Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome.

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.

Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance.

Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.

TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexplained. Our results show a concentration-dependent response to triclosan with different genes important in survival between static and cidal exposures. These genes include those previously reported to have a role in triclosan resistance as well as a new set of genes, including essential genes. Novel genes identified as being sensitive to triclosan exposure include those involved in barrier function, small molecule uptake, and integrity of transcription and translation. We anticipate the approach we show here, by allowing comparisons across multiple experimental conditions of TraDIS data, and including essential genes, will be a starting point for future work examining how different drug conditions impact bacterial survival mechanisms.

Genomic plasticity of pathogenic Escherichia coli mediates d-serine tolerance via multiple adaptive mechanisms.

The molecular environment of the host can have profound effects on the behavior of resident bacterial species. We recently established how the sensing and response of enterohemorrhagic Escherichia coli (EHEC) to d-serine (d-Ser) resulted in down-regulation of type 3 secretion system-dependent colonization, thereby avoiding unfavorable environments abundant in this toxic metabolite. However, this model ignores a key determinant of the success of bacterial pathogens, adaptive evolution. In this study, we have explored the adaptation of EHEC to d-Ser and its consequences for pathogenesis. We rapidly isolated multiple, independent, EHEC mutants whose growth was no longer compromised in the presence of d-Ser. Through a combination of whole-genome sequencing and transcriptomics, we showed that tolerance could be attributed to disruption of one of two d-Ser transporters and/or activation of a previously nonfunctional d-Ser deaminase. While the implication of cytoplasmic transport in d-Ser toxicity was unsurprising, disruption of a single transporter, CycA, was sufficient to completely overcome the repression of type 3 secretion system activity normally associated with exposure to d-Ser. Despite the fact that this reveals a mechanism by which evolution could drive a pathogen to colonize new niches, interrogation of sequenced E. coli O157:H7 genomes showed a high level of CycA conservation, highlighting a strong selective pressure for functionality. Collectively, these data show that CycA is a critically important conduit for d-Ser uptake that is central to the niche restriction of EHEC.

Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing.

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription­replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage.

DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3'-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3'-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

Streptomycin and nalidixic acid elevate the spontaneous genome-wide mutation rate in Escherichia coli.

Since antibiotic resistance is a growing public health problem worldwide, it is important to understand how antibiotics and spontaneous mutations cooperate and shape the genome-wide mutation rate and spectrum. Here, we quantitatively evaluate genome-wide mutational profiles of Escherichia coli after long-term subinhibitory exposure to a broad-spectrum (streptomycin) and a narrow-spectrum antibiotic (nalidixic acid), using a mutation accumulation design combined with whole-genome resequencing of replicate lines as a mutagenicity test. We determined that, while the genome-wide mutation rate is slightly higher in the streptomycin-treated lines compared to the control lines, there is a significant increase in the nalidixic acid-treated lines. Our findings suggest that both broad and narrow-spectrum antibiotics may elevate the mutation rates in E. coli, but mechanisms of action may affect the consequence, thus contribute to accelerating the rate of adaptation and conferring antibiotic resistance.

Characterization and complete genome analysis of Bacillus velezensis CB6 revealed ATP synthase subunit α against foodborne pathogens.

Given the serious threat of foodborne multidrug-resistant bacteria to animals and humans, finding an effective antibacterial compound has always been an important topic for scientists. Here, from the soil of Changbaishan, we have identified a bacterium that can inhibit the growth of Staphylococcus aureus. Nr genome database analysis and phylogenetic analysis showed that strain CB6 belongs to Bacillus velezensis. We found that the crude extract of strain CB6 has broad-spectrum antibacterial activity against foodborne pathogens. In addition, we showed that the crude extract loses antibacterial activity after treatment with papain. Next, strain CB6 was purified using ammonium sulfate precipitation, a Sephadex G-75 gel filtration column and high-performance liquid chromatography system (HPLC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the antibacterial compound was the protein ATP synthase subunit α (ATP-1), with a molecular weight of 55.397 KDa. Moreover, we reported the complete genome sequence of strain CB6, which is composed of a unique circular 3,963,507 bp chromosome with 3749 coding genes and a G + C content of 46.53%. The genome contained 12 gene clusters with antibacterial functions, which constituted over 20.947% of the complete genome. Of note, the amino acid sequence encoding the ATP-1 protein in the strain CB6 genome was identified. In addition to these findings, we speculate that the ATP-1 protein may provide energy for secondary metabolites, which in turn will improve the antibacterial activity of the secondary metabolites. All the above important features make the ATP-1 as a potential candidate for the development of new antibacterial drugs and food preservatives in the future.

The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori.

Helicobacter pylori encodes a large number of restriction-modification (R-M) systems despite its small genome. R-M systems have been described as 'primitive immune systems' in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H. pylori strain harbours a unique set of R-M systems resulting in a highly diverse methylome. We identified a highly conserved GCGC-specific m5C MTase (JHP1050) that was predicted to be active in all of 459 H. pylori genome sequences analyzed. Transcriptome analysis of two H. pylori strains and their respective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM-300. Ten genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the expression of genes directly, while methylation of other motifs might cause secondary effects. Thus, m5C methylation modifies the transcription of multiple genes, affecting important phenotypic traits that include adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and susceptibility to copper.

Genomic Information on Linezolid-Resistant Sequence-Type 398 Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolated from a Pig.

The frequent occurrence of sequence-type 398 (ST398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pigs has become a major public health concern owing to the increased zoonotic potential of the pathogen. Recently, a novel oxazolidinone resistance gene, chloramphenicol-florfenicol resistant (cfr), conferring multiresistance phenotypes to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (PhLOPSA), has been found among ST398 LA-MRSA strains isolated from pigs. In this study, we report the first in silico genome analysis of a linezolid-resistant ST398 LA-MRSA strain, designated PJFA-521M, recovered from a pig in Korea. Genomic analyses revealed that the presence of the cfr gene was responsible for the observed linezolid resistance in the PJFA-521M strain. Moreover, newer antimicrobial resistance genes, such as the dfrG, aadE, spw, lsa(E), lnu(B), and fexA genes, were found in the PJFA-521M strain. In addition to the genetic elements for antimicrobial resistance, the carriage of various virulence genes for adherence, invasion, and immunomodulation was identified in the genome, especially within several mobile genetic elements (MGEs). The presence of multiple antimicrobial resistance genes and virulence genes on MGEs in the genome of a linezolid-resistant ST398 LA-MRSA should raise awareness regarding the use of other antimicrobial agents in pig farms and may also provide selective pressure for the prevalence of the cfr gene and the associated multidrug-resistant phenotype.

Genomic analysis of Chromobacterium haemolyticum: insights into the species resistome, virulence determinants and genome plasticity.

The increasing number of Chromobacterium haemolyticum human infection reports, especially in tropical regions and connected with environmental sources, resulted in an urge to better describe this species. This study aimed to characterize the C. haemolyticum resistome, virulence determinants and genetic platforms related with genome plasticity. A comparative genomic analysis was conducted between clinical C. haemolyticum genomes publicly available and the genome of an environmental isolate obtained in this study. The pangenome of C. haemolyticum was calculated and a total of 3378 core genes were predicted in its core genome, corresponding to 51.7% of the pangenome. Genetic determinants putatively encoding resistance to beta-lactams, fosfomycin, aminoglycosides and trimethoprim were predicted in all genomes, possibly constituting the intrinsic resistome of this species. In terms of resistance to beta-lactams, 4 genes were predicted encoding beta-lactamases of classes A, C and D. Moreover, the analysis of Chromobacterium genomes and C. haemolyticum environmental isolates reinforced the role of this genus as progenitor of the blaKPC gene. Putative virulence factors (VFs) were predicted in all genomes, related to adherence, toxins production, colonization and cell invasion. Secretion systems, including type III, were detected. A significant number of transposases and genomic islands were predicted in C. haemolyticum, in some cases above the average reported for Gram-negative bacterial genomes. We conclude that C. haemolyticum strains, including those of environmental origin, present a noteworthy collection of antibiotic resistance genes and VFs. Furthermore, sequences related to gene mobility and genome plasticity suggest high adaptability potential and a possible role as disseminator of antibiotic resistance.

Stability of potential prophages in commercial strain Lactobacillus plantarum NCU116 under various stressors.

Genetic stability of bacterium as a starter culture is vital for product quality in fermentation industry. The commercial strain Lactobacillus plantarum NCU116 widely used in fruit and vegetable fermentation was induced with various stressors to investigate the stability of potential prophages. PHAge Search Tool (PHAST) identified three potential prophages in bacterial genome. By spectrophotometric analysis, mitomycin C (MMC), lactic acid, and bile salt were found to inhibit the growth of L. plantarum NCU116 while ethanol and hydrogen peroxide had no notable impacts. Transcriptions of four phage-synthesizing genes (phaR, phacap, phaada, phatail) and four phage-resistant genes (cas116, helR, hsd1, hsd2) under stressors were investigated by quantitative reverse transcription PCR. MMC was found to most significantly upregulated transcriptions of phage-synthesizing genes, followed by lactic acid and bile salt. By transmission electron microscopy, no virus particles from the lysates of strain NCU116 treated by MMC were observed, corresponding to the result that no phage nucleic acids could be extracted from the supernatants of strain NCU116 treated by MMC. This study suggested that no prophages could be induced from L. plantarum NCU116 by strong inducer MMC, indicating its genetic stability, which supports the comprehensive application of strain NCU116 in industry without causing fermentation failure.

Bacterial phylogeny structures soil resistomes across habitats.

Ancient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil, including genes identical to those in human pathogens. Despite the apparent overlap between soil and clinical resistomes, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown. General metagenome functions often correlate with the underlying structure of bacterial communities. However, ARGs are proposed to be highly mobile, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2,895 ARGs we discovered were mostly new, and represent all major resistance mechanisms. We demonstrate that distinct soil types harbour distinct resistomes, and that the addition of nitrogen fertilizer strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements (genes responsible for horizontal gene transfer between bacteria such as transposases and integrases) syntenic with ARGs were rare in soil by comparison with sequenced pathogens, suggesting that ARGs may not transfer between soil bacteria as readily as is observed between human pathogens. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny.

Genome-wide relationship between R-loop formation and antisense transcription in Escherichia coli.

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

Epidemiologic, Phenotypic, and Structural Characterization of Aminoglycoside-Resistance Gene aac(3)-IV.

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.

Heteroexpression of Mycobacterium leprae hypothetical protein ML0190 provides protection against DNA-alkylating agent methyl methanesulfonate.

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.

Genomic polymorphism of Trifolium repens root nodule symbionts from heavy metal-abundant 100-year-old waste heap in southern Poland.

In total, 77 rhizobial strains isolated from the root nodules of T. repens, inhabiting heavy metal-contaminated waste heap (36 isolates) and control grassland (41 ones) in southern Poland, were analyzed for genome polymorphism and strength of the heavy metals' (mainly Zn, Pb, Cd) selective pressure on bacterial genome polymorphism using two PCR-based techniques, ERIC- (enterobacterial repetitive intergenic consensus) and REP-PCR (repetitive extragenic palindromic) sequences. Both methods of different discriminatory power index (D) (ERIC-PCR D = 0.9737; REP-PCR D = 0.9826) allowed to distinguish 47 and 44 rhizobial strains, respectively. Combined analysis of ERIC-PCR and REP-PCR DNA amplicons differentiated all tested isolates. Both ERIC- and REP-PCR DNA fingerprinting techniques showed significant decline of the genome polymorphism (h) in rhizobial population from metalliferous waste heap (h = 0.89 ± 0.03; h = 0.90 ± 0.02, respectively) compared to rhizobia from control non-metalliferous area (h = 0.99 ± 0.01; h = 0.98 ± 0.02, respectively) as well as substantial differences in the genomic polymorphism between both these populations (FST = 0.162, p = 0.008; FST = 0.170, p = 0.000, respectively).

Combined Analysis of Variation in Core, Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations.

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug-resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

Antibiotic treatment enhances the genome-wide mutation rate of target cells.

Although it is well known that microbial populations can respond adaptively to challenges from antibiotics, empirical difficulties in distinguishing the roles of de novo mutation and natural selection have left several issues unresolved. Here, we explore the mutational properties of Escherichia coli exposed to long-term sublethal levels of the antibiotic norfloxacin, using a mutation accumulation design combined with whole-genome sequencing of replicate lines. The genome-wide mutation rate significantly increases with norfloxacin concentration. This response is associated with enhanced expression of error-prone DNA polymerases and may also involve indirect effects of norfloxacin on DNA mismatch and oxidative-damage repair. Moreover, we find that acquisition of antibiotic resistance can be enhanced solely by accelerated mutagenesis, i.e., without direct involvement of selection. Our results suggest that antibiotics may generally enhance the mutation rates of target cells, thereby accelerating the rate of adaptation not only to the antibiotic itself but to additional challenges faced by invasive pathogens.