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

The increasing resistance of genital mycoplasmas to tetracycline poses a problem because tetracycline is one of the few antimicrobial agents active against Mycoplasma hominis, Ureaplasma urealyticum, chlamydiae, gonococci, and other agents of genitourinary-tract disease. Since the quinolones are a promising group of antimicrobial agents, the susceptibilities of M. hominis and U. urealyticum to the newer 6-fluoroquinolones were determined by the agar dilution method. Ciprofloxacin, difloxacin, and ofloxacin had good activity against M. hominis, with the MIC for 50% of isolates tested (MIC50) being 1 microgram/ml. Fleroxacin, lomefloxacin, pefloxacin, and rosoxacin had MIC50s of 2 micrograms/ml. Enoxacin, norfloxacin, and amifloxacin had MIC50s of 8 to 16 micrograms/ml, and cinoxacin and nalidixic acid were inactive (MIC50, greater than or equal to 256 micrograms/ml). Overall, the activities of 6-fluoroquinolones for ureaplasmas were similar to those for M. hominis, with MICs being the same or twofold greater. The most active 6-fluoroquinolones against ureaplasmas were difloxacin, ofloxacin, and pefloxacin, with MIC50s of 1 to 2 micrograms/ml. Ciprofloxacin was unusual in that the MIC50 for M. hominis was 1 microgram/ml, whereas the MIC50 for ureaplasmas was 8 micrograms/ml. Since the MIC50s for the most active quinolones approximate achievable concentrations in blood and urine, quinolones have promise in treating mycoplasmal infections.

Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method.

We examined nine chloramphenicol-resistant (minimal inhibitory concentration, greater than or equal to 15 micrograms/ml) Haemophilus influenzae strains isolated in various parts of the world to characterize the genetic and biochemical bases of the resistance; four were type b. All nine contained conjugative plasmids, ranging in molecular weight from 34 x 10(6) to 46 x 10(6), which encoded for resistance to chloramphenicol and tetracycline or chloramphenicol, tetracycline, and ampicillin. Deoxyribonucleic acid homology studies showed that these plasmids were closely related to a previously described ampicillin-resistant plasmid, RSF007, and to each other. All nine isolates and their chloramphenicol-resistant transconjugants produced chloramphenicol acetyltransferase. We conclude that chloramphenicol resistance in these strains of H. influenzae is via plasmid-mediated production of chloramphenicol acetyltransferase.

Characterization of chloramphenicol-resistant Haemophilus influenzae.

Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students

The distributions of the nucleotide sequences related to the tetracycline resistance determinants Tet K, Tet L, Tet M, and Tet O were studied by dot blot hybridization with randomly chosen clinical urogenital tract isolates of viridans group streptococci, Streptococcus agalactiae, Enterococcus faecalis, Gardnerella vaginalis, Lactobacillus spp., Fusobacterium nucleatum, Peptostreptococcus spp., and Veillonella parvula. Among the Peptostreptococcus spp., 79% of the isolates hybridized with one (64%) or more (36%) of the probes for Tet K (27%), Tet L (30%), Tet M (75%) and Tet O (13%). Of the viridans group streptococci, 82% of the strains hybridized with one (34%) or more (66%) of the four probes. The distribution of the four determinants in this group was as follows: Tet K, 36%; Tet L, 31%; Tet M, 43%; Tet O, 61%. Twenty-nine percent of the enterococci and forty-six percent of the group B streptococci hybridized with the probes; however, the Tet K, Tet L, and Tet O determinants were found in only a few strains, while the Tet M determinant predominated. A total of 29% of the F. nucleatum isolates, 55% of the G. vaginalis isolates, and 26% of the V. parvula isolates hybridized with the Tet M determinant. In contrast, 43% of the Lactobacillus spp. hybridized with the Tet O determinant. The data indicate that tetracycline resistance determinants are common to many of the microorganisms isolated from the urogenital tract.

Genetic basis of tetracycline resistance in urogenital bacteria.

Objectives: To determine antibiotic resistance genes associated with 17 Nigerian CTX-M-positive Klebsiella pneumoniae plasmids from patients with community-acquired urinary tract infections. Methods: The size and restriction patterns of the plasmids were determined, and antibiotic resistance genes were identified using DNA-DNA hybridization, PCR assays, hybridization of PCR products with internal probes, and sequencing. Results: All CTX-M plasmids were large (58-320 kb) and carried the following genes: aac(6')-Ib (aminoglycoside resistance) which included aac(6')-Ib-cr (aminoglycoside-fluoroquinolone resistance), aadA2 (aminoglycoside resistance), erm(B) (macrolide-lincosamide-streptogramin B resistance), bla TEM-1 (ampicillin resistance), tet(A) (tetracycline resistance), sul1 (sulphonamide resistance), dfr (trimethoprim resistance) and intl1, an integrase associated with class 1 integrons. Eleven (65%) plasmids carried an mph(A) gene (macrolide resistance), seven (41%) plasmids carried a qnrB1 gene (low-level quinolone resistance) and four (24%) plasmids carried multiple cat genes (chloramphenicol resistance). catA2, catA3 and qnrB1 genes and a 6 kb PstI fragment, carrying the bla CTX-M gene, were sequenced. Conclusions: This is the first description of catA2 and catA3 genes in Klebsiella spp. and the first description of the erm(B) and floR genes associated with a CTX-M plasmid. This is also the first report of qnrB1 and aac(6')-Ib-cr in isolates from Africa and the first report of these two genes on the same plasmid.

Marilyn C. Roberts

Papers

Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method.

Characterization of chloramphenicol-resistant Haemophilus influenzae.

Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students

Genetic basis of tetracycline resistance in urogenital bacteria.

New antibiotic resistance genes associated with CTX-M plasmids from uropathogenic Nigerian Klebsiella pneumoniae.