Regulatory fine-tuning of mcr-1 increases bacterial fitness and stabilises antibiotic resistance in agricultural settings.

Antibiotic resistance tends to carry fitness costs, making it difficult to understand how resistance can be maintained in the absence of continual antibiotic exposure. Here we investigate this problem in the context of mcr-1, a globally disseminated gene that confers resistance to colistin, an agricultural antibiotic that is used as a last resort for the treatment of multi-drug resistant infections. Here we show that regulatory evolution has fine-tuned the expression of mcr-1, allowing E. coli to reduce the fitness cost of mcr-1 while simultaneously increasing colistin resistance. Conjugative plasmids have transferred low-cost/high-resistance mcr-1 alleles across an incredible diversity of E. coli strains, further stabilising mcr-1 at the species level. Regulatory mutations were associated with increased mcr-1 stability in pig farms following a ban on the use of colistin as a growth promoter that decreased colistin consumption by 90%. Our study shows how regulatory evolution and plasmid transfer can combine to stabilise resistance and limit the impact of reducing antibiotic consumption.

The evolution of colistin resistance increases bacterial resistance to host antimicrobial peptides and virulence.

Antimicrobial peptides (AMPs) offer a promising solution to the antibiotic resistance crisis. However, an unresolved serious concern is that the evolution of resistance to therapeutic AMPs may generate cross-resistance to host AMPs, compromising a cornerstone of the innate immune response. We systematically tested this hypothesis using globally disseminated mobile colistin resistance (MCR) that has been selected by the use of colistin in agriculture and medicine. Here, we show that MCR provides a selective advantage to Escherichia coli in the presence of key AMPs from humans and agricultural animals by increasing AMP resistance. Moreover, MCR promotes bacterial growth in human serum and increases virulence in a Galleria mellonella infection model. Our study shows how the anthropogenic use of AMPs can drive the accidental evolution of resistance to the innate immune system of humans and animals. These findings have major implications for the design and use of therapeutic AMPs and suggest that MCR may be difficult to eradicate, even if colistin use is withdrawn.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Pre-existing chromosomal polymorphisms in pathogenic E. coli potentiate the evolution of resistance to a last-resort antibiotic.

Bacterial pathogens show high levels of chromosomal genetic diversity, but the influence of this diversity on the evolution of antibiotic resistance by plasmid acquisition remains unclear. Here, we address this problem in the context of colistin, a 'last line of defence' antibiotic. Using experimental evolution, we show that a plasmid carrying the MCR-1 colistin resistance gene dramatically increases the ability of Escherichia coli to evolve high-level colistin resistance by acquiring mutations in lpxC, an essential chromosomal gene involved in lipopolysaccharide biosynthesis. Crucially, lpxC mutations increase colistin resistance in the presence of the MCR-1 gene, but decrease the resistance of wild-type cells, revealing positive sign epistasis for antibiotic resistance between the chromosomal mutations and a mobile resistance gene. Analysis of public genomic datasets shows that lpxC polymorphisms are common in pathogenic E. coli, including those carrying MCR-1, highlighting the clinical relevance of this interaction. Importantly, lpxC diversity is high in pathogenic E. coli from regions with no history of MCR-1 acquisition, suggesting that pre-existing lpxC polymorphisms potentiated the evolution of high-level colistin resistance by MCR-1 acquisition. More broadly, these findings highlight the importance of standing genetic variation and plasmid/chromosomal interactions in the evolutionary dynamics of antibiotic resistance.

Rationally designed foldameric adjuvants enhance antibiotic efficacy via promoting membrane hyperpolarization.

The negative membrane potential of bacterial cells influences crucial cellular processes. Inspired by the molecular scaffold of the antimicrobial peptide PGLa, we have developed antimicrobial foldamers with a computer-guided design strategy. The novel PGLa analogues induce sustained membrane hyperpolarization. When co-administered as an adjuvant, the resulting compounds - PGLb1 and PGLb2 - have substantially reduced the level of antibiotic resistance of multi-drug resistant Escherichia coli, Klebsiella pneumoniae and Shigella flexneri clinical isolates. The observed antibiotic potentiation was mediated by hyperpolarization of the bacterial membrane caused by the alteration of cellular ion transport. Specifically, PGLb1 and PGLb2 are selective ionophores that enhance the Goldman-Hodgkin-Katz potential across the bacterial membrane. These findings indicate that manipulating bacterial membrane electrophysiology could be a valuable tool to overcome antimicrobial resistance.

Evolutionary constraints on the acquisition of antimicrobial peptide resistance in bacterial pathogens.

Antimicrobial peptides (AMPs) offer a potential solution to the antibiotic resistance crisis. Recent studies have revealed important evolutionary constraints on the evolution and horizontal spread of AMP resistance in bacteria. Here, we summarize these advances and highlight their importance for therapeutic development of AMPs.

Rapid evolution and host immunity drive the rise and fall of carbapenem resistance during an acute Pseudomonas aeruginosa infection.

It is well established that antibiotic treatment selects for resistance, but the dynamics of this process during infections are poorly understood. Here we map the responses of Pseudomonas aeruginosa to treatment in high definition during a lung infection of a single ICU patient. Host immunity and antibiotic therapy with meropenem suppressed P. aeruginosa, but a second wave of infection emerged due to the growth of oprD and wbpM meropenem resistant mutants that evolved in situ. Selection then led to a loss of resistance by decreasing the prevalence of low fitness oprD mutants, increasing the frequency of high fitness mutants lacking the MexAB-OprM efflux pump, and decreasing the copy number of a multidrug resistance plasmid. Ultimately, host immunity suppressed wbpM mutants with high meropenem resistance and fitness. Our study highlights how natural selection and host immunity interact to drive both the rapid rise, and fall, of resistance during infection.

Chemical-genetic profiling reveals limited cross-resistance between antimicrobial peptides with different modes of action.

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.

Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance.

Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides.

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide-antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.

Genome Analysis of Staphylococcus capitis TE8 Reveals Repertoire of Antimicrobial Peptides and Adaptation Strategies for Growth on Human Skin.

Staphylococcus capitis TE8 was isolated from skin surface of a healthy human foot, and exhibited a strong antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus. Whole genome sequence of S. capitis TE8 was obtained by shotgun and paired-end pyrosequencing with a coverage of 109-fold. The draft genome contains 2,516,639 bp in 8 scaffolds with 209 total contigs. The genome contains 2319 protein coding sequences, 58 tRNA and 3 rRNA. Genome sequence analysis revealed 4 distinct gene loci with the ability to encode antimicrobial peptides: (i) an epidermicin gene cluster; (ii) a gallidermin gene cluster; (iii) a gene cluster encoding six phenol soluble modulin (PSM) ß-type peptides (PSMß1-ß6) and (iv) an additional gene that belonged to PSMß family and encoded a 44 residues long peptide, HTP2388. Synthetic peptides with sequence identical to seven PSMß-like peptides i.e. PSMß1-ß6 and peptide HTP2388 showed antibacterial activity. Genome sequence also revealed genes for adhesins, intracellular adhesins, osmoadaptation, oxidative and acid stress tolerance possibly responsible for initial attachment, colonization and survival of S. capitis TE8 on human skin. Comparative genome analysis revealed presence of a gamut of genes in S. capitis strains in comparison to Staphylococcus epidermidis and Staphylococcus caprae indicating towards their possible role in better adaptation and survival on human skin.

Insights into functional and evolutionary analysis of carbaryl metabolic pathway from Pseudomonas sp. strain C5pp.

Carbaryl (1-naphthyl N-methylcarbamate) is a most widely used carbamate pesticide in the agriculture field. Soil isolate, Pseudomonas sp. strain C5pp mineralizes carbaryl via 1-naphthol, salicylate and gentisate, however the genetic organization and evolutionary events of acquisition and assembly of pathway have not yet been studied. The draft genome analysis of strain C5pp reveals that the carbaryl catabolic genes are organized into three putative operons, 'upper', 'middle' and 'lower'. The sequence and functional analysis led to identification of new genes encoding: i) hitherto unidentified 1-naphthol 2-hydroxylase, sharing a common ancestry with 2,4-dichlorophenol monooxygenase; ii) carbaryl hydrolase, a member of a new family of esterase; and iii) 1,2-dihydroxy naphthalene dioxygenase, uncharacterized type-II extradiol dioxygenase. The 'upper' pathway genes were present as a part of a integron while the 'middle' and 'lower' pathway genes were present as two distinct class-I composite transposons. These findings suggest the role of horizontal gene transfer event(s) in the acquisition and evolution of the carbaryl degradation pathway in strain C5pp. The study presents an example of assembly of degradation pathway for carbaryl.

Draft Genome Sequence of Carbaryl-Degrading Soil Isolate Pseudomonas sp. Strain C5pp.

We report the draft genome sequence of carbaryl-degrading Pseudomonas sp. strain C5pp. Genes encoding salicylate and gentisate metabolism, large amounts of oxygenase, nitrogen metabolism, and heavy metal tolerance were identified. The sequence will provide further insight into the biochemical and evolutionary aspects of carbaryl degradation.

Genome sequence of a clinical isolate of dermatophyte, Trichophyton rubrum from India.

Trichophyton rubrum is one of the major causative agents of dermatophytosis in humans worldwide. We report the draft genome sequence of T. rubrum var. raubitschekii from Delhi, India, isolated from a patient presenting symptoms of onychomycosis. The total estimated genome size of the clinical isolate is 25.2 MB containing 8265 predicted protein-coding sequences, 91 tRNA and 15 rRNA genes. Sequence analysis of the secreted subtilases, one of the major virulence factors in dermatophytes, clusters them into three subfamilies with distinct sequence features. The genome sequence is a step in understanding diversity of dermatophytes worldwide and will aid in identification of virulence factors and dissecting mechanisms of pathogenesis among them.

Genome Sequence of Staphylococcus massiliensis Strain S46, Isolated from the Surface of Healthy Human Skin.

Staphylococcus massiliensis strain S46 was isolated from the surface of healthy human skin. Here, we report the draft genome sequence of S. massiliensis S46 (2,447,110 bp, with a G+C content of 36.3%).

Draft Genome Sequence of a Clinical Isolate of Multidrug-Resistant Mycobacterium tuberculosis East African Indian Strain OSDD271.

We describe the genome sequencing and analysis of a clinical isolate of Mycobacterium tuberculosis East African Indian (EAI) strain OSDD271 from India.

Draft Genome Sequence of Indibacter alkaliphilus Strain LW1T, Isolated from Lonar Lake, a Haloalkaline Lake in the Buldana District of Maharashtra, India.

We report the 5.0-Mb genome sequence of Indibacter alkaliphilus strain LW1(T), isolated from a haloalkaline crater lake in the Buldana district, Maharashtra, India.

Genome sequence of Nitratireductor aquibiodomus strain RA22.

The genus Nitratireductor represents nitrate-reducing bacteria from the family Phyllobacteriaceae. Here we report the draft genome sequence of Nitratireductor aquibiodomus strain RA22, which contains 4,592,790 bp, with a G+C content of 61.30%, and has 4,241 protein coding genes.