In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut. - Related Documents




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384101.0000In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut. The widespread sulfonamide resistance genes sul1, sul2, and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli, using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo. A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo. Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1, sul2, and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach.202439140732
380710.9996Antimicrobial drug resistance genes do not convey a secondary fitness advantage to calf-adapted Escherichia coli. Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.200616391076
380020.9996Alterations of Salmonella enterica Serovar Typhimurium Antibiotic Resistance under Environmental Pressure. Microbial horizontal gene transfer is a continuous process that shapes bacterial genomic adaptation to the environment and the composition of concurrent microbial ecology. This includes the potential impact of synthetic antibiotic utilization in farm animal production on overall antibiotic resistance issues; however, the mechanisms behind the evolution of microbial communities are not fully understood. We explored potential mechanisms by experimentally examining the relatedness of phylogenetic inference between multidrug-resistant Salmonella enterica serovar Typhimurium isolates and pathogenic Salmonella Typhimurium strains based on genome-wide single-nucleotide polymorphism (SNP) comparisons. Antibiotic-resistant S Typhimurium isolates in a simulated farm environment barely lost their resistance, whereas sensitive S Typhimurium isolates in soils gradually acquired higher tetracycline resistance under antibiotic pressure and manipulated differential expression of antibiotic-resistant genes. The expeditious development of antibiotic resistance and the ensuing genetic alterations in antimicrobial resistance genes in S Typhimurium warrant effective actions to control the dissemination of Salmonella antibiotic resistance.IMPORTANCE Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S Typhimurium isolates genetically shared relevancy with pathogenic S Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S Typhimurium in agricultural environments.201830054356
634030.9996Identification and functional analysis of novel protein-encoding sequences related to stress-resistance. Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.202337840709
891340.9996The gut environment regulates bacterial gene expression which modulates susceptibility to bacteriophage infection. Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.202235421351
895750.9996Transcriptome Profiling Reveals Interplay of Multifaceted Stress Response in Escherichia coli on Exposure to Glutathione and Ciprofloxacin. We have previously reported that supplementation of exogenous glutathione (GSH) promotes ciprofloxacin resistance in Escherichia coli by neutralizing antibiotic-induced oxidative stress and by enhancing the efflux of antibiotic. In the present study, we used a whole-genome microarray as a tool to analyze the system-level transcriptomic changes of E. coli on exposure to GSH and/or ciprofloxacin. The microarray data revealed that GSH supplementation affects redox function, transport, acid shock, and virulence genes of E. coli. The data further highlighted the interplay of multiple underlying stress response pathways (including those associated with the genes mentioned above and DNA damage repair genes) at the core of GSH, offsetting the effect of ciprofloxacin in E. coli. The results of a large-scale validation of the transcriptomic data using reverse transcription-quantitative PCR (RT-qPCR) analysis for 40 different genes were mostly in agreement with the microarray results. The altered growth profiles of 12 different E. coli strains carrying deletions in the specific genes mentioned above with GSH and/or ciprofloxacin supplementation implicate these genes in the GSH-mediated phenotype not only at the molecular level but also at the functional level. We further associated GSH supplementation with increased acid shock survival of E. coli on the basis of our transcriptomic data. Taking the data together, it can be concluded that GSH supplementation influences the expression of genes of multiple stress response pathways apart from its effect(s) at the physiological level to counter the action of ciprofloxacin in E. coli. IMPORTANCE The emergence and spread of multidrug-resistant bacterial strains have serious medical and clinical consequences. In addition, the rate of discovery of new therapeutic antibiotics has been inadequate in last few decades. Fluoroquinolone antibiotics such as ciprofloxacin represent a precious therapeutic resource in the fight against bacterial pathogens. However, these antibiotics have been gradually losing their appeal due to the emergence and buildup of resistance to them. In this report, we shed light on the genome-level expression changes in bacteria with respect to glutathione (GSH) exposure which act as a trigger for fluoroquinolone antibiotic resistance. The knowledge about different bacterial stress response pathways under conditions of exposure to the conditions described above and potential points of cross talk between them could help us in understanding and formulating the conditions under which buildup and spread of antibiotic resistance could be minimized. Our findings are also relevant because GSH-induced genome-level expression changes have not been reported previously for E. coli.201829468195
470860.9996Proteomic analysis of nalidixic acid resistance in Escherichia coli: identification and functional characterization of OM proteins. The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. To understand the mechanisms of the resistance is extremely important to the control of these bacteria. In the current study, proteomic methodologies were utilized to characterize OM proteome of Escherichia coli with nalidixic acid (NA) resistance. The OM proteins TolC, OmpT, OmpC and OmpW were found to be up-regulated, and FadL was down-regulated in the NA-resistant E. coli strains. The changes at the level of protein expression were validated using Western blotting. Furthermore, the possible roles these altered proteins played in regulation of NA resistance were investigated using genetically modified strains with the deletion of these genes. The results obtained from functional characterization of these genetically modified strains suggest that TolC and OmpC may play more important roles in the control of NA resistance than other OM proteins identified. To gain better understanding of the mechanisms of NA resistance, we also characterized the role of the two-component system EnvZ/OmpR which is responsible for the regulation of OmpC and OmpF expression in response to NA resistance using their genetically modified strains. Our results suggest that OmpF and the EnvZ/OmpR are also important participants of the pathways regulating the NA resistance of E. coli.200818438992
900470.9996Shedding light on the bacterial resistance to toxic UV filters: a comparative genomic study. UV filters are toxic to marine bacteria that dominate the marine biomass. Ecotoxicology often studies the organism response but rarely integrates the toxicity mechanisms at the molecular level. In this study, in silico comparative genomics between UV filters sensitive and resistant bacteria were conducted in order to unravel the genes responsible for a resistance phenotype. The genomes of two environmentally relevant Bacteroidetes and three Firmicutes species were compared through pairwise comparison. Larger genomes were carried by bacteria exhibiting a resistant phenotype, favoring their ability to adapt to environmental stresses. While the antitoxin and CRISPR systems were the only distinctive features in resistant Bacteroidetes, Firmicutes displayed multiple unique genes that could support the difference between sensitive and resistant phenotypes. Several genes involved in ROS response, vitamin biosynthesis, xenobiotic degradation, multidrug resistance, and lipophilic compound permeability were shown to be exclusive to resistant species. Our investigation contributes to a better understanding of UV filters resistance phenotypes, by identifying pivotal genes involved in key pathways.202134760358
838380.9996Novel insights into carbohydrate utilisation, antimicrobial resistance, and sporulation potential in Roseburia intestinalis isolates across diverse geographical locations. Roseburia intestinalis is one of the most abundant and important butyrate-producing human gut anaerobic bacteria that plays an important role in maintaining health and is a potential next-generation probiotic. We investigated the pangenome of 16 distinct strains, isolated over several decades, identifying local and time-specific adaptations. More than 50% of the genes in each individual strain were assigned to the core genome, and 77% of the cloud genes were unique to individual strains, revealing the high level of genome conservation. Co-carriage of the same enzymes involved in carbohydrate binding and degradation in all strains highlighted major pathways in carbohydrate utilization and reveal the importance of xylan, starch and mannose as key growth substrates. A single strain had adapted to use rhamnose as a sole growth substrate, the first time this has been reported. The ubiquitous presence of motility and sporulation gene clusters demonstrates the importance of these phenotypes for gut survival and acquisition of this bacterium. More than half the strains contained functional, potentially transferable, tetracycline resistance genes. This study advances our understanding of the importance of R. intestinalis within the gut ecosystem by elucidating conserved metabolic characteristics among different strains, isolated from different locations. This information will help to devise dietary strategies to increase the abundance of this species providing health benefits.202540089923
451090.9996Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli. Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.202337482346
6334100.9996Epigenetic inheritance based evolution of antibiotic resistance in bacteria. BACKGROUND: The evolution of antibiotic resistance in bacteria is a topic of major medical importance. Evolution is the result of natural selection acting on variant phenotypes. Both the rigid base sequence of DNA and the more plastic expression patterns of the genes present define phenotype. RESULTS: We investigated the evolution of resistant E. coli when exposed to low concentrations of antibiotic. We show that within an isogenic population there are heritable variations in gene expression patterns, providing phenotypic diversity for antibiotic selection to act on. We studied resistance to three different antibiotics, ampicillin, tetracycline and nalidixic acid, which act by inhibiting cell wall synthesis, protein synthesis and DNA synthesis, respectively. In each case survival rates were too high to be accounted for by spontaneous DNA mutation. In addition, resistance levels could be ramped higher by successive exposures to increasing antibiotic concentrations. Furthermore, reversion rates to antibiotic sensitivity were extremely high, generally over 50%, consistent with an epigenetic inheritance mode of resistance. The gene expression patterns of the antibiotic resistant E. coli were characterized with microarrays. Candidate genes, whose altered expression might confer survival, were tested by driving constitutive overexpression and determining antibiotic resistance. Three categories of resistance genes were identified. The endogenous beta-lactamase gene represented a cryptic gene, normally inactive, but when by chance expressed capable of providing potent ampicillin resistance. The glutamate decarboxylase gene, in contrast, is normally expressed, but when overexpressed has the incidental capacity to give an increase in ampicillin resistance. And the DAM methylase gene is capable of regulating the expression of other genes, including multidrug efflux pumps. CONCLUSION: In this report we describe the evolution of antibiotic resistance in bacteria mediated by the epigenetic inheritance of variant gene expression patterns. This provides proof in principle that epigenetic inheritance, as well as DNA mutation, can drive evolution.200818282299
9915110.9996Comparative Analysis of Transcriptome and Proteome Revealed the Common Metabolic Pathways Induced by Prevalent ESBL Plasmids in Escherichia coli. Antibiotic resistance has emerged as one of the most significant threats to global public health. Plasmids, which are highly efficient self-replicating genetic vehicles, play a critical role in the dissemination of drug-resistant genes. Previous studies have mainly focused on drug-resistant genes only, often neglecting the complete functional role of multidrug-resistant (MDR) plasmids in bacteria. In this study, we conducted a comprehensive investigation of the transcriptomes and proteomes of Escherichia coli J53 transconjugants harboring six major MDR plasmids of different incompatibility (Inc) groups, which were clinically isolated from patients. The RNA-seq analysis revealed that MDR plasmids influenced the gene expression in the bacterial host, in particular, the genes related to metabolic pathways. A proteomic analysis demonstrated the plasmid-induced regulation of several metabolic pathways including anaerobic respiration and the utilization of various carbon sources such as serine, threonine, sialic acid, and galactarate. These findings suggested that MDR plasmids confer a growth advantage to bacterial hosts in the gut, leading to the expansion of plasmid-carrying bacteria over competitors without plasmids. Moreover, this study provided insights into the versatility of prevalent MDR plasmids in moderating the cellular gene network of bacteria, which could potentially be utilized in therapeutics development for bacteria carrying MDR plasmids.202337762311
4383120.9996Importance of Core Genome Functions for an Extreme Antibiotic Resistance Trait. Extreme antibiotic resistance in bacteria is associated with the expression of powerful inactivating enzymes and other functions encoded in accessory genomic elements. The contribution of core genome processes to high-level resistance in such bacteria has been unclear. In the work reported here, we evaluated the relative importance of core and accessory functions for high-level resistance to the aminoglycoside tobramycin in the nosocomial pathogen Acinetobacter baumannii Three lines of evidence establish the primacy of core functions in this resistance. First, in a genome scale mutant analysis using transposon sequencing and validation with 594 individual mutants, nearly all mutations reducing tobramycin resistance inactivated core genes, some with stronger phenotypes than those caused by the elimination of aminoglycoside-inactivating enzymes. Second, the core functions mediating resistance were nearly identical in the wild type and a deletion mutant lacking a genome resistance island that encodes the inactivating enzymes. Thus, most or all of the core resistance determinants important in the absence of the enzymes are also important in their presence. Third, reductions in tobramycin resistance caused by different core mutations were additive, and highly sensitive double and triple mutants (with 250-fold reductions in the MIC) that retained accessory resistance genes could be constructed. Core processes that contribute most strongly to intrinsic tobramycin resistance include phospholipid biosynthesis, phosphate regulation, and envelope homeostasis.IMPORTANCE The inexorable increase in bacterial antibiotic resistance threatens to undermine many of the procedures that transformed medicine in the last century. One strategy to meet the challenge antibiotic resistance poses is the development of drugs that undermine resistance. To identify potential targets for such adjuvants, we identified the functions underlying resistance to an important class of antibiotics for one of the most highly resistant pathogens known.201729233894
8382130.9996Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline. Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.201830266728
8384140.9996In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors. Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.201425408687
6335150.9996Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.202134756069
8928160.9996Increased survival of antibiotic-resistant Escherichia coli inside macrophages. Mutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in the rpoB, rpsL, and gyrA genes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits-growth rate and survival ability-of 12 Escherichia coli K-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, all E. coli streptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival of E. coli in the context of an infection.201323089747
6318170.9996Phenotypic differences between Salmonella and Escherichia coli resulting from the disparate regulation of homologous genes. Phenotypic differences among closely related bacteria have been largely ascribed to species-specific genes, such as those residing in pathogenicity islands. However, we now report that the differential regulation of homologous genes is the mechanism responsible for the divergence of the enteric bacteria Salmonella enterica and Escherichia coli in their ability to make LPS modifications mediating resistance to the antibiotic polymyxin B. In S. enterica serovar Typhimurium, the PmrA/PmrB two-component system governing polymyxin B resistance is induced in low Mg(2+) in a process that requires the PmrD protein and by Fe(3+) in a PmrD-independent fashion. We establish that E. coli K-12 induces PmrA-activated gene transcription and polymyxin B resistance in response to Fe(3+), but that it is blind to the low Mg(2+) signal. The highly divergent PmrD protein is responsible for this phenotype as replacement of the E. coli pmrD gene by its Salmonella counterpart resulted in an E. coli strain that transcribed PmrA-activated genes and displayed polymyxin B resistance under the same conditions as Salmonella. Molecular analysis of natural isolates of E. coli and Salmonella revealed that the PmrD proteins are conserved within each genus and that selection might have driven the divergence between the Salmonella and E. coli PmrD proteins. Investigation of PmrD function demonstrated statistically different distributions for the Salmonella and E. coli isolates in PmrD-dependent transcription occurring in low Mg(2+). Our results suggest that the differential regulation of conserved genes may have ecological consequences, determining the range of niches a microorganism can occupy.200415569938
6341180.9996Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression. In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype.202337485524
9270190.9996Activation of class 1 integron integrase is promoted in the intestinal environment. Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.202235482826