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Journal of Medical Laboratory Science, 2019; 29 (3): 86 Journal of Medical Laboratory Science, 2019; 29 (3): 86-109 http://jomls.org ; [email protected] http://doi.org/10.5281/zenodo.4007804 Ndiokwere et al 16S rRNA Metagenomics of Seminal Fluids from Medical Microbiology Laboratory in a Tertiary Hospital, Southern Nigeria. Casmir Ndiokwere1 , Nkechi Augustina Olise2, Victoria Nmewurum3, Richard Omoregie1, Nneka Regina Agbakoba3, Kingsley C Anukam*3,4 1.Medical Microbiology Unit, Medical Laboratory Services, University of Benin Teaching Hospital, Benin City, Nigeria. 2. Department of Medical Laboratory Science, School of Basic Medical Sciences, College of Medical Sciences, University of Benin, Benin City, Nigeria. 3. Department of Medical Laboratory Sciences, Faculty of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Anambra State, Nigeria. 4. Uzobiogene Genomics, London, Ontario, Canada. ABSTRACT Background: Previous studies have relied on culture-dependent methods to determine microbial communities that may be found in the seminal fluids of men seeking reproductive health care. However, understanding the microbiome composition present in seminal fluids with the state-of-art next-generation sequencing technology is more germane than ever before, instead of culture methods which fails to identify over 99% of bacterial organisms present in biological samples. Methods: Forty semen samples were collected and after bacterial DNA extraction, 22 samples that passed quality check were used for amplification of the V4 region of the 16S rRNA using custom bar‑coded primers prior to sequencing with Illumina NextSeq 500 platform. Sequencing was performed in a pair-end modality rendering 2 x 150 base- pair sequences. Sequence reads were imported into Illumina BaseSpace Metagenomics pipeline for 16S rRNA recognition. Distribution of taxonomic categories at different levels of resolution was done using Greengenes database. Results: The taxonomic categories from the dataset produced phyla that ranges from 6 to 25; Class (9-49), Order (16-99), Family (42-214), Genus (55-555) and Species (56-1156). The taxonomic profiles represented 25 phyla, showing 39.5% of the total sequence reads were categorized to Proteobacteria as the most abundant. This was followed by Firmicutes (33.54%), Actinobacteria (20.77%) and Bacteroidetes (4.77%), Fusobacteria (0.613%), Tenericutes (0.31%) and Verrucomicrobia (0.12. At the species taxonomic level 1841 species were identified among the seminal fluid samples. Serratia marcescens (23.61% sequence reads) was the most abundant species found in 9/22 of the samples followed by Lactobacillus iners (18.22%) 13/22, Serratia entomophila (5.54%) 17/22, Haemophilus parainfluenzae (3.64%) 10/22, Corynebacterium tuberculostearicum (3.29%) 21/22, Gardnerella vaginalis 2.39%) 12/22, Lactobacillus taiwanensis (2.08%) 10/22, Enterobacter amnigenus (1.63%) 15/22, Corynebacterium genitalium (1.29%) 12/22, Neisseria lactamica (1.18%) 8/22, Finegoldia magna (1.17%) 16/22, Prevotella bivia (1.14%) 10/22, Corynebacterium imitans (1.12%) 15/22, Corynebacterium jeikeium (1.02%) 17/22 and Lactobacillus acidophilus (1.01%) found in 6/22 of the samples. Conclusions: We investigated the microbiome compositions from seminal fluids and showed that there are varying bacterial diversities that are unique in each sample in contrast to culture-dependent methods. Key words: Seminal fluid, microbiome, microbiota, metagenomics, sequencing, 16S rRNA. *Corresponding authors: Email: [email protected] ; ORCID: 0000-0002-2346-3345 Author’s contributions: This work was carried out and approved in collaboration between all the authors. KCA, NRA and CN designed the study; KCA sourced for funding, wrote the protocol and did literature search; CN, NAO and RO collected the samples; VN did taxonomic organization; KCA did bioinformatic analysis, drafted the manuscript, supervised the study and wrote the final manuscript; NRA and KCA proofread the manuscript. Received: September/15, 2019; Accepted: November/30, 2019; Published: December 28, 2019. Citation: Ndiokwere C , Olise NA, Nmewurum V, Omoregie R, Agbakoba NR, Anukam KC. 16S rRNA Metagenomics of Seminal Fluids from Medical Microbiology Laboratory in a Tertiary Hospital, Southern Nigeria. J Med Lab Sci, 2019; 29 (3): 86-109 A Publication of the Association of Medical Laboratory Scientists of Nigeria, under a Creative Commons Attribution Non-Commercial 4.0 International Public License (CC BY-NC 4.0). 86 Journal of Medical Laboratory Science, 2019; 29 (3): 86-109 http://jomls.org ; [email protected] Ndiokwere et al INTRODUCTION Staphylococcus saprophyticus (10%), Routine culture-dependent methods are Escherichia coli (11.4%), Klebsiella spp usually employed to isolate and determine (7.1%), Streptococcus pneumoniae (4.4%), microbial compositions of seminal fluids Pseudomonas aeruginosa (7.1%) and submitted to the microbiology laboratory of Candida spp (7.1%) were mainly teaching hospitals. These seminal fluids incriminated9, 10. Enwaru et al 11 reported originate from men who are seeking 49.4% Gram positive and 21% Gram reproductive health care as a result of either negative were isolated and Staphylococcus primary or secondary infertility, and from aureus (29.6%) and Escherichia coli (10.5%) other useful clinical pathologies. Male had the highest occurrence for each group infertility has been attributed to several respectively. factors, including but not limited to what is The objectives of this study are based out of referred to as “male factors” comprising of academic/professional curiosity by the infections affecting the genito-urinary tracts1, laboratory scientists that work on these hormonal imbalance, age factor, stress, seminal fluid samples and the opportunity of environmental pollution, some metabolic using culture-independent next-generation disorders2, 3 high throughput sequencing technology to Several microbiologists including laboratory determine microbial compositions of seminal scientists at the teaching hospital have at fluid samples submitted for culture in the many times tried to understand microbial laboratory. We compared bacterial organisms organisms that are present in seminal fluids. isolated by culture methods with the 16S Seminal fluid samples are normally cultured rRNA metagenomics sequencing results of on blood agar, chocolate agar, MacConkey the same samples. agar, nutrient agar, and sabouraud dextrose agar slants and incubated aerobically and in MATERIALS AND METHODS 5% CO2 at 37°C for 24 to 48 hours. It has been established that culture-dependent Ethics approval: Ethics approval was sought methods are now considered obsolete in the and approved at Nnamdi Azikiwe University 21st century microbial identification as it fails Teaching hospital, Nnewi as similar study to account for over 99.9% of the microbial involving large scale metagenomics compositions present in biological and sequencing from couples (Semen and vaginal environmental samples4. fluid) seeking reproductive health care is Some faculty research members have ongoing. attempted to isolate bacterial organisms present in semen. Onemu and Ibeh5 Study samples: previously reported by culture methods that Seminal fluid samples that were left over in Staphylococcus aureus constituted 43.7%, the laboratory after culturing for bacterial followed by Klebsiella species (28.2%), isolation were used. Forty semen samples Escherichia coli (11.5%), and Candida were collected and 100 microlitre from each albicans (7.7%). Parallel reports were sample was inoculated into a tube containing published subsequently by Momoh et al6, lysis and stabilization buffer that preserves Ibadin et al7, Ekhaise and Richard8. Other the DNA for transport at ambient authors from nearby institutions have temperature. The tubes were sent to uBiome presented similar findings whereby Inc. in California, United States America for Staphylococcus aureus (53%), DNA extraction and sequencing. The A Publication of the Association of Medical Laboratory Scientists of Nigeria, under a Creative Commons Attribution Non-Commercial 4.0 International Public License (CC BY-NC 4.0). 87 Journal of Medical Laboratory Science, 2019; 29 (3): 86-109 http://jomls.org ; [email protected] Ndiokwere et al sequencing results were analyzed with bioinformatic tools at Uzobiogene Genomics, Metagenomics sequence analysis: Raw London, Ontario, Canada. sequence reads were demultiplexed using Illumina’s BCL2FASTQ algorithm. Reads were filtered using an average Q-score > 30. Extraction of bacterial DNA from Seminal The 8 paired-end sequence FASTQ reads for fluid samples and Sequencing of the each sample were imported into MG-RAST amplified 16S rRNA region: pipeline for quality check (QC). Quantitative Bacterial DNA was extracted from the Insights into Microbial Ecology (QIIME) seminal fluids using an in-house protocol pipeline was used for 16S rRNA recognition. developed by uBiome Inc. Briefly, samples Sequences were pre-screened using QIIME- were lysed using bead-beating, and DNA was UCLUST algorithms for at least 97% identity extracted in a class 1000 clean room by a to ribosomal sequences from the RNA guanidine thiocyanate silica column-based databases. Reads passing all above filters purification method using a liquid-handling were aligned to the database of 16S rRNA robot. PCR amplification of the 16S rRNA gene sequences. Microbial taxonomy to genes was performed with primers containing species level was generated using the universal primers amplifying the V4 region Illumina BaseSpace Greengenes database. (515F: GTGCCAGCMGCCGCGGTAA and
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