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

Host range of theermF rRNA methylase gene in human and animal bacteria

Branhamella (Neisseria) catarrhalis wasidentified asaprobable respiratory tract pathogen insevenpatients, fourwithpneumonia andthree withbronchitis. FiveoftheB.catarrhalis isolates produced jf-lactamase. Production ofj8-lactamasecorrelated withpenicillin resistance bythestandardized diskdiffusion methodandalso withhighminimal inhibitory concentrations ofpenicillin forthe twostrains whichweretested. Inthepastyear, wehaveobserved twocharacteristics ofBranhamella (Neisseria) catarrhalis whichhavenotbeenwidely recognized. Thefirst istheapparent capacity toproduce acuterespiratory infections inpatients with chronic lungdisease, andthesecond istheabilitytoproduce ,8-lactamase. Insevenpatientsailofwhomhadunderlying pulmonary pathology-bronchitis orpneumonia wasdocumented byclinical presentation, roentgenographic findings, andsputumexamination. We usedone methodforplasmid extraction andfoundno plasmid deoxyribonucleic acidinthej3-lactamase-positive or-negative strains ofB.catarrhalis.

Branhamella (Neisseria) catarrhalis-a LowerRespiratory

Macrolides, lincosamides, streptogramins, ketolides, and oxazolidinones (MLSKO), though chemically distinct, share overlapping binding sites on the 50S subunit of the ribosome. Bacteria acquire resistance to one or more of these antibiotics by the acquisition of a gene(s) or with certain mutations. The ketolides are macrolide derivatives that have recently become available for human therapy. The lincosamides are a distinct group of antibiotics that are structurally unrelated to macrolides. The streptogramin B component shares an overlapping ribosomal binding site, on the 50S ribosomal subunit, with the macrolides and lincosamides and is impacted by erm methylation at A2058 (Escherichia coli numbering). The MLSKO antibiotics inhibit protein synthesis by binding to the 50S ribosomal subunit and blocking peptide bond formation and/or translation. The binding of 14-, 15-, and 16-membered macrolides, clindamycin, and two ketolides has been described in this chapter. The Mycobacterium erm genes are regulated at the translation step. These genes confer resistance to macrolides and lincosamides but not to streptogramin B. Mutations associated with resistance to macrolides, ketolides, streptogramins, or oxazolidinones have been described in the chapter. However, with the addition of the newer macrolides, streptogramins, ketolides, and oxazolidinones, use of this group of antibiotics has increased, and today they are important in the treatment of a variety of infectious diseases in both community and hospital environments. Ketolide resistance in streptococci and oxazolidinone resistance in gram-positive enterococci or staphylococci are surfacing with increased therapeutic use.

Macrolide, Lincosamide, Streptogramin, Ketolide, and Oxazolidinone Resistance

Twenty-two susceptible strains of Haemophilus influenzae were examined for mutation to rifampin resistance (minimal inhibitory concentration, greater than or equal to 10 micrograms/ml). All strains had detectable apparent mutation frequencies with a median minimal inhibitory concentration 2,000-fold greater than that of their wild-type parents. Of the type b strain mutants, 80% (8 of 10) expressed high-level resistance (minimal inhibitory concentration, 750 micrograms/ml) that was 75-fold greater than readily achievable serum concentrations.

Mutation frequency of Haemophilus influenzae to rifampin resistance.

E. coli was isolated from the Salish Sea (Puget Sound) ecosystem, including samples of marine and fresh water, and wildlife dependent on this environment. E. coli isolates were assessed for phenotypic and genotypic resistance to antibiotics. A total of 305 E. coli isolates was characterized from samples collected from: marine water obtained in four quadrants of the Salish Sea; select locations near beaches; fresh water from streams near marine beaches; and fecal samples from harbor porpoises (Phocoena phocoena), harbor seals (Phoca vitulina), river otters (Lontra canadensis), and English sole (Parophrys vetulus). Isolates were evaluated using antimicrobial susceptibility typing, whole-genome sequencing, fumC, and multilocus sequence typing. Resistance and virulence genes were identified from sequence data. Of the 305 isolates from Salish Sea samples, 20 (6.6%) of the E. coli were intermediate, and 31 (10.2%) were resistant to ≥1 class of antibiotics, with 26.9% of nonsusceptible (resistant and intermediate resistant) E. coli isolates from marine mammals and 70% from river otters. The proportion of nonsusceptible isolates from animals was significantly higher than samples taken from marine water (p < 0.0001). A total of 196 unique STs was identified including 37 extraintestinal pathogenic E. coli (ExPEC)-associated STs [ST10, ST38, ST58, ST69, ST73, ST117, ST131, and ST405]. The study suggests that animals may be potential sentinels for antibiotic-resistant and ExPEC E. coli in the Salish Sea ecosystem.

Marilyn C. Roberts

Papers

Host range of theermF rRNA methylase gene in human and animal bacteria

Branhamella (Neisseria) catarrhalis-a LowerRespiratory

Macrolide, Lincosamide, Streptogramin, Ketolide, and Oxazolidinone Resistance

Mutation frequency of Haemophilus influenzae to rifampin resistance.

Surveillance for Antibiotic-Resistant E. coli in the Salish Sea Ecosystem.