selection of better treatment regimens to manage ADLA attacks and disease progression.