Comparison of targeted metagenomics and IS-Pro methods for analysing the lung microbiome

BACKGROUND : Targeted metagenomics and IS-Pro method are two of the many methods that have been used to study the microbiome. The two methods target different regions of the 16 S rRNA gene. The aim of this study was to compare targeted metagenomics and IS-Pro methods for the ability to discern the microbial composition of the lung microbiome of COPD patients. METHODS : Spontaneously expectorated sputum specimens were collected from COPD patients. Bacterial DNA was extracted and used for targeted metagenomics and IS-Pro method. The analysis was performed using QIIME2 (targeted metagenomics) and IS-Pro software (IS-Pro method). Additionally, a laboratory cost per isolate and time analysis was performed for each method. RESULTS : Statistically significant differences were observed in alpha diversity when targeted metagenomics and ISPro methods’ data were compared using the Shannon diversity measure (p-value = 0.0006) but not with the Simpson diversity measure (p-value = 0.84). Distinct clusters with no overlap between the two technologies were observed for beta diversity. Targeted metagenomics had a lower relative abundance of phyla, such as the Proteobacteria, and higher relative abundance of phyla, such as Firmicutes when compared to the IS-Pro method. Haemophilus, Prevotella and Streptococcus were most prevalent genera across both methods. Targeted metagenomics classified 23 % (144/631) of OTUs to a species level, whereas IS-Pro method classified 86 % (55/64) of OTUs to a species level. However, unclassified OTUs accounted for a higher relative abundance when using the ISPro method (35 %) compared to targeted metagenomics (5 %). The two methods performed comparably in terms of cost and time; however, the IS-Pro method was more user-friendly. CONCLUSIONS : It is essential to understand the value of different methods for characterisation of the microbiome. Targeted metagenomics and IS-Pro methods showed differences in ability in identifying and characterising OTUs, diversity and microbial composition of the lung microbiome. The IS-Pro method might miss relevant species and could inflate the abundance of Proteobacteria. However, the IS-Pro kit identified most of the important lung pathogens, such as Burkholderia and Pseudomonas and may work in a more diagnostics-orientated setting. Both methods were comparable in terms of cost and time; however, the IS-Pro method was easier to use.SUPPLEMENTARY MATERIAL: Table S1. Inclusion and exclusion criteria for COPD patients in this study. Table S2. Clinical characteristic of patients. Table S3. Comparison of the number of amplicons and operational taxonomic units for each sample for the targeted metagenomics and IS-Pro methods. Figure S1. Relative abundance of specific phyla in the sputum microbiome of COPD participants as detected by targeted metagenomics and IS-Pro methods (n = 23). The dots represent the different abundances of each sample, according to the different phyla. Phyla that are depicted with a single line on the y-axis were not present in any samples for that method. Figure S2. Bar plots showing the relative abundance of genera in the sputum microbiome of COPD participants as characterised by targeted metagenomics and IS-Pro methods (n = 23). The operational taxonomic units that could not be classified at a genus level are indicated as NA on the graph. Figure S3. The distribution of the unclassified operational taxonomic units (OTUs) at a class level of the sputum microbiome of COPD participants for targeted metagenomics and IS-Pro methods by phyla. At a class level, all the OTUs from targeted metagenomics could be classified.National Health Laboratory Service of South Africa (NHLS) Research Trusthttps://bmcmicrobiol.biomedcentral.comam2022Internal MedicineMedical Microbiolog

Lung microbiome of chronic obstructive pulmonary disease patients with and without HIV infection in Pretoria, South Africa

Chronic obstructive pulmonary disease (COPD) is a leading cause of death and is highly prevalent in South Africa (19% in adults over the age of 40 years). Inflammation of the lungs in COPD impairs the immune response and allows colonisation and infection with bacteria and viruses, that may cause exacerbations of the disease. Culture-independent technologies have greatly increased the understanding of the lung microbiome. The most widely used method for targeted metagenomics is 16S rRNA sequencing. The IS-Pro (intergenic spacer profiling) method provides an alternative targeted metagenomics approach; however, the two methods have not been compared. There is limited data on the microbiome in the lungs of COPD patients in Africa. Due to local environmental conditions, immunological differences and clinical comorbidities, such as HIV, the microbiome may be different from that reported in studies from other countries. The purpose of this study was to identify the lung microbiome and lung virome in COPD patients in South Africa and to determine if the COPD disease states result in differences in its composition. Next-generation sequencing was used to determine the microbiome and virome of COPD patients from hospitals in Pretoria, South Africa and the IS-Pro method was compared to targeted metagenomics. Twenty-four patients over the age of 40 years with a confirmed COPD diagnosis and no Mycobacterium tuberculosis infection were included; eighteen were in the stable state of diseases and six were in the exacerbation state of disease. Sputum specimens were collected from all consenting participants and DNA and RNA were extracted directly from the specimens using commercial kits. The extracted bacterial DNA was sent for targeted metagenomics and the IS-Pro method and the extracted viral DNA and RNA were sent for shotgun metagenomics sequencing. The lung of the COPD participants showed a diverse microbiome with over 77 genera identified and the Firmicutes phylum predominating. When the stable and exacerbation states of COPD disease were compared, no significant differences in the alpha and beta diversity between the disease states were observed. However, during exacerbation state of the disease, the abundance of key phyla had decreased. Analysis of the virome showed a high prevalence of BeAn 58058, a close relative of the smallpox virus, with bacteriophages being the second most prevalent viruses. When comparing the IS-Pro method to targeted metagenomics, an increased relative abundance of Proteobacteria with the IS-Pro method was observed, which was attributed to known lung pathogens, such as Burkholderia. The IS-Pro method was able to classify more operational taxonomic units (OTUs) to a species level, however, the unclassified OTUs from the IS-Pro method could only be classified to a phylum level. To conclude, a diverse COPD microbiome was observed, with a virome that was dominated by the BeAn 58058 virus. The COPD disease states showed no variations in terms of diversity, however, the relative abundances of key phyla differed between disease states for the bacterial microbiome. Future studies should focus on longitudinal studies of the sputum microbiome in an African setting as well as functional metatranscriptomics studies with a focus on antibiotic resistance and virulence factors.Thesis (PhD (Medical Microbiology))--University of Pretoria, 2020.Medical MicrobiologyPhD (Medical Microbiology)Unrestricte

Lung microbiome of stable and exacerbated COPD patients in Tshwane, South Africa

Chronic obstructive pulmonary disease (COPD) is characterised by the occurrence of exacerbations triggered by infections. The aim of this study was to determine the composition of the lung microbiome and lung virome in patients with COPD in an African setting and to compare their composition between the stable and exacerbated states. Twenty-four adult COPD patients were recruited from three hospitals. Sputum was collected and bacterial DNA was extracted. Targeted metagenomics was performed to determine the microbiome composition. Viral DNA and RNA were extracted from selected samples followed by cDNA conversion. Shotgun metagenomics sequencing was performed on pooled DNA and RNA. The most abundant phyla across all samples were Firmicutes and Proteobacteria. The following genera were most prevalent: Haemophilus and Streptococcus. There were no considerable diferences for alpha and beta diversity measures between the disease states. However, a diference in the abundances between disease states was observed for: (i) Serratia (3% lower abundance in exacerbated state), (ii) Granulicatella (2.2% higher abundance in exacerbated state), (iii) Haemophilus (5.7% higher abundance in exacerbated state) and (iv) Veillonella (2.5% higher abundance in exacerbated state). Virome analysis showed a high abundance of the BeAn 58058 virus, a member of the Poxviridae family, in all six samples (90% to 94%). This study is among the frst to report lung microbiome composition in COPD patients from Africa. In this small sample set, no diferences in alpha or beta diversity between stable and exacerbated disease state was observed, but an unexpectedly high frequency of BeAn 58058 virus was observed. These observations highlight the need for further research of the lung microbiome of COPD patients in African settings.National Health Laboratory Service of South Africa (NHLS) Research Trusthttp://www.nature.com/srep/index.htmlpm2022Internal MedicineMedical Oncolog

Genetic relatedness of Staphylococcus aureus isolates obtained from cystic fibrosis patients at a tertiary academic hospital in Pretoria, South Africa

Cystic fibrosis (CF) is an inherited recessive disease that affects mucocillary clearance in the lung, allowing it to be colonised with bacteria such as Staphylococcus aureus. To survive in the CF lung S. aureus adapts both phenotypically and genotypically, through various mechanisms. In this study, multiple specimens were collected from the participants and were processed routinely and were additionally cultured in chromogenic media. Multiplex PCR assays were employed to detect methicillin resistance and selected virulence and quaternary ammonium compound (qac) genes. Genetic relatedness of the S. aureus was determined using agr, SCCmec and spa typing as well as pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Thirty-three S. aureus isolates were isolated, of which 51% (17/33) were methicillin resistant S. aureus (MRSA). The virulence and qac genes were more prevalent in MRSA than the methicillin sensitive S. aureus (MSSA) isolates. The PFGE analysis showed nine distinct pulsotypes while MLST showed eight sequence types. All the STs detected in this study, except for ST508 have been previously isolated from CF patients according to the literature. This study showed a genetically diverse S. aureus population with a high prevalence of virulence genes among the MRSA isolates from the CF clinic.The NHLS Research Trust, the NRF and RESCOM.http://www.nature.com/srepam2019Medical Microbiolog