Epigenetic inheritance based evolution of antibiotic resistance in bacteria. - Related Documents




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633401.0000Epigenetic 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
633510.9999Gene 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
899420.9999Bacteria can compensate the fitness costs of amplified resistance genes via a bypass mechanism. Antibiotic heteroresistance is a phenotype in which a susceptible bacterial population includes a small subpopulation of cells that are more resistant than the main population. Such resistance can arise by tandem amplification of DNA regions containing resistance genes that in single copy are not sufficient to confer resistance. However, tandem amplifications often carry fitness costs, manifested as reduced growth rates. Here, we investigated if and how these fitness costs can be genetically ameliorated. We evolved four clinical isolates of three bacterial species that show heteroresistance to tobramycin, gentamicin and tetracyclines at increasing antibiotic concentrations above the minimal inhibitory concentration (MIC) of the main susceptible population. This led to a rapid enrichment of resistant cells with up to an 80-fold increase in the resistance gene copy number, an increased MIC, and severely reduced growth rates. When further evolved in the presence of antibiotic, these strains acquired compensatory resistance mutations and showed a reduction in copy number while maintaining high-level resistance. A deterministic model indicated that the loss of amplified units was driven mainly by their fitness costs and that the compensatory mutations did not affect the loss rate of the gene amplifications. Our findings suggest that heteroresistance mediated by copy number changes can facilitate and precede the evolution towards stable resistance.202438485998
892830.9999Increased 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
633340.9999Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda. Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.201728210241
899550.9999Interaction between mutations and regulation of gene expression during development of de novo antibiotic resistance. Bacteria can become resistant not only by horizontal gene transfer or other forms of exchange of genetic information but also by de novo by adaptation at the gene expression level and through DNA mutations. The interrelationship between changes in gene expression and DNA mutations during acquisition of resistance is not well documented. In addition, it is not known whether the DNA mutations leading to resistance always occur in the same order and whether the final result is always identical. The expression of >4,000 genes in Escherichia coli was compared upon adaptation to amoxicillin, tetracycline, and enrofloxacin. During adaptation, known resistance genes were sequenced for mutations that cause resistance. The order of mutations varied within two sets of strains adapted in parallel to amoxicillin and enrofloxacin, respectively, whereas the buildup of resistance was very similar. No specific mutations were related to the rather modest increase in tetracycline resistance. Ribosome-sensed induction and efflux pump activation initially protected the cell through induction of expression and allowed it to survive low levels of antibiotics. Subsequently, mutations were promoted by the stress-induced SOS response that stimulated modulation of genetic instability, and these mutations resulted in resistance to even higher antibiotic concentrations. The initial adaptation at the expression level enabled a subsequent trial and error search for the optimal mutations. The quantitative adjustment of cellular processes at different levels accelerated the acquisition of antibiotic resistance.201424841263
892960.9999Interplay in the selection of fluoroquinolone resistance and bacterial fitness. Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug.200919662169
633070.9999Transcriptomic study of ciprofloxacin resistance in Streptomyces coelicolor A3(2). Soil organisms exhibit resistance to a wide range of antibiotics as they either need to protect themselves from endogenous antibiotics or from those present in their soil environment. The soil could serve as a reservoir for resistance mechanisms that have already emerged or have the potential to emerge in clinically important bacteria. Streptomyces coelicolor, a non-pathogenic soil-dwelling organism, is thus used as a model for the study of intrinsic resistance. Preliminary screening of several compounds showed that S. coelicolor had high intrinsic resistance for the fluoroquinolone group of antibiotics. We subjected the bacteria to sub-inhibitory concentrations of ciprofloxacin and studied the transcriptomic response using microarrays. The data were supported with various biochemical and phenotypic assays. Ciprofloxacin treatment leads to differential expression of many genes with enhanced mRNA expression of its target, DNA gyrase gene. High induction of DNA repair pathways was also observed and many transporters were upregulated. Ciprofloxacin was found to induce ROS formation in a dose dependent manner. Reduction of ROS via anti-oxidants increased the effective MIC of the drug in the bacteria. The regulation of antibiotic resistance in S. coelicolor was studied systematically and contribution of different mechanisms in the development of resistance was assessed. Our data suggest that multiple mechanisms work in coordination to facilitate the cell to combat the stress due to ciprofloxacin.201324100886
632880.9999Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis. Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.201828847541
380290.9999Exposure to One Antibiotic Leads to Acquisition of Resistance to Another Antibiotic via Quorum Sensing Mechanisms. The vancomycin-resistant Enterococci (VRE) have progressively become a severe medical problem. Although clinics have started to reduce vancomycin prescription, vancomycin resistance has not been contained. We found that the transfer of vancomycin resistance in Enterococcus faecalis increased more than 30-fold upon treatment by streptomycin. Notably, treatment with an antibiotic caused the bacteria to become resistant to another. The response was even stronger in the well-studied plasmid pCF10 and the number of transconjugants increased about 100,000-fold. We tested four different antibiotics, and all of them induced conjugal response. Through a mathematical model based on gene regulation, we found a plausible explanation. Via quorum sensing, the change of the cell density triggers the conjugation. Moreover, we searched for generality and found a similar strategy in Bacillus subtilis. The outcome of the present study suggests that even common antibiotics must not be overused.202033552007
6341100.9999Monitoring 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
8913110.9999The 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
8927120.9999Changes in Intrinsic Antibiotic Susceptibility during a Long-Term Evolution Experiment with Escherichia coli. High-level resistance often evolves when populations of bacteria are exposed to antibiotics, by either mutations or horizontally acquired genes. There is also variation in the intrinsic resistance levels of different bacterial strains and species that is not associated with any known history of exposure. In many cases, evolved resistance is costly to the bacteria, such that resistant types have lower fitness than their progenitors in the absence of antibiotics. Some longer-term studies have shown that bacteria often evolve compensatory changes that overcome these tradeoffs, but even those studies have typically lasted only a few hundred generations. In this study, we examine changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any antibiotic. On average, the evolved bacteria were more susceptible to most antibiotics than was their ancestor. The bacteria at 50,000 generations tended to be even more susceptible than after 2,000 generations, although most of the change occurred during the first 2,000 generations. Despite the general trend toward increased susceptibility, we saw diverse outcomes with different antibiotics. For streptomycin, which was the only drug to which the ancestral strain was highly resistant, none of the evolved lines showed any increased susceptibility. The independently evolved lineages often exhibited correlated responses to the antibiotics, with correlations usually corresponding to their modes of action. On balance, our study shows that bacteria with low levels of intrinsic resistance often evolve to become even more susceptible to antibiotics in the absence of corresponding selection.IMPORTANCE Resistance to antibiotics often evolves when bacteria encounter antibiotics. However, bacterial strains and species without any known exposure to these drugs also vary in their intrinsic susceptibility. In many cases, evolved resistance has been shown to be costly to the bacteria, such that resistant types have reduced competitiveness relative to their sensitive progenitors in the absence of antibiotics. In this study, we examined changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any drug. The evolved bacteria tended to become more susceptible to most antibiotics, with most of the change occurring during the first 2,000 generations, when the bacteria were undergoing rapid adaptation to their experimental conditions. On balance, our findings indicate that bacteria with low levels of intrinsic resistance can, in the absence of relevant selection, become even more susceptible to antibiotics.201930837336
4416130.9999Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. Tetracycline-resistant bacteria were first isolated in 1953 from Shigella dysenteriae, a bacterium which causes bacterial dysentery. Since then tetracycline-resistant bacterial have been found in increasing numbers of species and genera. This has resulted in reduced effectiveness of tetracycline therapy over time. Tetracycline resistance is normally due to the acquisition of new genes often associated with either a mobile plasmid or a transposon. These tetracycline resistance determinants are distinguishable both genetically and biochemically. Resistance is primarily due to either energy-dependent efflux of tetracycline or protection of the ribosomes from the action of tetracycline. Gram-negative tetracycline efflux proteins are linked to repressor proteins which in the absence of tetracycline block transcription of the repressor and structural efflux genes. In contrast, expression of the Gram-positive tetracycline efflux genes and some of the ribosomal protection genes appears to be regulated by attenuation of mRNA transcription. Specific tetracycline resistance genes have been identified in 32 Gram-negative and 22 Gram-positive genera. Tetracycline-resistant bacteria are found in pathogens, opportunistic and normal flora species. Tetracycline-resistant bacteria can be isolated from man, animals, food, and the environment. The nonpathogens in each of these ecosystems may play an important role as reservoirs for the antibiotic resistance genes. It is clear that if we are to reverse the trend toward increasingly antibiotic-resistant pathogenic bacteria we will need to change how antibiotics are used in both human and animal health and food production.19968916553
8957140.9999Transcriptome 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
4428150.9999Multidrug resistance in enteric and other gram-negative bacteria. In Gram-negative bacteria, multidrug resistance is a term that is used to describe mechanisms of resistance by chromosomal genes that are activated by induction or mutation caused by the stress of exposure to antibiotics in natural and clinical environments. Unlike plasmid-borne resistance genes, there is no alteration or degradation of drugs or need for genetic transfer. Exposure to a single drug leads to cross-resistance to many other structurally and functionally unrelated drugs. The only mechanism identified for multidrug resistance in bacteria is drug efflux by membrane transporters, even though many of these transporters remain to be identified. The enteric bacteria exhibit mostly complex multidrug resistance systems which are often regulated by operons or regulons. The purpose of this review is to survey molecular mechanisms of multidrug resistance in enteric and other Gram-negative bacteria, and to speculate on the origins and natural physiological functions of the genes involved.19968647368
3801160.9999Macrophage Cell Lines and Murine Infection by Salmonella enterica Serovar Typhi L-Form Bacteria. Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic resistance genes is well established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as that due to treatment with bacterial cell wall-inhibiting antibiotics such as ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin during the treatment of infectious diseases. This is different from in vitro drug testing, where the organism is exposed to fixed drug concentrations from the beginning until the end. To mimic the mode of antibiotic exposure of microorganisms within host tissues, we cultured the wild-type, ampicillin-sensitive Salmonella enterica serovar Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that displayed several features of the so-called L-form of bacteria, including the absence of the cell wall, altered shape, and lower growth rate compared with the parental form. Studies of the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental form. These results suggested that L-form generation following the initiation of treatment with antibiotics could lead to drug escape of S. Typhi and cell to cell (macrophages) spread of the bacteria, which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through the fecal-oral route, highlighting the need for new approaches to decrease the reservoir of infection.202235587200
8930170.9999Repeated Exposure of Escherichia coli to High Ciprofloxacin Concentrations Selects gyrB Mutants That Show Fluoroquinolone-Specific Hyperpersistence. Recent studies have shown that not only resistance, but also tolerance/persistence levels can evolve rapidly in bacteria exposed to repeated antibiotic treatments. We used in vitro evolution to assess whether tolerant/hyperpersistent Escherichia coli ATCC25922 mutants could be selected under repeated exposure to a high ciprofloxacin concentration. Among two out of three independent evolution lines, we observed the emergence of gyrB mutants showing an hyperpersistence phenotype specific to fluoroquinolones, but no significant MIC increase. The identified mutation gives rise to a L422P substitution in GyrB, that is, outside of the canonical GyrB QRDR. Our results indicate that mutations in overlooked regions of quinolone target genes may impair the efficacy of treatments via an increase of persistence rather than resistance level, and support the idea that, in addition to resistance, phenotypes of tolerance/persistence of infectious bacterial strains should receive considerations in the choice of antibiotic therapies.202235707170
6332180.9999Search and analysis of genes involved in antibiotic resistance in Chilean strains of Piscirickettsia salmonis. Piscirickettsia salmonis is the pathogen causing Piscirickettsiosis. For treatment, the industry mainly uses oxytetracycline and florfenicol, so it is essential to understand the degree of susceptibility of this pathogen to these drugs. But this is still unknown for a large number of P. salmonis strains, as are the molecular mechanisms responsible for greater or lesser susceptibility. However, genes that confer resistance to these antimicrobials have been reported and characterized for this and other bacterial species, among which are membrane proteins that take out the drug. Our results identified differences in the degree of susceptibility to both antibiotics among different Chilean isolated of these bacteria. We analysed 10 available genomes in our laboratory and identified ~140 genes likely to be involved in antibiotic resistance. We analysed six specific genes, which suggests that some of them would eventually be relevant in conferring resistance to both antibiotics, as they encode for specific transporter proteins, which increase the number of transcripts when grown in media with these antibiotics. Our results were corroborated with EtBr permeability analysis, which revealed that the LF-89 strain accumulates this compound and has a reduced capacity to expulse it compared with the field strains.201727982445
6314190.9999Identification of genes involved in the resistance of mycobacteria to killing by macrophages. The survival of M. leprae and M. tuberculosis in the human host is dependent upon their ability to produce gene products that counteract the bactericidal activities of macrophages. To identify such mycobacterial genes and gene products, recombinant DNA libraries of mycobacterial DNA in E. coli were passed through macrophages to enrich for clones carrying genes that endow the normally susceptible E. coli bacteria with an enhanced ability to survive within macrophages. Following three cycles of enrichment, 15 independent clones were isolated. Three recombinants were characterized in detail, and each confers significantly enhanced survival on E. coli cells carrying them. Two of the cloned genetic elements also confer enhanced survival onto M. smegmatis cells. Further characterization of these genes and gene products should provide insights into the survival of mycobacteria within macrophages and may identify new approaches of targets for combatting these important pathogens.19948080180