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

Prevalence of Mycoplasma Genitalium Determined by DNA Probe in Men with Urethritis

Sir, The incidence of multidrug resistance (i.e. resistance to two or more antibiotic classes) amongst clinical isolates of Streptococcus pneumoniae has increased markedly over the last 10 years; most of these strains belong to one of five serogroups (6, 9, 14, 19 and 23). This situation has prompted a search for alternative antibiotics, especially for the treatment of patients with invasive pneumococcal diseases. The present study was undertaken to evaluate the in-vitro activities of some of the newer agents against isolates of S. pneumoniae recovered from patients in Washington State. The organisms studied included 62 non-replicate pneumococci isolated from patients with sterile site infections during a Washington State Surveillance Study (1995–1996). Of these strains, 12 belonged to serogroup 6, 13 to serogroup 9, nine to serogroup 14, 17 to serogroup 19 and eight to serogroup 23; three were non-typeable. Additional non-replicate strains (three of which belonged to serogroup 19 and nine of which were non-typeable) were recovered in the course of a Paediatric Carriage Study (1996), and one further strain (serogroup 19F) was isolated from a patient in 1997. The 75 isolates were selected because the MICs of penicillin for them were >0.06 mg/L; 14 were categorized as susceptible (MICs 5 0.06 mg/L), 52 exhibited intermediate susceptibility (MICs 5 0.1–1 mg/L) and nine were resistant (MICs > 2 mg/L). The antibiotics tested were as follows: clindamycin, erythromycin and penicillin (Sigma Chemical Co., St Louis, MO, USA), ciprofloxacin and sparfloxacin (Bayer, Lansing, IL, USA), cefprozil (Bristol-Myers Squibb, Princeton, NJ, USA), grepafloxacin (GlaxoWellcome Co., Triangle Park, NC, USA), levofloxacin and the novel ketolide, HMR 3647 (Hoechst Marion Roussel, France), the oxazolidinone, linezolid (Pharmacia and Upjohn, Kalamazoo, MI, USA), and trovafloxacin and azithromycin (Pfizer, Groton, CT, USA). MICs were determined by an agar dilution method recommended by the National Committee for Clinical Laboratory Standards. The medium used was Mueller– Hinton agar (Difco, Detroit, MI, USA) supplemented with 5% sheep blood, and inocula of 10 cfu were delivered to the surfaces of the plates with a Steers replicator. S. pneu moniae ATCC 6305 and ATCC 40619 were included as controls. The in-vitro activities of the 11 antibiotics against the 75 isolates are shown in the Table. Most of the penicillinsusceptible isolates were susceptible to all of the antibiotics tested, the exception being ciprofloxacin to which all but one strain were resistant. Of the 52 isolates exhibiting intermediate susceptibility to penicillin, at least 51 were susceptible to each of the newer fluoroquinolones and linezolid. The susceptibilities of these strains to azithromycin, erythromycin and clindamycin were variable (51.9–86.5%), and only 7.7% and 3.8% were susceptible to cefprozil and ciprofloxacin, respectively. The susceptibility patterns of the penicillin-resistant isolates were broadly similar to those of the strains with intermediate susceptibilities, with the novel fluoroquinolones and linezolid being highly active, the macrolides and clindamycin having variable activities (44.4–100% of isolates being susceptible) and all of the isolates being resistant to cefprozil and ciprofloxacin. As neither validated nor tentative MIC breakpoints for HMR 3647 were available at the time the study was undertaken, susceptibility categories could not be assigned. However, the MICs of this drug are very low (<0.03 mg/L) and it is likely that all of the isolates tested, irrespective of their penicillin susceptibilities, would be regarded as susceptible. We also determined the MICs of the two macrolides and clindamycin for the study isolates following induction, i.e. after incubation of the strains in the presence of erythromycin at a concentration of 0.5 mg/L, and compared them with those determined without induction. The MICs of all three antibiotics for six of the strains (one penicillinsusceptible and five exhibiting intermediate susceptibility) increased by at least two two-fold dilutions following exposure to erythromycin. These six isolates harboured the ermB gene, which encodes methylation of 23S rRNA, were resistant to both the macrolides and clindamycin, and exhibited inducible resistance. The remaining 22 erythromycinresistant isolates were not phenotypically inducible, exhib-

In-vitro activities of 11 antibiotics against 75 strains of Streptococcus pneumoniae with reduced susceptibilities to penicillin isolated from patients in Washington State.

In the years between the introduction of antibiotic therapy and the mid-1970s, antibiotic-resistant bacteria were generally restricted to the hospital setting and epidemic diseases which were not major issues in much of the industrialized world. In this chapter, the development of tetracycline-resistant (Tcr) fish pathogens and bacteria associated with aquaculture environments is discussed. Antibiotic residues may be found in a variety of foods produced around the world. Currently, unlabeled but high levels of cephalosporins (ceftiofur) are allowed in some foods. Rules exist which aim to minimize the level of antibiotic residues found in food products; however, not all foods are tested, nor is it clear that the standards required by farms in North America and Europe are followed when the food is produced for overseas consumption. The majority of the novel genes were mobile elements carrying antibiotic resistance and virulence genes. Enterococci are normal inhabitants of the intestinal flora of most mammals, birds, and humans, as well as from soil, surface waters, plants, vegetables, raw foods such as milk and meat, and fermented meats such as Italian salami or raw sausage. Studies have shown that fish foods, even unlabeled ones, may contain antibiotic-resistant bacteria and/or antibiotic residues. This chapter also illustrates a few examples in which specific antibiotic resistance genes, regions of DNA, and/or plasmids have been found in bacteria from very different ecosystems, from different parts of the world, and in bacteria that are host species-specific.

The evolution of antibiotic-resistant microbes in foods and host ecosystems.

Sir, Haemophilus influenzae is an opportunistic pathogen that is found in the nasopharynx of 75% of healthy adults. It is able to cause a variety of different infections and is commonly found in cystic fibrosis patients. – 3 Recently, 106 non-typeable H. influenzae samples isolated from cystic fibrosis children in 2007 and 2008 were shown to carry mobile acquired Grampositive macrolide resistance genes. Three of these unrelated isolates (CHH062, CHH082 and CHH126) were resistant to both macrolides (Em) and tetracycline (Tc). Previously all Tc H. influenzae carried the tet(B) gene generally on Haemophilus plasmids with host range restricted to within the genus. With the isolation of the tet(M) gene on a Haemophilus ducreyi plasmid from a strain isolated in 1986, we predicted that the tet(M) gene would move into other species of Haemophilus. More recently, Tristram et al. predicted that in the future Tc H. influenzae would carry the mobile broad-host range tet(M) gene associated with or without the tet(B) gene. This study found that all three Em Tc H. influenzae carried both the tet(M) and tet(B) genes and the two isolates tested could conjugally transfer the tet(M) gene to an unrelated Grampositive Enterococcus faecalis JH2-2 recipient, while both tet genes could be transferred to a H. influenzae recipient. The data suggest that H. influenzae are now reservoirs for both broad-host range mobile antibiotic resistance genes, which is a shift from that of a recipient of mobile antibiotic resistance genes from unrelated genera in the 1970s, and today these strains are able to act as donors of these genes to unrelated genera. The three Em Tc H. influenzae strains were screened for the presence of the tet(B) and tet(M) genes using previously described PCR assays with appropriate controls, and all three isolates were positive for both genes. Plasmid extractions were done and 60 kb plasmids were identified that carried the tet(B) gene. The plasmids could be conjugally transferred at a frequency ranging from 1.08×10 to 5.0×10/recipient to the H. influenzae Rd, which is in the range previously reported for transfer of Haemophilus plasmids. All the transconjugants examined carried the plasmid and the tet(B) gene, while 10%–15% of the transconjugants carried the tet(M) gene, suggesting that the tet(M) gene was not located on the plasmid. Conjugation was performed with the H. influenzae donors and E. faecalis JH2-2 recipient at 25:1 ratio, as previously described. The tet(M) gene transferred at a frequency ranging between 2.39×10 and 3.15×10/recipient, while the tet(B) gene did not transfer. This is the first report of the tet(M) gene in H. influenzae. Although the three isolates came from cystic fibrosis patients in North America, it is unlikely that they are unique to Tc H. influenzae strains from North America. However, it is unclear if H. influenzae from people other than cystic fibrosis patients, who normally are treated with antibiotics, also carry strains that harboured the tet(M) gene. Nevertheless, it is of interest that the tet(M) gene, which has the widest host range of the tet genes and has been identified in 37 Gram-negative genera and 35 Gram-positive genera, was recognized in Tc H. influenzae isolated .30 years after Tc H. influenzae carrying the tet(B) genes were described. The presence of the mobile tet(M) genes along with the previous identification of mobile erm(A), erm(B), erm(C), erm(F) and mef(A) macrolide resistance genes in H. influenzae indicates a major shift for H. influenzae from simply being able to acquire antibiotic resistance genes from both Gram-positive and Gramnegative bacteria that could be transferred between the genus Haemophilus to now having mobile elements that are able to conjugally transfer antibiotic resistance genes to unrelated genera within the bacterial community. The presence of the Tn916-like family of conjugative transposons in H. influenzae suggests the potential for antibiotic resistance genes often associated with the Tn916-like elements, aminoglycoside, chloramphenicol and macrolide resistance genes to become more common in the H. influenzae population. Whether surveillance of these mobile genes in the H. influenzae population has value will require further study.

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Marilyn C. Roberts

Papers

Prevalence of Mycoplasma Genitalium Determined by DNA Probe in Men with Urethritis

In-vitro activities of 11 antibiotics against 75 strains of Streptococcus pneumoniae with reduced susceptibilities to penicillin isolated from patients in Washington State.

The evolution of antibiotic-resistant microbes in foods and host ecosystems.

tet(M)-carrying Haemophilus influenzae as a potential reservoir for mobile antibiotic resistance genes

Cloning andCharacterization oftetMGenefroma Ureaplasma urealyticum Strain