Culture-independent Sequencing Research Articles

The Effect of Face Mask Wear on the Ocular Surface and Contact Lens Microbiome.

As face mask wear can result in the redirection of nasal and oral exhalation toward the ocular region, this study investigated the impact of face mask wear on the conjunctiva, eyelid margin, and contact lens (CL) surface microbiome. In this prospective, cross-over study, experienced CL wearers (N=20) were randomized to wear a face mask for 6 hr/day (minimum) for a week or no mask for a week. The conjunctiva, eyelid, and CLs were then sampled. After a 1-week washout period, participants were crossed over into the alternate treatment for 1 week and sampling was repeated. Sampling was bilateral and randomly assigned to be processed for culturing or 16S ribosomal(r) RNA gene sequencing. Culturing showed no effect of mask wear on the average number of bacterial colonies isolated on the conjunctiva, eyelid, or CL, but there was increased isolation of Staphylococcus capitis on CL samples with mask wear (P=0.040). Culture-independent sequencing found differences in the taxonomic complexity and bacterial composition between the three sites (P<0.001), but there was no effect of bacterial diversity within and between sites. Mask wear did not impact dry eye or CL discomfort, but increased ocular surface staining was reported (P=0.035). Mask wear did not substantially alter the microbiome of the conjunctiva, eyelid margin, or CL surfaces in uncompromised healthy eyes.

Uncovering Endolysins against Methicillin-Resistant Staphylococcus aureus Using a Microbial Single-Cell Genome Database.

Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of these endolysins is vital. We propose here microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof of concept, we analyzed and recovered endolysin genes within prophage regions of Staphylococcus single-amplified genomes in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against Staphylococcus aureus. One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties, such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target and boost the development of novel antimicrobial agents.

Microbial community analysis offers insight into the complex origins of plant disease in a smallholder farm context

Classical approaches to plant disease diagnosis assume a single pathogen/single disease paradigm. Here we revisit the presumed role of Fusarium oxysporum as the causal agent of wilting and yellowing of chickpea plants on small holder farms in Ethiopia. Contrary to expectations, detection of Fusarium DNA using conserved PCR primers failed to associate the pathogen with symptomatic plants. Instead, culture-independent sequencing of microbial communities nominated unexpected pathogens and revealed patchiness in the assembly of common microbial consortia. Surprisingly, tests of differential enrichment identified Phytophthora as the most common disease-associated taxon. More generally, across all field sites, multi-level pattern analysis identified indicator taxa whose patterns of co-occurrence demarcate discrete microbial communities and are consistent with a range of specific interactions, including mutualism and antagonism. Taken together, these data indicate that soil-borne chickpea disease in Ethiopia has heterogeneous origins, and that despite decades of emphasis and disease resistance breeding, the role of Fusarium as the frequent agent of chickpea disease in Ethiopia remains enigmatic.

Culture-independent Multilocus sequence typing screening for Haemophilus influenzae cross-infection in non-cystic fibrosis bronchiectasis

Whether cross-infection of respiratory pathogens between patients with non-cystic fibrosis bronchiectasis occurs is debated. Investigation with traditional microbiological culture risks simplifying the lung microbiome. We demonstrate the use of culture-independent Multilocus sequence typing to screen for Haemophilus influenzae strain types in a cohort of twenty-eight patients with non-cystic fibrosis bronchiectasis.

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Microbial diversity analysis of Chumathang geothermal spring, Ladakh, India.

In light of their unique and challenging environment, the high-altitude Chumathang geothermal springs in Ladakh, India, are undeniably intriguing for microbiological study. The purpose of this study was to employ a culture-independent sequencing approach to give a comprehensive characterization of the unknown bacterial and archaeal community structure, composition and networks in water and soil from the Chumathang geothermal spring. A total of 50%, and 42.86% bacterial phyla were found in the water, and soil samples respectively and this analysis also showed a total of 9.62% and 7.94% of archaeal phyla in both the samples, respectively. Further, the presence of unclassified (derived from other sequences, water: 17.31%, and soil: 19.05%) and unclassified (derived from bacteria, water: 13.46%, and soil: 12.70%) were also observed in the current metagenomics investigation. Firmicutes and Proteobacteria were the most abundant bacterial phyla in water, whereas Proteobacteria and Bacteroidetes were the most abundant bacterial phyla in geothermal soil. Crenarchaeota and Euryarchaeota dominated archeal communities in soil and water, respectively. This metagenomic study gave a detailed insight into the microbial diversity found in Chumathang geothermal spring and surrounding area, located in Ladakh, India. Surprisingly, this finding indicated the existence of geographically distinct microbial communities that were suited to various geothermal water habitats along the Himalayan Geothermal Belt. Future studies must take into account the metabolic pathways of these microbial communities that exist in these extreme environments. This will allow us to obtain a better knowledge of the microbial metabolisms that are common at these geothermal locations, which have a lot of potential for biotechnological applications. They will also enable us to establish links between the microbial community composition and the physicochemical environment of geothermal water and area.

Insights into Endophytic and Rhizospheric Bacteria of Five Sugar Beet Hybrids in Terms of Their Diversity, Plant-Growth Promoting, and Biocontrol Properties

Sugar beet is the most important crop for sugar production in temperate zones. The plant microbiome is considered an important factor in crop productivity and health. Here, we investigated the bacterial diversity of seeds, roots, and rhizosphere of five sugar beet hybrids named Eduarda (ED), Koala (KO), Tibor (T), Tajfun (TF), and Cercospora-resistant (C). A culture-independent next-generation sequencing approach was used for the further investigation of seed-borne endophytes. Hybrid-associated bacteria were evaluated for their plant growth–promoting (PGP) characteristics, antagonistic activity towards Cercospora beticola and several Fusarium strains in dual culture assays, and drought and salinity tolerance. High-throughput sequencing revealed that the Proteobacteria phylum was most dominant in the seeds of all hybrids, followed by Cyanobacteria and Actinobacteriota. The predominant genus in all hybrids was Pantoea, followed by Pseudomonas, Acinetobacter, Chalicogloea, Corynebacterium, Enterobacter, Enterococcus, Glutamicibacter, Kosakonia, and Marinilactibacillus. Unique genera in the hybrids were Pleurocapsa and Arthrobacter (T), Klebsiella (TF), Apibacter (ED), and Alloscardovia (KO). The genera that were most represented in one hybrid were Weissella and Staphylococcus (TF); Streptococcus (T); Gardnerella, Prevotella, and Rothia (KO); and Gilliamella, Lactobacillus, and Snodgrassella (ED). Thirty-two bacteria out of 156 isolates from the rhizosphere, roots, and seeds were selected with respect to various plant growth–promoting activities in vitro, i.e., nitrogen fixation, phosphate solubilization, siderophore production, indole-3-acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase activity, hydrogen cyanide production, exoenzymatic activity (amylase, protease, lipase, cellulase, xylanase, mannanases, gelatinase, and pectinase), mitigation of environmental stresses, and antifungal activity. Mixta theicola KO3-44, Providencia vermicola ED3-10, Curtobacterium pusillum ED2-6, and Bacillus subtilis KO3-18 had the highest potential to promote plant growth due to their multiple abilities (nitrogen fixation, phosphate solubilization, production of siderophores, and IAA). The best antagonistic activity towards phytopathogenic fungi was found for Bacillus velezensis C3-19, Paenibacillus polymyxa C3-36 and Bacillus halotolerans C3-16/2.1. Only four isolates B. velezensis T2-23, B. subtilis T3-4, B. velezensis ED2-2, and Bacillus halotolerans C3-16/2.1 all showed enzymatic activity, with the exception of xylanase production. B. halotolerans C3-16/2.1 exhibited the greatest tolerance to salinity, while two B. subtilis strains (C3-62 and TF2-1) grew successfully at the maximum concentration of PEG. The current study demonstrates that sugar beet–associated bacteria have a wide range of beneficial traits and are therefore highly promising for the formulation of biological control and PGP agents.

Characterization of the semen microbiota of healthy stud dogs using 16S RNA sequencing

The reproductive microbiota of male dogs has never been investigated using culture-independent sequencing techniques. The purpose of the present study was to get seminal knowledge on the microbiota of the ejaculate. Specifically, factors as the fraction of the ejaculate, the sperm quality (normospermia, teratozoospermia), and the living environment were evaluated. The sperm-rich and the prostatic fractions of the ejaculate were collected from healthy stud dogs. Following the sperm analysis, samples from twenty animals (normospermic n = 10 and teratozoospermic n = 10) were stored at – 80 °C until further processing including DNA extraction and 16S rRNA sequencing. Alpha- (Shannon index) and beta- (Bray-Curtis, Unweighted UniFrac) diversities were assessed and compared (PERMANOVA) based on the group of samples (biological samples from the ejaculate and controls), the fraction of the ejaculate (sperm-rich and prostatic fractions), the animal group (normospermia and teratozoospermia), and the living environment of the animal (kennel or pet living in-house). The most abundant bacterial phyla in canine semen samples were Proteobacteria, Firmicutes, and Actinobacteria. Overall, the dominant bacterial family was that of Pasteurellaceae The genus Mycoplasma was never detected. No differences in terms of bacterial composition were found based on the fraction of the ejaculate and based on the animal group (P > 0.05). On the other hand, differences in alpha and beta diversities were highlighted based on the living environment (P = 0.001).Overall, the results of the present study provide preliminary insights on dog semen microbiota, opening a new chapter in the field of canine andrology. Our results suggest that the environment may play a role in influencing the reproductive microbiota of male dogs and that the prostatic fraction of the ejaculate can be used for further research as a representative of the semen microbiota.

Draft genomes of novel avian Chlamydia abortus strains from Australian Torresian crows (Corvus orru) shed light on possible reservoir hosts and evolutionary pathways.

Chlamydia abortus, an obligate intracellular bacterium, is a major causative agent of reproductive loss in ruminants, with zoonotic potential. Though this pathogen is primarily known to infect livestock, recent studies have detected and isolated genetically distinct avian strains of C. abortus from wild birds globally. Before this study, only five avian C. abortus genomes were publicly available. Therefore, we performed culture-independent probe-based whole-genome sequencing on clinical swabs positive for avian C. abortus obtained from Australian Torresian crows (Corvus orru) in 2019 and 2020. We successfully obtained draft genomes for three avian C. abortus strains (C1, C2 and C3), each comprising draft chromosomes with lengths of 1 115 667, 1 120 231 and 1 082 115 bp, and associated 7 553 bp plasmids, with a genome completeness exceeding 92 %. Molecular characterization revealed that these three strains comprise a novel sequence type (ST333), whilst phylogenetic analyses placed all three strains in a cluster with other avian C. abortus genomes. Interestingly, these three strains share a distant genomic relation (2693 single nucleotide variants) with the reference strain 15-58d/44 (ST152), isolated from a Eurasian magpie (Pica pica) in Poland, highlighting the need for more publicly available genomes. Broad comparative analyses with other avian C. abortus genomes revealed that the three draft genomes contain conserved Chlamydia genomic features, including genes coding for type III secretion system and polymorphic membrane proteins, and potential virulence factors such as the large chlamydial cytotoxin, warranting further studies. This research provides the first avian C. abortus draft genomes from Australian birds, highlighting Torresian crows as novel reservoir hosts for these potential pathogens, and demonstrates a practical methodology for sequencing novel Chlamydia genomes without relying on traditional cell culture.

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

The altered sputum microbiome profile in patients with moderate and severe COPD exacerbations, compared to the healthy group in the Indian population

Background: Microbial culture-independent sequencing techniques have advanced our understanding of host-microbiome interactions in health and disease. The purpose of this study was to explore the dysbiosis of airway microbiota in patients with moderate or severe chronic obstructive pulmonary disease (COPD) and compare them with healthy controls. Methods: The COPD patients were investigated for disease severity based on airflow limitations and divided into moderate (50%≤FEV1&lt;80% predicted) and severe groups (FEV1&lt;50% predicted). Spontaneous sputum samples were collected and, the V3-V4 regions of the 16S rRNA coding gene were sequenced to examine the microbiome profile of COPD and healthy participants. Results: A total of 45 sputum samples were collected from 17 severe COPD, 12 moderate COPD cases, and 16 healthy volunteers. The bacterial alpha diversity (Shannon and Simpson’s index) significantly decreased in the moderate and severe COPD groups, compared to healthy samples. A significantly higher proportion of Firmicutes and Actinobacteria were present in moderate COPD, and Proteobacteria numbers were comparatively increased in severe COPD. In healthy samples, Bacteroidetes and Fusobacteria were more abundant in comparison to both the COPD groups. Among the most commonly detected 20 bacterial genera, Streptococcus was predominant among the COPD sputum samples, whereas Prevotella was the top genus in healthy controls. Linear discriminant analysis (LDA&gt;2) revealed that marker genera like Streptococcus and Rothia were abundant in moderate COPD. For severe COPD, the genera Pseudomonasand Leptotrichia were most prevalent, whereas Fusobacterium and Prevotella were dominant in the healthy group. Conclusions: Our findings suggest a significant dysbiosis of the respiratory microbiome in COPD patients. The decreased microbial diversity may influence the host immune response and provide microbiological biomarkers for the diagnosis and monitoring of COPD.

The altered sputum microbiome profile in patients with moderate and severe COPD exacerbations, compared to the healthy group in the Indian population.

Background: Microbial culture-independent sequencing techniques have advanced our understanding of host-microbiome interactions in health and disease. The purpose of this study was to explore the dysbiosis of airway microbiota in patients with moderate or severe chronic obstructive pulmonary disease (COPD) and compare them with healthy controls. Methods: The COPD patients were investigated for disease severity based on airflow limitations and divided into moderate (50%≤FEV1<80% predicted) and severe groups (FEV1<50% predicted). Spontaneous sputum samples were collected and, the V3-V4 regions of the 16S rRNA coding gene were sequenced to examine the microbiome profile of COPD and healthy participants. Results: A total of 45 sputum samples were collected from 17 severe COPD, 12 moderate COPD cases, and 16 healthy volunteers. The bacterial alpha diversity (Shannon and Simpson's index) significantly decreased in the moderate and severe COPD groups, compared to healthy samples. A significantly higher proportion of Firmicutes and Actinobacteria were present in moderate COPD, and Proteobacteria numbers were comparatively increased in severe COPD. In healthy samples, Bacteroidetes and Fusobacteria were more abundant in comparison to both the COPD groups. Among the most commonly detected 20 bacterial genera, Streptococcus was predominant among the COPD sputum samples, whereas Prevotella was the top genus in healthy controls. Linear discriminant analysis (LDA>2) revealed that marker genera like Streptococcus and Rothia were abundant in moderate COPD. For severe COPD, the genera Pseudomonasand Leptotrichia were most prevalent, whereas Fusobacterium and Prevotella were dominant in the healthy group. Conclusions: Our findings suggest a significant dysbiosis of the respiratory microbiome in COPD patients. The decreased microbial diversity may influence the host immune response and provide microbiological biomarkers for the diagnosis and monitoring of COPD.

Genomic markers of drug resistance in Mycobacterium tuberculosis populations with minority variants.

Minority variants of Mycobacterium tuberculosis harboring mutations conferring resistance can become dominant populations during tuberculosis (TB) treatment, leading to treatment failure. Our understanding of drug-resistant within-host subpopulations and the frequency of resistance-conferring mutations in minority variants remains limited. M. tuberculosis sequences recovered from liquid cultures of culture-confirmed TB cases notified between January 2017 and December 2021 in New South Wales, Australia were examined. Potential drug resistance-conferring minority variants were identified using LoFreq, and mixed populations of different M. tuberculosis strains (≥100 SNPs apart) were examined using QuantTB. A total of 1831 routinely sequenced M. tuberculosis strains were included in the analysis. Drug resistance-conferring minority variants were detected in 3.5% (65/1831) of sequenced cultures; 84.6% (55/65) had majority strains that were drug susceptible and 15.4% (10/65) had majority strains that were drug resistant. Minority variants with high-confidence drug resistance-conferring mutations were 1.5 times more common when the majority strains were drug resistant. Mixed M. tuberculosis strain populations were documented in 10.0% (183/1831) of specimens. Minority variants with high-confidence drug resistance-conferring mutations were more frequently detected in mixed M. tuberculosis strain populations (2.7%, 5/183) than in single strain populations (0.6%, 10/1648; P = 0.01). Drug-resistant minority variants require monitoring in settings that implement routine M. tuberculosis sequencing. The frequency with which drug-resistant minority variants are detected is likely influenced by pre-culture requirement. Culture-independent sequencing methods should provide a more accurate reflection of drug-resistant subpopulations.

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 μg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

The altered sputum microbiome profile in patients with moderate and severe COPD compared to the healthy group in the Indian population

Background: Microbial culture-independent sequencing techniques have advanced our understanding of host-microbiome interactions in health and disease. The purpose of this study was to explore the dysbiosis of airway microbiota in patients with moderate or severe chronic obstructive pulmonary disease (COPD) and compare them with healthy controls. Methods: The COPD patients were investigated for disease severity based on airflow limitations and divided into moderate (50%≤FEV1&lt;80% predicted) and severe groups (FEV1&lt;50% predicted). Spontaneous sputum samples were collected and, the V3-V4 regions of the 16S rRNA coding gene were sequenced to examine the microbiome profile of COPD and healthy participants. Results: A total of 45 sputum samples were collected from 17 severe COPD, 12 moderate COPD cases, and 16 healthy volunteers. The bacterial alpha diversity (Shannon and Simpson’s index) significantly decreased in the moderate and severe COPD groups, compared to healthy samples. A significantly higher proportion of Firmicutes and Actinobacteria were present in moderate COPD, and Proteobacteria numbers were comparatively increased in severe COPD. In healthy samples, Bacteroidetes and Fusobacteria were more abundant in comparison to both the COPD groups. Among the most commonly detected 20 bacterial genera, Streptococcus was predominant among the COPD sputum samples, whereas Prevotella was the top genus in healthy controls. Linear discriminant analysis (LDA&gt;2) revealed that marker genera like Streptococcus and Rothia were abundant in moderate COPD. For severe COPD, the genera Pseudomonas and Leptotrichia were most prevalent, whereas Fusobacterium and Prevotella were dominant in the healthy group. Conclusions: Our findings suggest a significant dysbiosis of the respiratory microbiome in COPD patients. The decreased microbial diversity may influence the host immune response and provide microbiological biomarkers for the diagnosis and monitoring of COPD.

Microbiome of Clothing Items Worn for a Single Day in a Non-Healthcare Setting

When worn, clothing acquires a microbiome of bacteria and fungi derived from the wearer’s skin and from the environment. The types of bacteria and fungi that may be recovered from clothing in healthcare settings have been well characterized, but less is known regarding the microbiome of clothing worn in non-healthcare settings and the possible roles that such clothing may play in microbial exchange. Culture-based methods and culture-independent genomic sequencing were used to enumerate and identify bacteria and fungi recovered from T-shirts, baby onesies, socks, and underwear worn for a single day after having been purchased new, washed, and dried. The highest bacterial loads were recovered from socks, underwear, and onesies (&gt;106 colony-forming units [cfu]/sample) and the highest fungal loads were obtained from socks and underwear (&gt;5 × 102 cfu/sample). The sequencing method identified opportunistic pathogens present in the samples, including members of genus Staphylococcus and Corynebacterium, as well as anaerobic members of the family Clostridiales. The opportunistic fungal pathogen Candida parapsilosis was identified in a high proportion of worn clothing samples. These results suggest that clothing may represent a pathogen reservoir and a vector for microbial exchange between household occupants or the community outside of the home.

Diversity and distribution of microbial communities on the surface of External Thermal Insulation Composite Systems (ETICS) facades in residential buildings

External Thermal Insulation Composite Systems (ETICS) are frequently used to enhance the energy efficiency of the built environment. However, stains of presumable biological nature are often detected shortly after application, causing cladding defacement and altering the building aesthetics. To address which microbiota could contribute to these biodeterioration related color/aesthetic anomalies, samples collected from stains detected on the surface of building facades with ETICS in three residential sites in Lisbon, Portugal, were analyzed through microbiological culture-dependent technique and culture-independent amplicon DNA high throughput sequencing taxonomic profiling. The obtained data provided a comprehensive description of microbial communities assigned to diverse taxa of the major microbial groups of heterotrophic bacteria, fungi, cyanobacteria, and microalgae (through DNA plastid detection) in the sampled stains. Based on that, we propose that new microorganisms could be added to the list of bio-susceptibility testing organisms in ETICS. Furthermore, microbiota diversity depended more on facade location and cardinal orientation than on ETICS material composition. Overall, this study reveals the unique microbial communities of color/aesthetic biodeterioration stains in ETICS facades, unlike those of other surfaces, and the associated environmental dynamics.

Whole-genome sequencing of Chlamydia psittaci from Australasian avian hosts: A genomics approach to a pathogen that still ruffles feathers.

Chlamydia psittaci is a globally distributed veterinary pathogen with zoonotic potential. Although C. psittaci infections have been reported in various hosts, isolation and culture of Chlamydia is challenging, hampering efforts to produce contemporary global C. psittaci genomes. This is particularly evident in the lack of avian C. psittaci genomes from Australia and New Zealand. In this study, we used culture-independent probe-based whole-genome sequencing to expand the global C. psittaci genome catalogue. Here, we provide new C. psittaci genomes from two pigeons, six psittacines, and novel hosts such as the Australian bustard (Ardeotis australis) and sooty shearwater (Ardenna grisea) from Australia and New Zealand. We also evaluated C. psittaci genetic diversity using multilocus sequence typing (MLST) and major outer membrane protein (ompA) genotyping on additional C. psittaci-positive samples from various captive avian hosts and field isolates from Australasia. We showed that the first C. psittaci genomes sequenced from New Zealand parrots and pigeons belong to the clonal sequence type (ST)24 and diverse 'pigeon-type' ST27 clade, respectively. Australian parrot-derived strains also clustered in the ST24 group, whereas the novel ST332 strain from the Australian bustard clustered in a genetically diverse clade of strains from a fulmar, parrot, and livestock. MLST and ompA genotyping revealed ST24/ompA genotype A in wild and captive parrots and a sooty shearwater, whilst 'pigeon-types' (ST27/35 and ompA genotypes B/E) were found in pigeons and other atypical hosts, such as captive parrots, a little blue penguin/Kororā (Eudyptula minor) and a zebra finch (Taeniopygia guttata castanotis) from Australia and New Zealand. This study provides new insights into the global phylogenomic diversity of C. psittaci and further demonstrates the multi-host generalist capacity of this pathogen.

Metatranscriptome Analysis of Nasopharyngeal Swabs across the Varying Severity of COVID-19 Disease Demonstrated Unprecedented Species Diversity.

The recent global emergence of the SARS-CoV-2 pandemic has accelerated research in several areas of science whose valuable outputs and findings can help to address future health challenges in the event of emerging infectious agents. We conducted a comprehensive shotgun analysis targeting multiple aspects to compare differences in bacterial spectrum and viral presence through culture-independent RNA sequencing. We conducted a comparative analysis of the microbiome between healthy individuals and those with varying degrees of COVID-19 severity, including a total of 151 participants. Our findings revealed a noteworthy increase in microbial species diversity among patients with COVID-19, irrespective of disease severity. Specifically, our analysis revealed a significant difference in the abundance of bacterial phyla between healthy individuals and those infected with COVID-19. We found that Actinobacteria, among other bacterial phyla, showed a notably higher abundance in healthy individuals compared to infected individuals. Conversely, Bacteroides showed a lower abundance in the latter group. Infected people, regardless of severity and symptoms, have the same proportional representation of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteriales. In addition to SARS-CoV-2 and numerous phage groups, we identified sequences of clinically significant viruses such as Human Herpes Virus 1, Human Mastadenovirus D, and Rhinovirus A in several samples. Analyses were performed retrospectively, therefore, in the case of SARS-CoV-2 various WHO variants such as Alpha (B.1.1.7), Delta (B.1.617.2), Omicron (B.1.1.529), and 20C strains are represented. Additionally, the presence of specific virus strains has a certain effect on the distribution of individual microbial taxa.