Accurate and robust inference of microbial growth dynamics from metagenomic sequencing reveals personalized growth rates

Patterns of sequencing coverage along a bacterial genome---summarized by a peak-to-trough ratio (PTR)---have been shown to accurately reflect microbial growth rates, revealing a new facet of microbial dynamics and host-microbe interactions. Here, we introduce CoPTR (Compute PTR): a tool for computing PTRs from complete reference genomes and assemblies. Using simulations and data from growth experiments in simple and complex communities, we show that CoPTR is more accurate than the current state-of-the-art, while also providing more PTR estimates overall. We further develop theory formalizing a biological interpretation for PTRs. Using a reference database of 2935 species, we applied CoPTR to a case-control study of 1304 metagenomic samples from 106 individuals with inflammatory bowel disease. We show that growth rates are personalized, are only loosely correlated with relative abundances, and are associated with disease status. We conclude by demonstrating how PTRs can be combined with relative abundances and metabolomics to investigate their effect on the microbiome.

Genome Analysis of the Enterococcus faecium Entfac.YE Prophage

Background: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. Methods: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. Results: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. Conclusion: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.

Forensic Human Identification for Cutaneous Microbiome, a Brief Review

Forensic Science compounds many study areas in context of solving crimes, one of which is the forensic microbiology. Combined with genomic approaches, microbiology has shown strong performance in studies regarding the relationship between microorganisms present on human skin and environment. The Human Microbiome Project (HMP) has contributed significantly to characterization of microbial complexity and their connection to human being. The purpose of this work consists of a historical overview of scientific articles, demonstrating the growth and possibility of using skin microbiome in forensic identification. Studies about use of cutaneous microbiome in human identification, as well its forensic approaches, were looked into for writing of this review. Comparisons among cutaneous microbial communities and manipulated objects have been tested using 16S rRNA, as well as a thorough sequencing of the bacterial genome. From use of ecological measures of distance to genetic markers with nucleotide variants and predictive algorithms, research has shown promising results for advances in field of forensic identification. The development of metagenomic microbial panel markers, named hidSkinPlax for targeted sequencing has been designed and tested with great results. Research results show satisfactory potential in human identification by cutaneous microbiome and the possibility for contributive use in elucidating crimes.

Prediction of prophages and their host ranges in pathogenic and commensal Neisseria species

The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one other, primarily via transformation and homologous recombination, and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from other prophages and MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE: Drug-resistant N. gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus, and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) have been identified and characterized in Neisseria species. In this study, we identified a large number of candidate phages integrated within the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.

Comparative Analysis and Data Provenance for 1,113 Bacterial Genome Assemblies

The quality and traceability of microbial genomics data in public databases is deteriorating as they rapidly expand and struggle to cope with data curation challenges. While the availability of public genomic data has become essential for modern life sciences research, the curation of the data is a growing area of concern that has significant real-world impacts on public health epidemiology, drug discovery, and environmental biosurveillance research. While public microbial genome databases such as NCBI's RefSeq database leverage the scalability of crowd sourcing for growth, they do not require data provenance to the original biological source materials or accurate descriptions of how the data was produced. Here, we describe the de novo assembly of 1,113 bacterial genome references produced from authenticated materials sourced from the American Type Culture Collection (ATCC), each with full data provenance. Over 98% of these ATCC Standard Reference Genomes (ASRGs) are superior to assemblies for comparable strains found in NCBI's RefSeq database. Comparative genomics analysis revealed significant issues in RefSeq bacterial genome assemblies related to genome completeness, mutations, structural differences, metadata errors, and gaps in traceability to the original biological source materials. For example, nearly half of RefSeq assemblies lack details on sample source information, sequencing technology, or bioinformatics methods. We suggest there is an intrinsic connection between the quality of genomic metadata, the traceability of the data, and the methods used to produce them with the quality of the resulting genome assemblies themselves. Our results highlight common problems with "reference genomes" and underscore the importance of data provenance for precision science and reproducibility. These gaps in metadata accuracy and data provenance represent an "elephant in the room" for microbial genomics research, but addressing these issues would require raising the level of accountability for data depositors and our own expectations of data quality.

Pseudomarimonas arenosa gen. nov., sp. nov. isolated from marine sand

A novel Gram-negative, aerobic, non-motile, rod-shaped, bacterial strain (CAU 1598T) was isolated from marine sand. Strain CAU 1598T grew well at 30 °C, pH 6.5–7.0 and with 3 % NaCl (w/v). Phylogeny results based on 16S rRNA gene sequencing indicated that the identified strain had the highest similarity (94.3%) to Pseudoxanthomonas putridarboris , indicating that strain CAU 1598T belongs to the family Xanthomonadaceae . Further, the fatty acid profile of the strain was primarily composed of C16:0, iso-C15 : 0, iso-C16 : 0, summed feature 3 (consisting of C16 : 1 ω7c/iso-C15 : 0 2-OH) and summed feature 9 (consisting of iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), with ubiquinone-8 as the major isoprenoid quinone. The polar lipid profile included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphoglycolipid, an unidentified aminolipid and an unidentified lipid. The G+C content of the bacterial genome was 62.6 mol% and its 5.4 Mb length encompassed 144 contigs and 4236 protein-coding genes. These phenotypic, chemotaxonomic and phylogenetic data indicate that CAU 1598T belongs to a new genus and species, for which the name Pseudomarimonas arenosa gen. nov., sp. nov. is proposed. The type strain is CAU 1598T (=KCTC 82406T=MCCC 1K05673T).

Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System

Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S.enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S.enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S.enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S.enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S.enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonellaenterica.

Characterization of Staphylococcus pseudintermedius Isolates from Skin Infections of Dogs

Background: Staphylococcus pseudintermedius is a part of the canine skin microflora and an opportunistic pathogen. It plays a central role in canine pyoderma, otitis and surgical wound infections. These conditions correlate with virulence genes distributed in the bacterial genome. These genes determine strain variability on typing, in turn aiding epidemiological surveillance. The aim of this study was to isolate, identify and characterize Staphylococcus pseudintermedius (SP) and Methicillin resistant Staphylococcus pseudintermedius (MRSP) from dogs with skin infections in Chennai, India. Methods: SP and MRSP positive isolates were identified by multiplex PCR for nuc and mecA genes respectively. Characterization of the isolates for virulence genes responsible for biofilm formation (icaA, icaD), cell wall adherence (SpsO, SpsK, SpsP, SpsQ, SpsF), toxins (ExpA, ExpB, SIET, Sel, Se-int, LukS, LukF) and gene regulation (Agr, SarA) was performed. Result: Out of 275 samples, 120 SP and 8 MRSP positive isolates were identified. Only one isolate could be typed as SCCmec Type V whereas other MRSP isolates were non typeable. Agr typing of MRSP isolates revealed type II in 7 isolates and type III in one isolate. Our study revealed that there was no significant difference in the detection of virulence genes between MSSP and MRSP.