In bacterial genome and metagenome sequencing, Illumina sequencers are most frequently used due to their high throughput capacity, and multiple library preparation kits have been developed for Illumina platforms. Here, we systematically analysed and compared the sequencing bias generated by currently available library preparation kits for Illumina sequencing. Our analyses revealed that a strong sequencing bias is introduced in low-GC regions by the Nextera XT kit. The level of bias introduced is dependent on the level of GC content; stronger bias is generated as the GC content decreases. Other analysed kits did not introduce this strong sequencing bias. The GC content-associated sequencing bias introduced by Nextera XT was more remarkable in metagenome sequencing of a mock bacterial community and seriously affected estimation of the relative abundance of low-GC species. The results of our analyses highlight the importance of selecting proper library preparation kits according to the purposes and targets of sequencing, particularly in metagenome sequencing, where a wide range of microbial species with various degrees of GC content is present. Our data also indicate that special attention should be paid to which library preparation kit was used when analysing and interpreting publicly available metagenomic data.
Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.
Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the ‘ S. marcescens complex’), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila , S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum β-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.
Bloodstream infection (BSI) is a well-known cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. Here, we conducted a retrospective study to assess the morbidity, etiology, risk factors, and outcomes of BSI in the postengraftment period (PE-BSI) after allo-HSCT. Forty-three of 316 patients (13.6%) developed 57 PE-BSI episodes, in which 62 pathogens were isolated: Gram-positive bacteria, gram-negative bacteria, and fungi, respectively, accounted for 54.8%, 35.5%, and 9.7% of the isolates. Multivariate analysis revealed methylprednisolone use for graft-versus-host disease (GVHD) prophylaxis (odds ratio [OR], 6.49; 95% confidence interval [CI], 1.49 to 28.2; P = .013) and acute gastrointestinal GVHD (GI-GVHD) (OR, 8.82; 95% CI, 3.99 to 19.5; P < .0001) as risk factors for developing PE-BSI. This finding suggested that GI-GVHD increases the risk of bacterial translocation and subsequent septicemia. Moreover, among patients with GI-GVHD, insufficient response to corticosteroids, presumably related to an intestinal dysbiosis, significantly correlated with this complication. Patients with PE-BSI presented worse outcome compared with those without (3-year overall survival, 47.0% versus 18.6%; P < .001). Close microbiologic monitoring for BSIs and minimizing intestinal dysbiosis may be crucial to break the vicious cycle between GI-GVHD and bacteremia and to improve transplant outcomes especially in patients who require additional immunosuppressants.
Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi -like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola , little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola -like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi , H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I–IV) in H. cinaedi/canicola/‘magderbugensis’ (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.
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