Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space. - Related Documents




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884301.0000Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space. Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease.IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.201931409659
884210.9998Transcriptomic study of Salmonella enterica subspecies enterica serovar Typhi biofilm. BACKGROUND: Typhoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi). In chronic carriers, the bacteria survive the harsh environment of the gallbladder by producing biofilm. The phenotype of S. Typhi biofilm cells is significantly different from the free-swimming planktonic cells, and studies have shown that they are associated with antibiotic resistance, immune system evasion, and bacterial persistence. However, the mechanism of this transition and the events leading to biofilm formation are unknown. High throughput sequencing was performed to identify the genes involved in biofilm formation and to postulate the mechanism of action. RESULTS: Planktonic S. Typhi cells were cultured using standard nutrient broth whereas biofilm cells were cultured in a stressful environment using high shearing-force and bile to mimic the gallbladder. Sequencing libraries were prepared from S. Typhi planktonic cells and mature biofilm cells using the Illumina HiSeq 2500 platform, and the transcriptome data obtained were processed using Cufflinks bioinformatics suite of programs to investigate differential gene expression between the two phenotypes. A total of 35 up-regulated and 29 down-regulated genes were identified. The identities of the differentially expressed genes were confirmed using NCBI BLAST and their functions were analyzed. The results showed that the genes associated with metabolic processes and biofilm regulations were down-regulated while those associated with the membrane matrix and antibiotic resistance were highly up-regulated. CONCLUSIONS: It is proposed that the biofilm phenotype of S. Typhi allows the bacteria to increase production of the membrane matrix in order to serve as a physical shield and to adhere to surfaces, and enter an energy conservation state in response to the stressful environment. Conversely, the planktonic phenotype allows the bacteria to produce flagella and increase metabolic activity to enable the bacteria to migrate and form new colonies of infection. This data provide a basis for further studies to uncover the mechanism of biofilm formation in S. Typhi and to discover novel genes or pathways associated with the development of the typhoid carrier state.201729089020
627920.9997Comparative transcriptomics analyses of the different growth states of multidrug-resistant Acinetobacter baumannii. Multidrug-resistant (MDR) Acinetobacter baumannii is an important bacterial pathogen commonly associated with hospital acquired infections. A. baumannii can remain viable and hence virulent in the environment for a long period of time due primarily to its ability to form biofilms. A total of 459 cases of MDR A. baumannii our hospital collected from March 2014 to March 2015 were examined in this study, and a representative isolate selected for high-throughput mRNA sequencing and comparison of gene expression profiles under the biofilm and exponential growth conditions. Our study found that the same bacteria indeed exhibited differential mRNA expression under different conditions. Compared to the rapidly growing bacteria, biofilm bacteria had 106 genes upregulated and 92 genes downregulated. Bioinformatics analyses suggested that many of these genes are involved in the formation and maintenance of biofilms, whose expression also depends on the environment and specific signaling pathways and transcription factors that are absent in the log phase bacteria. These differentially expressed mRNAs might contribute to A. baumannii's unique pathogenicity and ability to inflict chronic and recurrent infections.201727916419
629030.9997Transcriptomic profiling of ceftriaxone-tolerant phenotypes of Neisseria gonorrhoeae reveals downregulation of ribosomal genes - a pilot study. Antibiotic tolerance is associated with failure of antibiotic treatment and accelerates the development of antimicrobial resistance. The molecular mechanisms underlying antimicrobial tolerance remain poorly understood. Tolerant bacteria can slow metabolism by extending the lag phase without altering antimicrobial susceptibility. We recently induced ceftriaxone (CRO) tolerance in the Neisseria gonorrhoeae reference strain WHO P. In the current study, we characterized the transcriptomic profiles of these CRO-tolerant phenotypes. To induce tolerance, WHO P strains were grown under 3-h intermittent CRO exposure (10× the MIC), followed by overnight growth in gonococcal (GC) broth for seven consecutive days, with cultures maintained in sextuplicate. Two control cultures were maintained without CRO exposure. The tolerance and CRO susceptibility of the isolates were assessed using a modified tolerance disc (TD) test. Total RNA was isolated from tolerant isolates (n = 12) and control (n = 3) strains, followed by Ribo depletion, Illumina Library preparation, and sequencing. Transcriptomic analysis revealed no differentially expressed genes after 1 day of CRO exposure. However, after 3 days of CRO exposure, 13 genes were found to be significantly downregulated, including tRNA-Ser (C7S06_RS03100) and tRNA-Leu (C7S06_RS04945) and ribosomal RNA genes (16S and 23S rRNA). Following 7 days of exposure, 51 genes were differentially expressed, with most downregulated, such as SecB (Protein-export chaperone SecB) and tRNA-Ser (C7S06_RS01850) and the 16S and 23S ribosomal RNA genes. The development of CRO-tolerance in N. gonorrhoeae was associated with the downregulation of various ribosomal genes and associated genes, reflecting a potential mechanism for bacterial survival under antibiotic stress. IMPORTANCE: Antibiotic tolerance allows some bacteria to survive antibiotic treatment, contributing to treatment failure and creating conditions that promote resistance. In this study, we showed that Neisseria gonorrhoeae, the bacteria that causes gonorrhea, can become tolerant to ceftriaxone-the last-line treatment used. By repeatedly exposing the bacteria to high doses of ceftriaxone, we observed the development of tolerance over several days. Using transcriptomic analysis, we found that tolerant bacteria consistently reduced the activity of genes involved in protein synthesis, including ribosomal RNAs and transfer RNAs. This suggests that N. gonorrhoeae may survive antibiotic stress by entering a low-metabolic state that makes the antibiotic less effective. These findings highlight a survival mechanism that does not rely on genetic resistance. Understanding this tolerance response is vital for improving current treatment approaches and could inform the development of new strategies to prevent antibiotic failure in gonorrhea and other infections.202540622217
627840.9997Genome evolution drives transcriptomic and phenotypic adaptation in Pseudomonas aeruginosa during 20 years of infection. The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lungs of patients with cystic fibrosis (CF). During infection the bacteria evolve and adapt to the lung environment. Here we use genomic, transcriptomic and phenotypic approaches to compare multiple isolates of P. aeruginosa collected more than 20 years apart during a chronic infection in a CF patient. Complete genome sequencing of the isolates, using short- and long-read technologies, showed that a genetic bottleneck occurred during infection and was followed by diversification of the bacteria. A 125 kb deletion, an 0.9 Mb inversion and hundreds of smaller mutations occurred during evolution of the bacteria in the lung, with an average rate of 17 mutations per year. Many of the mutated genes are associated with infection or antibiotic resistance. RNA sequencing was used to compare the transcriptomes of an earlier and a later isolate. Substantial reprogramming of the transcriptional network had occurred, affecting multiple genes that contribute to continuing infection. Changes included greatly reduced expression of flagellar machinery and increased expression of genes for nutrient acquisition and biofilm formation, as well as altered expression of a large number of genes of unknown function. Phenotypic studies showed that most later isolates had increased cell adherence and antibiotic resistance, reduced motility, and reduced production of pyoverdine (an iron-scavenging siderophore), consistent with genomic and transcriptomic data. The approach of integrating genomic, transcriptomic and phenotypic analyses reveals, and helps to explain, the plethora of changes that P. aeruginosa undergoes to enable it to adapt to the environment of the CF lung during a chronic infection.202134826267
889350.9997Transcriptome of uropathogenic Escherichia coli during urinary tract infection. A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.200415501767
461560.9997Effect of conditioned media from Aeromonas caviae on the transcriptomic changes of the porcine isolates of Pasteurella multocida. BACKGROUND: Pasteurella multocida is an opportunistic pathogen causing porcine respiratory diseases by co-infections with other bacterial and viral pathogens. Various bacterial genera isolated from porcine respiratory tracts were shown to inhibit the growth of the porcine isolates of P. multocida. However, molecular mechanisms during the interaction between P. multocida and these commensal bacteria had not been examined.  METHODS: This study aimed to investigate the interaction between two porcine isolates of P. multocida (PM2 for type D and PM7 for type A) with Aeromonas caviae selected from the previously published work by co-culturing P. multocida in the conditioned media prepared from A. caviae growth and examining transcriptomic changes using RNA sequencing and bioinformatics analysis.  RESULTS: In total, 629 differentially expressed genes were observed in the isolate with capsular type D, while 110 genes were significantly shown in type A. High expression of genes required for energy metabolisms, nutrient uptakes, and quorum sensing were keys to the growth and adaptation to the conditioned media, together with the decreased expression of those in the unurgent pathways, including translation and antibacterial resistance. CONCLUSION: This transcriptomic analysis also displayed the distinct capability of the two isolates of P. multocida and the preference of the capsular type A isolate in response to the tough environment of the A. caviae conditioned media. Therefore, controlling the environmental sensing and nutrient acquisition mechanisms of P. multocida would possibly prevent the overpopulation of these bacteria and reduce the chance of becoming opportunistic pathogens.202236368971
633270.9997Search 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
470880.9997Proteomic 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
631990.9996Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica. Heteroresistance, a phenomenon where subpopulations of a bacterial isolate exhibit different susceptibilities to an antibiotic, is a growing clinical problem where the underlying genetic mechanisms in most cases remain unknown. We isolated colistin resistant mutants in Escherichia coli and Salmonella enterica serovar Typhimurium at different concentrations of colistin. Genetic analysis showed that genetically stable pmrAB point mutations were responsible for colistin resistance during selection at high drug concentrations for both species and at low concentrations for E. coli. In contrast, for S. Typhimurium mutants selected at low colistin concentrations, amplification of different large chromosomal regions conferred a heteroresistant phenotype. All amplifications included the pmrD gene, which encodes a positive regulator that up-regulates proteins that modify lipid A, and as a result increase colistin resistance. Inactivation and over-expression of the pmrD gene prevented and conferred resistance, respectively, demonstrating that the PmrD protein is required and sufficient to confer resistance. The heteroresistance phenotype is explained by the variable gene dosage of pmrD in a population, where sub-populations with different copy number of the pmrD gene show different levels of colistin resistance. We propose that variability in gene copy number of resistance genes can explain the heteroresistance observed in clinically isolated pathogenic bacteria.201627381382
8414100.9996Patterns of Piscirickettsia salmonis load in susceptible and resistant families of Salmo salar. The pathogen Piscirickettsia salmonis produces a systemic aggressive infection that involves several organs and tissues in salmonids. In spite of the great economic losses caused by this pathogen in the Atlantic salmon (Salmo salar) industry, very little is known about the resistance mechanisms of the host to this pathogen. In this paper, for the first time, we aimed to identify the bacterial load in head kidney and muscle of Atlantic salmon exhibiting differential familiar mortality. Furthermore, in order to assess the patterns of gene expression of immune related genes in susceptible and resistant families, a set of candidate genes was evaluated using deep sequencing of the transcriptome. The results showed that the bacterial load was significantly lower in resistant fish, when compared with the susceptible individuals. Based on the candidate genes analysis, we infer that the resistant hosts triggered up-regulation of specific genes (such as for example the LysC), which may explain a decrease in the bacterial load in head kidney, while the susceptible fish presented an exacerbated innate response, which is unable to exert an effective response against the bacteria. Interestingly, we found a higher bacterial load in muscle when compared with head kidney. We argue that this is possible due to the availability of an additional source of iron in muscle. Besides, the results show that the resistant fish could not be a likely reservoir of the bacteria.201525862974
4807110.9996Age influences resistance of Caenorhabditis elegans to killing by pathogenic bacteria. Caenorhabditis elegans has previously been proposed as an alternative host for models of infectious disease caused by human pathogens. When exposed to some human pathogenic bacteria, the life span of nematodes is significantly reduced. We have shown that mutations in the age-1, and/or age-2 genes of C. elegans, that normally enhance life expectancy, can also increase resistance to killing by the bacterial pathogens Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium, Burkholderia cepacia or Yersinia pseudotuberculosis. We also found that the rate at which wild-type C. elegans was killed by the bacterial pathogens tested increased as nematodes aged. In the case of P. aeruginosa infection, the difference in life span of wild type and age-1 mutants of C. elegans was not due to differences in the level of bacterial colonisation of the gut.200415135534
4703120.9996Positive adaptive state: microarray evaluation of gene expression in Salmonella enterica Typhimurium exposed to nalidixic acid. The emergence of antimicrobial resistance among foodborne bacteria associated with food animal production is an important global issue. We hypothesised that antibiotics generate a positive adaptive state in Salmonella that actively contributes to the development of antimicrobial resistance. This is opposed to common views that antimicrobials only act as a passive selective pressure. Microarray analysis was used to evaluate changes in gene expression that occur upon exposure of Salmonella enterica Typhimurium ATCC 14028 to 1.6 microg/mL of nalidixic acid. The results showed a significant (P < 0.02) difference (fold expression differences >2.0) in the expression of 226 genes. Comparatively repressed transcripts included Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Induced genes included efflux pumps representing all five families of multidrug-resistance efflux pumps, outer membrane lipoproteins, and genes involved in regulating lipopolysaccharide chain length. This profile suggests both enhanced antimicrobial export from the cell and membrane permeability adaptations to limit diffusion of nalidixic acid into the cell. Finally, increased expression of the error-prone DNA repair mechanisms were also observed. From these data we show a highly integrated genetic response to nalidixic acid that places Salmonella into a positive adaptive state that elicits mutations. Evaluation of gene expression profile changes that occur during exposure to antibiotics will continue to improve our understanding of the development of antibiotic resistance.200717600486
6345130.9996Transfer RNA gene numbers may not be completely responsible for the codon usage bias in asparagine, isoleucine, phenylalanine, and tyrosine in the high expression genes in bacteria. It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β', respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.201223053196
6320140.9996Identification of the Extracytoplasmic Function σ Factor σ(P) Regulon in Bacillus thuringiensis. Bacillus thuringiensis and other members of the Bacillus cereus family are resistant to many β-lactams. Resistance is dependent upon the extracytoplasmic function sigma factor σ(P). We used label-free quantitative proteomics to identify proteins whose expression was dependent upon σ(P). We compared the protein profiles of strains which either lacked σ(P) or overexpressed σ(P). We identified 8 members of the σ(P) regulon which included four β-lactamases as well as three penicillin-binding proteins (PBPs). Using transcriptional reporters, we confirmed that these genes are induced by β-lactams in a σ(P)-dependent manner. These genes were deleted individually or in various combinations to determine their role in resistance to a subset of β-lactams, including ampicillin, methicillin, cephalexin, and cephalothin. We found that different combinations of β-lactamases and PBPs are involved in resistance to different β-lactams. Our data show that B. thuringiensis utilizes a suite of enzymes to protect itself from β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is major concern for public health. β-Lactams remain an important treatment option for many diseases. However, the spread of β-lactam resistance continues to rise. Many pathogens acquire antibiotic resistance from environmental bacteria. Thus, understanding β-lactam resistance in environmental strains may provide insights into additional mechanisms of antibiotic resistance. Here, we describe how a single regulatory system, σ(P), in B. thuringiensis controls expression of multiple genes involved in resistance to β-lactams. Our findings indicate that some of these genes are partially redundant. Our data also suggest that the large number of genes controlled by σ(P) results in increased resistance to a wider range of β-lactam classes than any single gene could provide.202235080471
6328150.9996Inactivation 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
6277160.9996A large-scale whole-genome comparison shows that experimental evolution in response to antibiotics predicts changes in naturally evolved clinical Pseudomonas aeruginosa. Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants could tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study we analysed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479kb arose in experimentally evolved meropenem resistant mutants and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.201931570397
3801170.9996Macrophage 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
8928180.9996Increased 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
8943190.9996Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses. BACKGROUND: Many Gram-positive and Gram-negative bacteria produce large quantities of indole as an intercellular signal in microbial communities. Indole demonstrated to affect gene expression in Escherichia coli as an intra-species signaling molecule. In contrast to E. coli, Salmonella does not produce indole because it does not harbor tnaA, which encodes the enzyme responsible for tryptophan metabolism. Our previous study demonstrated that E. coli-conditioned medium and indole induce expression of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium for inter-species communication; however, the global effect of indole on genes in Salmonella remains unknown. RESULTS: To understand the complete picture of genes regulated by indole, we performed DNA microarray analysis of genes in the S. enterica serovar Typhimurium strain ATCC 14028s affected by indole. Predicted Salmonella phenotypes affected by indole based on the microarray data were also examined in this study. Indole induced expression of genes related to efflux-mediated multidrug resistance, including ramA and acrAB, and repressed those related to host cell invasion encoded in the Salmonella pathogenicity island 1, and flagella production. Reduction of invasive activity and motility of Salmonella by indole was also observed phenotypically. CONCLUSION: Our results suggest that indole is an important signaling molecule for inter-species communication to control drug resistance and virulence of S. enterica.201222632036