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

Tetracyclines are antimicrobial agents that interact with bacterial ribosomes and block protein synthesis. They have activity against a wide range of gram positive, gram-negative, anaerobic and aerobic bacteria, cell-wall free myco plasmas, chlamydiae, mycobacteria, rickettsia, Helicobacter, Listeria, and protozoan parasites, such as Entamoeba histolytica, Gzardia lamblia, and Plas modiumfalciparum 1-6. Tetracyclnes have been extenstvely used in humans for therapy of bacterial respiratory and urogenital tract diseases, as well as perrodontal, Lyme, and rtckettsial diseases. They are also used for therapy in animals and as growth promoters for animal food produciton (1), Tetracyclines were the first major group of antibrotrcs to which the term "broad-spectrum" was applied. Because of this spectrum of actrvity, their relative safety and low cost, tetracyclines have been widely used throughout the world, and are second after the penicillins in world consumption (7).

Resistance to tetracyclines, macrolides, trimethoprim and sulfonamides.

The majority of people in developing nations rely on untreated or minimally treated surface and shallow groundwater sources which are prone to faecal contamination. This study evaluated the utility of EC 3M Petrifilm and sanitary inspection forms (SIFs) as tools to assess 47 water sources and identify hazards of contamination in two rural Ugandan villages (90% were surface sources). Water samples were cultured on EC 3M Petrifilm, which are intended for the enumeration of E. coli and total coliforms following 24 h incubation at 37°C. Isolated bacteria were cultured on MacConkey agar and identified using standard biochemical tests, while selected isolates were verified by sequencing 16S rRNA genes. From 105 Petrifilms, 110 presumptive E. coli were isolated and identified to genus level. However, only 33 presumptive E. coli isolates from 14 water sources (representing 27 distinct strains as determined by PFGE) were confirmed E. coli. The other presumptive E. coli isolates were identified as Citrobacter, Enterobacter, Proteus, Salmonella and Yersinia species. SIFs used an adapted survey designed for urban water sources of Uganda. The form yielded an SIF score based on binary data and characterized potential sources of contamination. SIF scores alone offered little information to distinguish between contamination levels of surface water sources, but the information collected in the surveys could be used to identify ways to improve sources. The results of this study suggest that the use of sanitary surveys may assist in identifying potential pollution sources that may be targeted to protect water sources. Bacterial monitoring using EC 3M Petrifilms may be effective for the screening of relative levels of contamination of source waters, including surface sources.

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Utility of EC 3M Petrifilm and sanitary surveys for source water assessment in Nyabushozi County, south-western Uganda

All the macrolides, lincosamides, streptogramins, ketolides, and oxazolidinones (MLSKO) antibiotics inhibit protein synthesis by binding to the 50S bacterial ribosomal subunit, close to the peptidyltransferase center. The last group of acquired genes, and the focus of this chapter, are those that encode an adenine-N6-methyltransferase (rRNA methylase). Currently, 32 different genes coding for enzymes that modify the 23S rRNA have been described in the chapter. This modification blocks the binding of the MLSK group of antibiotics and allows the bacterial ribosomes to continue to produce protein in the presence of macrolides, lincosamides, and streptogramin B. Mobile elements often carry multiple different genes which confer antibiotic resistance to a variety of different antibiotic classes. Association of the erm genes with mobile elements (plasmids, conjugative transposons, and transposons) provides the potential to move from one species to another, one genus to another, and one ecosystem to another, between bacterial hosts, and from food to man and the environment. If DNA-DNA hybridization is used, confirmation by a PCR assay is advisable. One can speculate that as the newer macrolide derivatives became available in the 1980s this influenced the increase in carriage of macrolide resistance genes. It can be predicted that new innate genes may be found, which when mutated could provide increased levels of resistance to all the MSLKO antibiotics. It is even possible that new mechanisms of MLS resistance will be described in the coming years.

rRNA Methylases and Resistance to Macrolide, Lincosamide, Streptogramin, Ketolide, and Oxazolidinone (MLSKO) Antibiotics

(2006). Tetracycline Resistant Plasmids from Uropathogenic Escherichia coli from Southwestern Nigeria. Journal of Chemotherapy: Vol. 18, No. 1, pp. 112-114.

Tetracycline Resistant Plasmids from Uropathogenic Escherichia coli from Southwestern Nigeria

Antibiotic use has steadily increased since these agents were introduced in the 1940s. Millions of metric tons of antibiotics have been produced and used worldwide, which has led to contamination of both natural and man-made environments. Antibiotics are used on domestic animals, aquacultured fish, crops, and man. Because of the widespread use of antibiotics, wildlife is exposed either directly or indirectly through contamination of their food. This widespread contamination by antibiotics and their residues has influenced and selected for increasing numbers of antibiotic-resistant bacteria and antibiotic-resistant genes over time. It is widely recognized that a global “One Health” approach is needed to understand how antibiotic-resistant bacteria and resistance genes spread among and between animals, humans, and the environment.

Marilyn C. Roberts

Papers

Resistance to tetracyclines, macrolides, trimethoprim and sulfonamides.

Utility of EC 3M Petrifilm and sanitary surveys for source water assessment in Nyabushozi County, south-western Uganda

rRNA Methylases and Resistance to Macrolide, Lincosamide, Streptogramin, Ketolide, and Oxazolidinone (MLSKO) Antibiotics

Tetracycline Resistant Plasmids from Uropathogenic Escherichia coli from Southwestern Nigeria

Antibiotics and Resistance in the Environment