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)

Gene families are growing rapidly, but standard methods for inferring phylogenies do not scale to alignments with over 10,000 sequences. We present FastTree, a method for constructing large phylogenies and for estimating their reliability. Instead of storing a distance matrix, FastTree stores sequence profiles of internal nodes in the tree. FastTree uses these profiles to implement neighbor-joining and uses heuristics to quickly identify candidate joins. FastTree then uses nearest-neighbor interchanges to reduce the length of the tree. For an alignment with N sequences, L sites, and a different characters, a distance matrix requires O(N^2) space and O(N^2 L) time, but FastTree requires just O( NLa + N sqrt(N) ) memory and O( N sqrt(N) log(N) L a ) time. To estimate the tree's reliability, FastTree uses local bootstrapping, which gives another 100-fold speedup over a distance matrix. For example, FastTree computed a tree and support values for 158,022 distinct 16S ribosomal RNAs in 17 hours and 2.4 gigabytes of memory. Just computing pairwise Jukes-Cantor distances and storing them, without inferring a tree or bootstrapping, would require 17 hours and 50 gigabytes of memory. In simulations, FastTree was slightly more accurate than neighbor joining, BIONJ, or FastME; on genuine alignments, FastTree's topologies had higher likelihoods. FastTree is available at http://microbesonline.org/fasttree.

/pdf/fast-tree-computing-large-minimum-evolution-trees-with-4grq0gwfpy.pdf

Fast Tree: Computing Large Minimum-Evolution Trees with Profiles instead of a Distance Matrix

Tigecycline is a last-resort antibiotic that is used to treat severe infections caused by extensively drug-resistant bacteria. tet(X) has been shown to encode a flavin-dependent monooxygenase that modifies tigecycline1,2. Here, we report two unique mobile tigecycline-resistance genes, tet(X3) and tet(X4), in numerous Enterobacteriaceae and Acinetobacter that were isolated from animals, meat for consumption and humans. Tet(X3) and Tet(X4) inactivate all tetracyclines, including tigecycline and the newly FDA-approved eravacycline and omadacycline. Both tet(X3) and tet(X4) increase (by 64-128-fold) the tigecycline minimal inhibitory concentration values for Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. In addition, both Tet(X3) (A. baumannii) and Tet(X4) (E. coli) significantly compromise tigecycline in in vivo infection models. Both tet(X3) and tet(X4) are adjacent to insertion sequence ISVsa3 on their respective conjugative plasmids and confer a mild fitness cost (relative fitness of >0.704). Database mining and retrospective screening analyses confirm that tet(X3) and tet(X4) are globally present in clinical bacteria-even in the same bacteria as blaNDM-1, resulting in resistance to both tigecycline and carbapenems. Our findings suggest that both the surveillance of tet(X) variants in clinical and animal sectors and the use of tetracyclines in food production require urgent global attention.

Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans

A Specific Oxidative Catastrophe Three different classes of antibiotics induce bacterial cell death by the production of hydroxyl radicals. Hydroxyl radicals are powerful oxidizing agents in living cells and will oxidize the nucleic acid base, guanine, to form 8-oxoguanine, which is potentially mutagenic because it can pair with both cytosine and adenine and form lethal double-strand DNA breaks. Foti et al. (p. 315) discovered that overproduction of the nucleotide sanitizer MutT, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, gives striking protection against cell death. Several antibiotics kill bacteria by causing oxidative damage to guanine nucleotides, which then damage nucleic acids. A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

Oxidation of the Guanine Nucleotide Pool Underlies Cell Death by Bactericidal Antibiotics

ROS-scavenging hydrogel to promote healing of bacteria infected diabetic wounds.

The rapid emergence and dissemination of multidrug-resistant (MDR) bacterial pathogens pose a serious threat to global healthcare. One particular concern is the carbapenem-resistant Enterobacteriaceae (CRE), a group of Gram-negative bacteria that have evolved resistance to all or nearly all available antibiotics. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. We found a short linear antibacterial peptide (SLAP)-S25 carrying four non-natural amino acids of 2,4-diaminobutanoic acid (Dab), which solely showed weak antibacterial activity but boosted the efficacy of antibiotics covering all major classes, including cefepime, colistin, ofloxacin, rifampicin, tetracycline and vancomycin, against MDR Gram-negative pathogens. Mechanistic studies showed that SLAP-S25 triggers membrane damage by binding to both lipopolysaccharide (LPS) in the outer membrane and phosphatidylglycerol (PG) in bacterial cytoplasmic membrane, to potentiate antibiotic efficacy through collaborative strategies. Lastly, SLAP-S25 effectively enhanced the activity of colistin against MDR Escherichia coli-associated infections in three animal models. Our findings provide a potential therapeutic option using existing antibiotics in combination with broad-spectrum antibiotic adjuvants, to address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

A broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens

Widespread multidrug resistance caused by the abuse of antibiotics calls for novel strategies and materials. Gold nanoclusters (AuNCs) are scarcely explored for combating multidrug-resistant (MDR) bacteria in vivo. We herein synthesized a novel class of AuNCs, namely quaternary ammonium (QA) capped AuNCs (QA-AuNCs) as potent antibiotics selectively targeting MDR Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), in vivo. QA-AuNCs kill bacteria through a combined physicochemical mechanism, and show excellent therapeutic effects in both a skin infection model and a bacteremia model induced by MRSA. In addition, owing to their intense fluorescence, QA-AuNCs can be used for the discrimination of live/dead bacteria and bacteria counting, suggesting their potential for clinical theranostics.

Gold Nanoclusters for Targeting Methicillin-Resistant Staphylococcus aureus In Vivo.

Antibiotic resistance in Gram-negative pathogens has emerged and constituted a global crisis, thereby novel antibiotics and other anti-infective strategies are urgently needed. However, the growing...

Antibiotic adjuvants: an alternative approach to overcome multi-drug resistant Gram-negative bacteria.

Highly persistent incidence of multidrug resistant (MDR) bacterial pathogens constitutes a global burden for public health. An alternative strategy to alleviate such a crisis is to identify promising compounds to restore antibiotics activity against MDR bacteria. It is reported that the antidiabetic drug metformin exhibits the potentiation effect on tetracycline antibiotics, particularly doxycycline and minocycline, against MDR S. aureus, E. faecalis, E. coli, and S. enteritidis. Mechanistic studies demonstrate that metformin promotes intracellular accumulation of doxycycline in tetracycline-resistant E. coli. In addition, metformin boosts the immune response and alleviates the inflammatory responses in vitro. Last, metformin fully restores the activity of doxycycline in three animal infection models. Collectively, these results reveal the potential of metformin as a novel tetracyclines adjuvant to circumvent MDR bacterial pathogens and to improve the treatment outcome of recalcitrant infections.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/advs.201902227

Metformin Restores Tetracyclines Susceptibility against Multidrug Resistant Bacteria.

https://pubs.rsc.org/en/content/articlepdf/2019/NP/C8NP00031J

Yuan Liu

Papers

A broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens

Gold Nanoclusters for Targeting Methicillin-Resistant Staphylococcus aureus In Vivo.

Antibiotic adjuvants: an alternative approach to overcome multi-drug resistant Gram-negative bacteria.

Metformin Restores Tetracyclines Susceptibility against Multidrug Resistant Bacteria.

Nonribosomal antibacterial peptides that target multidrug-resistant bacteria.