The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

https://www.clinicalmicrobiologyandinfection.com/article/S1198-743X(14)61632-3/pdf

Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance

Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

/pdf/detection-of-2019-novel-coronavirus-2019-ncov-by-real-time-5an3azdech.pdf

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.

Objectives
Identification of antimicrobial resistance genes is important for understanding the underlying mechanisms and the epidemiology of antimicrobial resistance. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available in routine diagnostic laboratories and is anticipated to substitute traditional methods for resistance gene identification. Thus, the current challenge is to extract the relevant information from the large amount of generated data.

/pdf/identification-of-acquired-antimicrobial-resistance-genes-3jqc4799a3.pdf

Identification of acquired antimicrobial resistance genes

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Two isolates of a new Salmonella serovar, Salmonella Lamphun were discovered from animal feeds in Thailand, in 2003, which belongs to group C, with antigenic formula 6,8:y: 1,2. Both isolates were susceptible to all antimicrobials tested. The pulsed field gel electrophoresis pattern of both isolates comprises 11 DNA fractions sized 48,65,77, 105, 110, 170, 244, 330, 337, 453 and 1,135 kbp. Up to April 2005, no human or animal infection by this new Salmonella serovar was reported.

Salmonella lamphun: first isolation of a new Salmonella serovar in Thailand.

/pdf/reply-to-catry-and-threlfall-4yvabnuck3.pdf

Reply to Catry and Threlfall

The rapid spread of antimicrobial resistance (AMR) is a threat to global health, and the nature of co-occurring antimicrobial resistance genes (ARGs) may cause collateral AMR effects once antimicrobial agents are used. Therefore, it is essential to identify which pairs of ARGs co-occur. Given the wealth of NGS data available in public repositories, we have investigated the correlation between ARG abundances in a collection of 214,095 metagenomic datasets. Using more than 6.76·108 read fragments aligned to ARGs to infer pairwise correlation coefficients, we found that more ARGs correlated with each other in human and animal sampling origins than in soil and water environments. Furthermore, we showed that the correlations serve as risk profiles of resistance co-occurring to critically important antimicrobials. Using these profiles, we found several key ARGs indirectly but strongly selecting for ARGs of critical importance, such as tetracycline ARGs correlating with most forms of resistances. In conclusion, this study highlights the important ARG players indirectly involved in shaping the resistomes of various environments that can serve as monitoring targets in AMR surveillance programs.

Utilizing co-abundances of antimicrobial resistance genes to identify potential co-selection in the resistome

SamplesDirectly from Clinical Detection and Characterization of Rapid Whole-Genome Sequencing for

Plasmids are infectious double stranded DNA molecules that are found within bacteria. Horizontal gene transfer promotes successful spread of different types of plasmids within or among bacteria species, making their detection an important task for guiding clinical treatment. We used whole genome sequenced data to determine the prevalence of plasmid replicons in clinical bacterial isolates, the presence of resistance and virulence genes in plasmids, and the relationship between resistance and virulence genes within each plasmid. All bacterial sequences were de novo assembled using Unicycler before extraction of plasmids. Assembly graphs were submitted to Gplas+plasflow for plasmid prediction. The predicted plasmid components were validated using PlasmidFinder. A total of 159 (56.2%) out of 283 bacterial isolates were found to carry plasmids, with E. coli, K. pneumoniae and S. aureusbeing the most prevalent plasmid carriers. A total of 27 (87.1%) combined plasmids were found to carry both resistance and virulence genes compared to 4 (12.9%) single plasmids. No statistically significant correlation was found between the number of antimicrobial resistance and virulence genes in plasmids (r =-0.25, p > 0.05). Our findings show a relatively high proportion of plasmid-carrying isolates suggesting selection pressure due to antibiotic use in the hospital. Co-occurrence of antibiotic resistance and virulence genes in clinical isolates is a public health relevant problem needing attention.

Frank Møller Aarestrup

Papers

Salmonella lamphun: first isolation of a new Salmonella serovar in Thailand.

Reply to Catry and Threlfall

Utilizing co-abundances of antimicrobial resistance genes to identify potential co-selection in the resistome

SamplesDirectly from Clinical Detection and Characterization of Rapid Whole-Genome Sequencing for

Plasmid characterization in bacterial isolates of public health relevance in a tertiary healthcare facility in Kilimanjaro, Tanzania