Comparison of DNA extraction methods for 16S rRNA gene sequencing in the analysis of the human gut microbiome.

The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to compare DNA extraction protocols for 16S sequencing. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) upstream DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and 16S sequenced. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates. SPD improved overall efficiency of three of the four tested protocols compared with their commercial version, in terms of DNA extraction yield, sample alpha-diversity, and recovery of Gram-positive bacteria. The best overall performance was obtained for the S-DQ protocol, SPD combined with the DNeasy PowerLyser PowerSoil protocol from QIAGEN. Based on this evaluation, we strongly believe that the use of such stool preprocessing device improves both the standardization and the quality of the DNA extraction in the human gut microbiome studies.

Species-specific recruitment of transcription factors dictates toxin expression.

Tight and coordinate regulation of virulence determinants is essential for bacterial biology and involves dynamic shaping of transcriptional regulatory networks during evolution. The horizontally transferred two-partner secretion system ExlB-ExlA is instrumental in the virulence of different Pseudomonas species, ranging from soil- and plant-dwelling biocontrol agents to the major human pathogen Pseudomonas aeruginosa. Here, we identify a Cro/CI-like repressor, named ErfA, which together with Vfr, a CRP-like activator, controls exlBA expression in P. aeruginosa. The characterization of ErfA regulon across P. aeruginosa subfamilies revealed a second conserved target, the ergAB operon, with functions unrelated to virulence. To gain insights into this functional dichotomy, we defined the pan-regulon of ErfA in several Pseudomonas species and found ergAB as the sole conserved target of ErfA. The analysis of 446 exlBA promoter sequences from all exlBA+ genomes revealed a wide variety of regulatory sequences, as ErfA- and Vfr-binding sites were found to have evolved specifically in P. aeruginosa and nearly each species carries different regulatory sequences for this operon. We propose that the emergence of different regulatory cis-elements in the promoters of horizontally transferred genes is an example of plasticity of regulatory networks evolving to provide an adapted response in each individual niche.

Insertion sequences drive the emergence of a highly adapted human pathogen.

Pseudomonas aeruginosa is a highly adaptive opportunistic pathogen that can have serious health consequences in patients with lung disorders. Taxonomic outliers of P. aeruginosa of environmental origin have recently emerged as infectious for humans. Here, we present the first genome-wide analysis of an isolate that caused fatal haemorrhagic pneumonia. In two clones, CLJ1 and CLJ3, sequentially recovered from a patient with chronic pulmonary disease, insertion of a mobile genetic element into the P. aeruginosa chromosome affected major virulence-associated phenotypes and led to increased resistance to the antibiotics used to combat the infection. Comparative genome, proteome and transcriptome analyses revealed that this ISL3-family insertion sequence disrupted the genes for flagellar components, type IV pili, O-specific antigens, translesion polymerase and enzymes producing hydrogen cyanide. Seven-fold more insertions were detected in the later isolate, CLJ3, than in CLJ1, some of which modified strain susceptibility to antibiotics by disrupting the genes for the outer-membrane porin OprD and the regulator of ß-lactamase expression AmpD. In the Galleria mellonella larvae model, the two strains displayed different levels of virulence, with CLJ1 being highly pathogenic. This study revealed insertion sequences to be major players in enhancing the pathogenic potential of a P. aeruginosa taxonomic outlier by modulating both its virulence and its resistance to antimicrobials, and explains how this bacterium adapts from the environment to a human host.

Genome Sequence of Rickettsia hoogstraalii, a Geographically Widely Distributed Tick-Associated Bacterium.

Rickettsia hoogstraalii is a tick-associated member of the spotted fever group rickettsiae that is geographically widely distributed. We report here the draft genome of R. hoogstraalii strain Croatica(T) (=DSM 22243 = UTMB 00003), which was isolated from Haemaphysalis sulcata ticks collected in Croatia.

Genome Sequence of Rickettsia tamurae, a Recently Detected Human Pathogen in Japan.

Rickettsia tamurae is a member of the spotted fever group rickettsiae, which was reported in 2011 to cause human infections in Japan. We report the draft genome sequence of R. tamurae strain AT-1(T), isolated from Amblyomma testudinarium ticks.

Draft Genome Sequence of Rickettsia aeschlimannii, Associated with Hyalomma marginatum Ticks.

Rickettsia aeschlimannii is a tick-associated human pathogen. We report here the draft genome of R. aeschlimannii strain MC16, isolated from Hyalomma marginatum marginatum ticks collected in Morocco.

A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species.

Currently, bacterial taxonomy relies on a polyphasic approach based on the combination of phenotypic and genotypic characteristics. However, the current situation is paradoxical in that the genetic criteria that are used, including DNA-DNA hybridization, 16S rRNA gene sequence nucleotide similarity and phylogeny, and DNA G+C content, have significant limitations, but genome sequences that contain the whole genetic information of bacterial strains are not used for taxonomic purposes, despite the decreasing costs of sequencing and the increasing number of available genomes. Recently, we diversified bacterial culture conditions with the aim of isolating uncultivated bacteria. To classify the putative novel species that we cultivated, we used a polyphasic strategy that included phenotypic as well as genomic criteria (genome characteristics as well as genomic sequence similarity). Herein, we review the pros and cons of genome sequencing for taxonomy and propose that the incorporation of genome sequences in taxonomic studies has the advantage of using reliable and reproducible data. This strategy, which we name taxono-genomics, may contribute to the taxonomic classification of bacteria.

Genome Sequence of Rickettsia gravesii, Isolated from Western Australian Ticks.

Rickettsia gravesii is a new Rickettsia species closely related to the human pathogen Rickettsia massiliae. Here, we describe the genome sequence of R. gravesii strain BWI-1, isolated from Amblyomma triguttatum triguttatum ticks collected from humans on Barrow Island, Western Australia.

Genome sequence of Rickettsia conorii subsp. caspia, the agent of Astrakhan fever.

Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.

Genome sequence of Rickettsia conorii subsp. israelensis, the agent of Israeli spotted fever.

Rickettsia conorii subsp. israelensis is the agent of Israeli spotted fever. The present study reports the draft genome of Rickettsia conorii subsp. israelensis strain ISTT CDC1, isolated from a Rhipicephalus sanguineus tick collected in Israel.

Genome sequence of Rickettsia conorii subsp. indica, the agent of Indian tick typhus.

Rickettsia conorii subsp. indica is the agent of Indian tick typhus. The present study reports the draft genome of Rickettsia conorii subsp. indica strain ITTR (ATCC VR-597).

Sequence and annotation of Rickettsia sibirica sibirica genome.

Rickettsia sibirica sibirica is the causative agent of Siberian or North Asian tick typhus, a tick-borne rickettsiosis known to exist in Siberia and eastern China. Here we present the draft genome of Rickettsia sibirica sibirica strain BJ-90 isolated from Dermacentor sinicus ticks collected in Beijing, China.

Genome sequence of "Rickettsia sibirica subsp. mongolitimonae".

"Rickettsia sibirica subsp. mongolitimonae" is the agent of lymphangitis-associated rickettsiosis, an emerging human disease that has been diagnosed in Europe and Africa. The present study reports the draft genome of Rickettsia sibirica subsp. mongolitimonae strain HA-91.