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.

/pdf/antibacterial-resistance-worldwide-causes-challenges-and-1cbvr301rt.pdf

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.

/pdf/chronic-obstructive-pulmonary-disease-4ipss13czj.pdf

Chronic obstructive pulmonary disease

Summary: Of 261 strains of Neisseria gonorrhoeae examined for plasmids, 6 were plasmid-free, 217 contained only a small multicopy 2.6 × 106 dalton plasmid and 38 carried a large 24.5 × 106 dalton plasmid. Restriction enzyme digests and DNA-DNA hybridization studies revealed that the large plasmids isolated between 1940 and 1978 share a common core of DNA sequences (70 to 100%) and represent a group of closely related molecules.

/pdf/the-ecology-of-gonococcal-plasmids-22sug3pqhn.pdf

The ecology of gonococcal plasmids.

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group

Vibrio salmonicida is the causative agent of cold-water vibriosis in farmed Atlantic salmon. We cloned a 6.9-kb PstI fragment from the 170-MDa plasmid (pRVS1) containing a tetracycline resistance determinant. A subcloned 1.96-kb HindIII fragment was found to mediate the tetracycline resistance. The 2.5-kb ClaI-PvuI fragment carrying the Tet E determinant from Escherichia coli hybridized under stringent conditions with the cloned 1.96-kb HindIII fragment from V. salmonicida. The 1.96-kb HindIII fragment codes for a protein of 26.5 kDa which represents a candidate for the structural TetA protein of the class E determinant. Deletion of a 0.28-kb BalI fragment from the middle of the HindIII fragment resulted in the loss of tetracycline resistance. We were able to show that when E. coli carries the cloned 6.9-kb PstI fragment, expression of tetracycline resistance is regulated by the concentration of tetracycline in the medium. In contrast, tetracycline resistance was constitutively expressed in the E. coli isolate carrying the 1.96-kb HindIII fragment. The tetracycline resistance gene isolated from the 170-MDa R plasmid of the marine fish pathogen V. salmonicida was characterized and shown to be a class E determinant.

Identification and cloning of a tetracycline resistance gene from the fish pathogen Vibrio salmonicida.

a These species were shown to carry plasmids of various sizes which hybridized with probes made from the 2.6-MDa plasmid. b Related plasmids are found in the genus Haemophilus (4). Identical plasmids are found in clinical isolates of K. denitrificans and Eikenella corrodens (24). d Related plasmids are found in enteric bacteria and Eikenella corrodens

/pdf/plasmids-of-neisseria-gonorrhoeae-and-other-neisseria-41yizraxuw.pdf

Plasmids of Neisseria gonorrhoeae and other Neisseria species.

A commentary on

http://www.frontiersin.org/Antimicrobials,_Resistance_and_Chemotherapy/10.3389/fmicb.2011.00203/abstract

Acquired antibiotic resistance genes: an overview

by van Hoek, A. H. A. M., Mevius, D., Guerra, B., Mullany, P., Roberts, A. P., and Aarts, H. J. M. (2011). Front. Microbio. 2:203. doi: 10.3389/fmicb.2011.00203

Dr. Marilyn C. Roberts and Dr. Stefan Schwarz have contacted the authors of the original publication with several comments and suggestions to better harmonize the correct nomenclature of the antibiotic resistance genes, as the gene names were not always correctly presented in the various tables given.

Authors often pick their own gene names which in many cases have been approved for use for other genetically distinct genes or give names to determinants which were already given an approved designated name. Therefore, we (Dr. Marilyn C. Roberts and Dr. Stefan Schwarz and Dr. Henk J. M. Aarts on behalf of the authors of the original publication) would like to present here the correct nomenclature and mechanistic features of the antibiotic resistance genes belonging to the following classes: Aminoglycosides (Table ​(Table1),1), Phenicols (Table ​(Table3),3), Macrolides–Lincosamides–Streptogramin B (Table ​(Table4),4), Quinolones (Table ​(Table5),5), Tetracyclines (Table ​(Table6),6), and Trimethoprim (Table ​(Table7).7). In addition some additional information is given on the various classes of antibiotic resistance genes as also a section regarding the antibiotic class Oxazolidinones has been added. Table ​Table22 was correctly displayed by van Hoek et al. (2011) but has been updated.



Table 1

Acquired aminoglycoside resistance genes*.





Table 2

β-lactamases and ESBLs families.





Table 3

Acquired phenicol resistance genes*.





Table 4

Acquired macrolide-lincosamide-streptogramin B (MLS) resistance genes*.





Table 5

Acquired quinolone resistance genes*.





Table 6

Acquired tetracycline resistance genes*.





Table 7

Acquired trimethoprim resistance genes*.



To the subsection dealing with the “Resistance mechanisms” of the AMINOGLYCOSIDES we would like to add that to date six additional methylases have been reported, i.e., npmA, rmtA, rmtB, rmtC, rmtD, and rmtE (Courvalin, 2008; Doi et al., 2008; Davis et al., 2010). Futhermore, that within the three major classes (AAC, ANT, and APH) an additional subdivision can be made based on the enzymes' target sites within the aminoglycoside molecules: i.e., there are four acetyltransferases: AAC(1), AAC(2′), AAC(3), and AAC(6′); five nucleotidyltransferases: ANT(2″), ANT(3″), ANT(4′), ANT(6), and ANT(9); and seven phosphotransferases: APH(2″), APH(3′), APH(3″), APH(4), APH(6), APH(7″), and APH(9).

To the subsection β-LACTAM, Resistance, mechanisms we would like to add that in recent years acquired genes encoding ESBLs have become a major concern (Bradford, 2001). Over time, the genes for the parent enzymes blaTEM−1, blaTEM−2, and blaSHV−1 have undergone point mutations which resulted in amino acid substitutions that changed the substrate spectrum to that of ESBLs, starting with blaTEM−3 and blaSHV−2 (Bradford, 2001).

Because chloramphenicol is not an actual antibiotic class the subsection of CHLORAMPHENICOL should be called PHENICOLS. Concerning the history of PHENICOLS, it is worthwhile to know the first antibiotic, chloramphenicol, originally referred to as chloromycetin, was isolated already in 1947 from Streptomyces venezuelae (Ehrlich et al., 1947).

Besides the inactivating enzymes (chloramphenicol acetyltransferases), there are also reports on other phenicol resistance systems, such as the inactivation by phosphotransferases, mutations of the target site, permeability barriers, and efflux systems (Schwarz et al., 2004). Of the latter mechanism, cmlA and floR are the most commonly known genes in Gram-negative bacteria (Bissonnette et al., 1991; Briggs and Fratamico, 1999).

The macrolides (subsection MACROLIDES–LINCOSAMIDES–STREPTOGRAMIN B) have a similar mode of antibacterial action, comparable antibacterial spectra and in part overlapping binding sites at the ribosome as two other antibiotic classes, i.e., lincosamides and streptogramin antibiotics (comprising streptogramin A and B compounds that act synergistically). Consequently, these antibiotics, although chemically distinct, have been clustered together as MLS antibiotics (Roberts, 1996). Macrolides, lincosamides and streptogramins all inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria (Weisblum, 1995; Roberts, 2002).

To Resistance mechanisms of the subsection MACROLIDES–LINCOSAMIDES–STREPTOGRAMIN B. Shortly after the introduction of erythromycin into clinical setting in the 1950s, bacterial resistance to this antibiotic was reported for the first time in staphylococci (Weisblum, 1995). Since then a large number of bacteria have been identified that are resistant to MLS due to the presence of various different genes. The resistance determinants responsible include rRNA methylases that modify the ribosomal target sites, ABC transporters, and efflux proteins of the Major Facilitator Superfamily, as well as genes for inactivating enzymes (Roberts et al., 1999; Roberts, 2008). The latter group can be further subdivided into esterases, lyases, phosphorylases, and transferases (Table ​(Table44).

The most common mechanism of MLSB resistance is due to the presence of rRNA methylases, encoded by the erm genes. These enzymes methylate the adenine residue(s) resulting in MLSB resistance. The methylated adenine(s) prevents the drugs from binding to the 50S ribosomal subunit. The other two mechanisms efflux and enzymatic inactivation result in resistance to only 1 or 2 classes of antibiotics belonging to the MLS group.

There are currently 77 MLS resistance genes recognized. A new MLS gene must have <79% amino acid identity with all previously characterized MLS genes before receiving a unique name (Roberts et al., 1999; Roberts, 2008). For an actual list of the MLS acquired resistance genes we refer to the website of Dr. Marilyn Roberts, http://faculty.washington.edu/marilynr/.

In addition to the subsection of QUINOLONES currently five families of qnr genes have been reported; qnrA (7 subtypes), qnrB (59 subtypes), qnrC (1 subtype), qnrD (1 subtype), and qnrS (8 subtypes) (Jacoby et al., 2008; Cattoir and Nordmann, 2009; Cavaco et al., 2009; Strahilevitz et al., 2009; Torpdahl et al., 2009).

Another mechanism of conferring resistance to quinolones is represented by the plasmid-borne gene qepA, which codes for an efflux pump that can export hydrophilic fluoroquinolones, e.g., ciprofloxacin and enrofloxacin (Perichon et al., 2007; Yamane et al., 2007). A variant of this resistance pump, QepA2, was identified in an E. coli isolate from France (Cattoir et al., 2008).

Regarding TETRACYCLINE, Resistance mechanisms, currently there are 45 different acquired tetracycline resistance determinants recognized (Roberts, 1996, 2005; Brown et al., 2008) (Table ​(Table6).6). For an up-to-date list of the acquired tetracycline resistance genes, we refer to the website of Dr. Marilyn Roberts, http://faculty.washington.edu/marilynr/. Among these, 26 of the tet genes, 2 of the otr genes and the only tcr determinant code for efflux pumps, whereas 11 tet genes and 1 otr gene code for ribosomal protection proteins (RPPs). The enzymatic inactivation mechanism can be attributed to 3 tet genes. The tet(U) determinant represents an unknown tetracycline resistance mechanism since its sequence does not appear to be related to either efflux or RPPs, nor to the inactivation enzymes. The efflux and RPP encoding genes are found in members of Gram-positive, Gram-negative, aerobic, as well as anaerobic bacteria. In contrast, the enzymatic tetracycline inactivation mechanism has so far only been identified in Gram-negative bacteria. The tet(M) has the broadest host range of all tetracycline resistance genes, whereas tet(B) gene has the widest range among the Gram-negative bacteria. In recent years published data indicate that there are increasing numbers of Gram-negative bacteria that carry tet genes originally identified in Gram-positive bacteria (Roberts, 2002).

To the subsection TRIMETHOPRIM, Resistance mechanisms. Initially, the acquired DHFRs fell into two distinct families A and B, encoded by the dfrA and dfrB genes (Howell, 2005). Up to now 6 plasmid-mediated families can be distinguished with relatively few dfr determinants originating from Gram-positive bacteria (Table ​(Table7).7). The dfrK and dfrA28 genes are the newest additions to the trimethoprim resistance determinant family (Kadlec and Schwarz, 2009; Kadlec et al., 2011). In contrast to the latest reported DHFRs, the oldest families, dfrA and dfrB, each contain several members (Roberts, 2002; Levings et al., 2006). For example, the dfrA group accomodates over 30 published genes; however, unpublished, dfrA variants are also present in the public DNA libraries and some genes apparently have changed nomenclature (Table ​(Table77).

Furthermore, we suggest an additional section concerning oxazolidinones.

Marilyn C. Roberts

Papers

The ecology of gonococcal plasmids.

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group

Identification and cloning of a tetracycline resistance gene from the fish pathogen Vibrio salmonicida.

Plasmids of Neisseria gonorrhoeae and other Neisseria species.

Erratum: Acquired antibiotic resistance genes: an overview