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)

Antibiotics have always been considered one of the wonder discoveries of the 20th century. This is true, but the real wonder is the rise of antibiotic resistance in hospitals, communities, and the environment concomitant with their use. The extraordinary genetic capacities of microbes have benefitted from man's overuse of antibiotics to exploit every source of resistance genes and every means of horizontal gene transmission to develop multiple mechanisms of resistance for each and every antibiotic introduced into practice clinically, agriculturally, or otherwise. This review presents the salient aspects of antibiotic resistance development over the past half-century, with the oft-restated conclusion that it is time to act. To achieve complete restitution of therapeutic applications of antibiotics, there is a need for more information on the role of environmental microbiomes in the rise of antibiotic resistance. In particular, creative approaches to the discovery of novel antibiotics and their expedited and controlled introduction to therapy are obligatory.

Origins and Evolution of Antibiotic Resistance

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

The optimism of the early period of antimicrobial discovery has been tempered by the emergence of bacterial strains with resistance to these therapeutics. Today, clinically important bacteria are characterized not only by single drug resistance but also by multiple antibiotic resistance--the legacy of past decades of antimicrobial use and misuse. Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs.

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Antibacterial resistance worldwide: causes, challenges and responses.

Chronic obstructive pulmonary disease (COPD) is a common problem in the elderly. The disease is characterised by intermittent worsening of symptoms and these episodes are called acute exacerbations. The best estimate, based on several lines of evidence, is that approximately half of all exacerbations are caused by bacteria. These lines of evidence include studies of lower respiratory tract bacteriology during exacerbations, correlation of airways’ inflammation with results of sputum cultures during exacerbations, analysis of immune responses to bacterial pathogens, and the observation in randomised, prospective, placebo-controlled trials that antibacterial therapy is of benefit. The most important bacterial causes of exacerbations of COPD are nontypeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Chlamydia pneumoniae.

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Chronic obstructive pulmonary disease

We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the beta-lactamase bla(TEM-1) gene, and a 16S ribosomal DNA gene (Escherichia coli) were generated from known controls by PCR. The resulting products (ca. 550 bp) were applied as spots onto epoxy-silane-derivatized, Teflon-masked slides by using a robotic spotter. DNA was extracted from test strains, biotinylated, hybridized overnight to individual microarrays at 65 degrees C, and detected with Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a detection threshold of 3x the standard deviation, we correctly identified tet genes carried by 39 test strains. Nine additional strains were not known to harbor any genes represented on the microarray, and these strains were negative for all 17 tet probes as expected. We verified that R741a, which was originally thought to carry a novel tet gene, tet(I), actually harbored a tet(G) gene. Microarray technology has the potential for screening a large number of different antibiotic resistance genes by the relatively low-cost methods outlined in this paper.

Identifying Antimicrobial Resistance Genes with DNA Microarrays

Xylitol is promoted in caries-preventive strategies, yet its effective dose range is unclear This study determined the dose-response of mutans streptococci in plaque and unstimulated saliva to xylitol gum Participants (n = 132) were randomized: controls (G1) (sorbitol/maltitol), or combinations giving xylitol 344 g/day (G2), 688 g/day (G3), or 1032 g/day (G4) Groups chewed 3 pellets/4 times/d Samples were taken at baseline, 5 wks, and 6 mos, and were cultured on modified Mitis Salivarius agar for mutans streptococci and on blood agar for total culturable flora At 5 wks, mutans streptococci levels in plaque were 10x lower than baseline in G3 and G4 (P = 0007/0003) There were no differences in saliva At 6 mos, mutans streptococci in plaque for G3 and G4 remained 10x lower than baseline (P = 0007/004) Saliva for G3 and G4 was lower than baseline by 8 to 9x (P = 0011/0038) Xylitol at 644 g/day and 1032 g/day reduces mutans streptococci in plaque at 5 wks, and in plaque and unstimulated sal

Mutans Streptococci Dose Response to Xylitol Chewing Gum

Tetracycline resistance determinants are widespread and distinguishable genetically and biochemically. The nomenclature for this increasing number of determinants has been varied and inconsistent. This communication suggests ways of naming these determinants and their genes and gene products consistent with current genetic terminology.

Nomenclature for tetracycline resistance determinants.

Clinical isolates of Mycoplasma hominis resistant to high levels of tetracycline contained DNA sequences homologous to the streptococcal tetracycline determinant, tetM. In contrast, none of the susceptible M. hominis isolates tested carried this determinant. This is the first description of tetM in an unrelated genus and suggests the spread of tetM from Streptococcus spp. to Mycoplasma spp.

Tetracycline-resistant Mycoplasma hominis strains contain streptococcal tetM sequences.

The molecular nature of two distinct gonococcal R plasmids, 44 X 10(6) and 32 X 10(6) daltons, encoding beta-lactamase activity were examined Both plasmids contained about 40% of the transposable ampicillin resistance sequence Tn2 Deoxyribonucleic acid-deoxyribonucleic acid polynucleotide sequence studies have shown that the two gonococcal plasmids share about 70% of their sequences and are closely related to RSF0885, a 41 X 10(6)-dalton plasmid found in a beta-lactamase-producing strain of Haemophilus influenzae All three of these R plasmids possess a guanine-plus-cytosine content of 040 to 041 mol fraction and are present as multicopy gene pools in their bacterial hosts

Marilyn C. Roberts

Papers

Identifying Antimicrobial Resistance Genes with DNA Microarrays

Mutans Streptococci Dose Response to Xylitol Chewing Gum

Nomenclature for tetracycline resistance determinants.

Tetracycline-resistant Mycoplasma hominis strains contain streptococcal tetM sequences.

Molecular characterization of two beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae.