The expression regulation of recA gene and bacterial class 2 integron-associated genes induced by antibiotics. - Related Documents




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575701.0000The expression regulation of recA gene and bacterial class 2 integron-associated genes induced by antibiotics. OBJECTIVE: To investigate the effects and mechanisms of common antibiotics induction on the expression of class 2 integron integrase and variable region resistance genes in bacteria, as well as potential structural mutations. METHODS: Clinical isolates containing non-functional class 2 integrons and functional class 2 integrons were selected. Strains containing non-functional class 2 integrons or functional class 2 integrons were constructed using isolated DNA templates. These strains were subjected to continuous induction with drug concentrations of 1/2 MIC and 1/4 MIC (ciprofloxacin, ampicillin, and kanamycin) and a concentration of 0.2 μg/ml (mitomycin C) over 8 days. The relative expression levels of relevant genes were measured on days 1, 3, and 8. Drug resistance in the experimental strains was assessed before and after induction to identify any differences. Finally, the sequence of the non-functional class 2 integron integrase gene was analyzed for structural changes that occurred as a result of induction. RESULTS: All drugs selected in this study increased the relative expression levels of recA, intI2, dfrA1, sat2, and aadA1. Significant differences in inductive abilities were observed among the drugs. The 1/2 MIC concentrations were more effective than 1/4 MIC concentrations in increasing the relative expression levels of target genes and enhancing the resistance of the experimental strains. The relative expression levels of recA, intI2, and dfrA1 rose on day 1, peaked on day 3, and slightly declined by day 8. Induced strains exhibited increased resistance to the drugs, with the most significant changes observed in the clinical isolates, particularly concerning CIP resistance. Notably, clinical isolate 7b induced with 1/2 MIC KAN exhibited the loss of one base at position 12bp in the integrase sequence. However, none of the four drugs induced mutations at the 444 bp position of class 2 integrons. CONCLUSION: Sub-MIC concentrations of drugs have been shown to induce an increase in the relative expression level of the SOS response-related gene recA, as well as the integrase and resistance genes of class 2 integrons. Continuous induction leads to sustained upregulation of these genes, which stabilizes or slightly decreases upon reaching a plateau. However, the capacity of different drugs to induce expression varies significantly. Short-term antibiotic exposure did not result in critical mutations that convert class 2 integrons into functional forms.202540950603
576210.9998Evolution of antimicrobial resistance in E. coli biofilm treated with high doses of ciprofloxacin. The evolution of antimicrobial resistance (AMR) has mainly been studied in planktonic bacteria exposed to sub-inhibitory antimicrobial (AM) concentrations. However, in a number of infections that are treated with AMs the bacteria are located in biofilms where they tolerate high doses of AM. In the present study, we continuously exposed biofilm residing E. coli at body temperature to high ciprofloxacin (CIP) concentrations increasing from 4 to 130 times the minimal inhibitory concentration (MIC), i.e., from 0.06 to 2.0 mg/L. After 1 week, the biofilms were full of CIP resistant bacteria. The evolutionary trajectory observed was the same as described in the literature for planktonic bacteria, i.e., starting with a single mutation in the target gene gyrA followed by mutations in parC, gyrB, and parE, as well as in genes for regulation of multidrug efflux pump systems and outer membrane porins. Strains with higher numbers of these mutations also displayed higher MIC values. Furthermore, the evolution of CIP resistance was more rapid, and resulted in strains with higher MIC values, when the bacteria were biofilm residing than when they were in a planktonic suspension. These results may indicate that extensive clinical AM treatment of biofilm-residing bacteria may not only fail to eradicate the infection but also pose an increased risk of AMR development.202337731931
575620.9998Chlorogenic acid attenuates tet (X)-mediated doxycycline resistance of Riemerella anatipestifer. INTRODUCTION: The increasing resistance of R. anatipestifer has posed a significant threat to the poultry industry in recent years. The tet gene is the primary determinant of tetracycline resistance in numerous bacteria, and the enzyme modification gene tet(X) is predominantly detected in tetracycline-resistant R. anatipestifer strains. METHODS: In this study, we evaluated the susceptibility of both the standard strain and clinical isolates of R. anatipestifer to doxycycline. And the expression levels of tet(X), tet(A), and tet(O) genes were detected. To assess drug susceptibility, shuttle plasmids were constructed to transfer the tet(X) gene into the standard strain of R. anatipestifer followed by treatment with chlorogenic acid. RESULTS AND DISCUSSION: The results revealed that the minimum inhibitory concentration of doxycycline for the standard strain was 0.25μg/mL, whereas it exceeded 8μg/mL for the clinical isolates. Furthermore, there was a significant upregulation observed in expression levels of tet(X), tet(A), and tet(O) genes among induced strains. Interestingly, when transferring the tet(X) gene into the standard strain, its sensitivity to doxycycline decreased; however, MIC values for chlorogenic acid remained consistent between both standard and drug-resistant strains of R. anatipestifer. Moreover, we made a surprising discovery that screening passage with chlorogenic acid resulted in increased sensitivity of R. anatipestifer to doxycycline. Further analysis demonstrated a reversal in expression trends among three differentially expressed genes within induced drug resistance group after intervention with chlorogenic acid. The main objective behind this study is to investigate both killing effect exerted by chlorogenic acid on drug-resistant R. anatipestifer as well as its regulatory impact on drug resistance genes. This will provide novel insights and theoretical basis towards development of chlorogenic acid as a promising drug for treatment and control of drug resistance in R. anatipestifer.202438764851
778530.9998Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine. The practice of urine source-separation for fertilizer production necessitates an understanding of the presence and impact of extracellular DNA in the urine. This study examines the fate of plasmid DNA carrying ampicillin and tetracycline resistance genes in aged urine, including its ability to be taken up and expressed by competent bacteria. Plasmid DNA incubated in aged urine resulted in a >2 log loss of bacterial transformation efficiency in Acinetobacter baylyi within 24 h. The concentration of ampicillin and tetracycline resistance genes, as measured with quantitative polymerase chain reaction, did not correspond with the observed transformation loss. When the plasmid DNA was incubated in aged urine that had been filtered (0.22 μm) or heated (75 °C), the transformation efficiencies were more stable than when the plasmids were incubated in unfiltered and unheated aged urine. Gel electrophoresis results indicated that plasmid linearization by materials larger than 100 kDa in the aged urine caused the observed transformation efficiency decreases. The results of this study suggest that extracellular DNA released into aged urine poses a low potential for the spread of antibiotic resistance genes to bacteria once it is released to the environment.202031965791
457340.9997High pressure processing, acidic and osmotic stress increased resistance to aminoglycosides and tetracyclines and the frequency of gene transfer among strains from commercial starter and protective cultures. This study analyzed the effect of food-related stresses on the expression of antibiotic resistance of starter and protective strains and resistance gene transfer frequency. After exposure to high-pressure processing, acidic and osmotic stress, the expression of genes encoding resistance to aminoglycosides (aac(6')Ie-aph(2″)Ia and aph(3')-IIIa) and/or tetracyclines (tetM) increased. After cold stress, a decrease in the expression level of all tested genes was observed. The results obtained in the gene expression analysis correlated with the results of the phenotype patterns. After acidic and osmotic stresses, a significant increase in the frequency of each gene transfer was observed. To the best of the authors' knowledge, this is the first study focused on changes in antibiotic resistance associated with a stress response among starter and protective strains. The results suggest that the physicochemical factors prevailing during food production and storage may affect the phenotype of antibiotic resistance and the level of expression of antibiotic resistance genes among microorganisms. As a result, they can contribute to the spread of antibiotic resistance. This points to the need to verify strains used in the food industry for their antibiotic resistance to prevent them from becoming a reservoir for antibiotic resistance genes.202235953184
626250.9997Potential of Tetracycline Resistance Proteins To Evolve Tigecycline Resistance. Tigecycline is a glycylcycline antibiotic active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M), and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant, since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines.201626596936
457260.9997Effect of high pressure processing on changes in antibiotic resistance genes expression among strains from commercial starter cultures. This study analyzed the effect of high-pressure processing on the changes in resistance phenotype and expression of antibiotic resistance genes among strains from commercial starter cultures. After exposure to high pressure the expression of genes encoding resistance to aminoglycosides (aac(6')Ie-aph(2″)Ia and aph(3')-IIIa) decreased and the expression of genes encoding resistance to tetracyclines (tetM and tetW), ampicillin (blaZ) and chloramphenicol (cat) increased. Expression changes differed depending on the pressure variant chosen. The results obtained in the gene expression analysis correlated with the results of the phenotype patterns. To the best of the authors' knowledge, this is one of the first studies focused on changes in antibiotic resistance associated with a stress response among strains from commercial starter cultures. The results suggest that the food preservation techniques might affect the phenotype of antibiotic resistance among microorganisms that ultimately survive the process. This points to the need to verify strains used in the food industry for their antibiotic resistance as well as preservation parameters to prevent the further increase in antibiotic resistance in food borne strains.202336462825
457170.9997Growth of soil bacteria, on penicillin and neomycin, not previously exposed to these antibiotics. There is growing evidence that bacteria, in the natural environment (e.g. the soil), can exhibit naturally occurring resistance/degradation against synthetic antibiotics. Our aim was to assess whether soils, not previously exposed to synthetic antibiotics, contained bacterial strains that were not only antibiotic resistant, but could actually utilize the antibiotics for energy and nutrients. We isolated 19 bacteria from four diverse soils that had the capability of growing on penicillin and neomycin as sole carbon sources up to concentrations of 1000 mg L(-1). The 19 bacterial isolates represent a diverse set of species in the phyla Proteobacteria (84%) and Bacteroidetes (16%). Nine antibiotic resistant genes were detected in the four soils but some of these genes (i.e. tetM, ermB, and sulI) were not detected in the soil isolates indicating the presence of unculturable antibiotic resistant bacteria. Most isolates that could subsist on penicillin or neomycin as sole carbon sources were also resistant to the presence of these two antibiotics and six other antibiotics at concentrations of either 20 or 1000 mg L(-1). The potentially large and diverse pool of antibiotic resistant and degradation genes implies ecological and health impacts yet to be explored and fully understood.201424956077
576380.9997Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa. Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA(83) mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance.202032758289
565190.9997Class 1 integron causes vulnerability to formaldehyde in Escherichia coli. In this study, the relationships of integron 1 element, formaldehyde dehydrogenase, and orfF genes with the level of formaldehyde resistance of isolated E. coli were investigated. E. coli bacteria were isolated from apparently healthy and colibacillosis-affected broilers of Fars Province, Iran. Formaldehyde resistance level and the presence of genetic markers were measured using MIC, and PCR tests, respectively. The prevalence of integron 1 element, orfF, and formaldehyde dehydrogenase genes in E. coli isolates were 61%, 8%, and 94%, respectively. In addition, according to our cut off definition, 15% and 85% of isolates were resistant and sensitive to formaldehyde, respectively. None of the genes had a statistically significant relationship with the formaldehyde resistance; however, the isolates containing integron 1 were significantly more sensitive to formaldehyde in the MIC test than those without integron 1. Integron 1 gene cassette could carry some bacterial surface proteins and porins with different roles in bacterial cells. Formaldehyde could also interfere with the protein functions by alkylating and cross-linking, and this compound would affect bacterial cell surface proteins in advance. Through an increase in the cell surface proteins, the presence of integron 1 gene cassette might make E. coli more sensitive to formaldehyde. As integron 1 was always involved in increasing bacterial resistance to antibiotics and disinfectants such as QACs, this is the first report of bacterial induction of sensitivity to a disinfectant through integron 1. Finally, integron 1 does not always add an advantage to E. coli bacteria, and it could be assumed as a cause of vulnerability to formaldehyde.202134148112
3610100.9997Quantitative real-time PCR study of the expression and regulation of the tetracycline resistance gene in Riemerella anatipestifer. Riemerella anatipestifer (RA) is one of the most important pathogens of 1- to 8-wk-old ducklings that severely affects the development of the duck industry in China. Every year, antibiotic medicines including tetracycline and doxycycline are used in the duck industry. Few reports compare the expression of multidrug-resistant genes in RA before and after addition of chemical drugs. With this in mind, the direct effects of gradient concentration of tetracyclines on the expression of tetracycline resistance genes (TETr) in RA at the cDNA level were studied by using a quantitative real-time PCR method. The expression of TETr, tetA, tetC, and tetM was investigated in ATCC11845 and in 30 RA isolated from different samples. Using a range of doxycycline concentrations up to 50% of the minimum inhibitory concentration (MIC), the optimal induction concentration of 0.0625 μg/mL was selected. Under the optimal inducible expression, concentrations of TETr, tetC, and tetM cDNA were detected in all isolates, and the highest mRNA expression level of TETr genes was shown. Additionally, the expression levels of 3 TETr genes in RA14 (tetA and tetC) and RA17 (tetM and tetC) were compared. Both tetC and tetA found in isolate RA14 was found to express both tetC and tetA, and tetC cDNA was detected in isolate RA17 at all doxycycline concentrations tested, whereas tetM cDNA was not detected at any concentration. We can conclude that resistance pump is the main mechanism of tetracycline antibiotic resistance, and under the action of drug resistance pump tetC, the expression of tetM was not activated in RA17. These data suggest that the mRNA expression level of TETr genes was correlated with the MIC values, indicating that the degree of drug resistance is determined by the expression levels of TETr genes. Also, the induction of TETr is the major tetracycline resistance mechanism in RA, especially the resistance pump. However, lower concentrations of doxycycline induced higher TETr expression, and higher concentrations inhibited TETr expression. Maybe that is the reason for selection mutation to make tolerated bacteria survive.201323687151
3586110.9997Comparison of Plasmid Curing Efficiency across Five Lactic Acid Bacterial Species. With the recent stringent criteria for antibiotic susceptibility in probiotics, the presence of antibiotic resistance genes and plasmids associated with their transfer has become a limiting factor in the approval of probiotics. The need to remove genes related to antibiotic resistance and virulence through plasmid curing for the authorization of probiotics is increasing. In this study, we investigated the curing efficiency of ethidium bromide, acridine orange, and novobiocin at different concentrations and durations in five strains of plasmid-bearing lactic acid bacteria and examined the curing characteristics in each strain. Limosibacillus reuteri and Lacticaseibacillus paracasei exhibited curing efficiencies ranging from 5% to 44% following treatment with ethidium bromide (10-50 μg/ml) for 24-72 h, while Lactobacillus gasseri showed the highest efficiency at 14% following treatment with 10 μg/ml novobiocin for 24 h. Lactiplantibacillus plantarum, which harbors two or more plasmids, demonstrated curing efficiencies ranging from 1% to 8% after an additional 72-h treatment of partially cured strains with 10 μg/ml novobiocin. Plasmid curing in strains with larger plasmids exhibited lower efficiencies and required longer durations. In strains harboring two or more plasmids, a relatively low curing efficiency with a single treatment and a high frequency of false positives, wherein recovery occurred after curing, were observed. Although certain strains exhibited altered susceptibilities to specific antibiotics after curing, these outcomes could not be attributed to the loss of antibiotic resistance genes. Furthermore, the genomic data from the cured strains revealed minimal changes throughout the genome that did not lead to gene mutations.202439403731
3601120.9997R factors mediate resistance to mercury, nickel, and cobalt. Fifty-five clinical isolates and laboratory stocks of Escherichia coli and Salmonella were studied for resistance to each of ten metals. Eleven clinical isolates carrying R factors were resistant to mercury, and, in each case, the resistance was mediated by a previously undefined R-factor gene. The gene was phenotypically expressed within 2 to 4 minutes after entry into sensitive bacteria, but the basis for the resistance remains undefined. Fourteen strains, 12 infected with R factors, were resistant to cobalt and nickel, but these resistances were mediated by R-factor genes in only two strains; separate R-factor genes mediated the resistances to nickel and cobalt. These and other results indicate that the genetic composition of R factors is greater than that originally defined.19675337360
5836130.9997Identification of Pseudomonas aeruginosa genes associated with antibiotic susceptibility. Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria's high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates containing antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at 1/2 MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in susceptibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resistance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold increase in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.201020953948
2801140.9997Principal component analysis exploring the association between antibiotic resistance and heavy metal tolerance of plasmid-bearing sewage wastewater bacteria of clinical relevance. This paper unravels the occurrence of plasmid-mediated antibiotic resistance in association with tolerance to heavy metals among clinically relevant bacteria isolated from sewage wastewater. The bacteria isolated were identified following conventional phenotypic and/or molecular methods, and were subjected to multiple-antibiotic resistance (MAR) profiling. The isolates were tested against the heavy metals Hg(2+), Cd(2+), Cr(2+) and Cu(2+). SDS-PAGE and agarose gel electrophoretic analyses were performed, respectively, for the characterization of heavy metal stress protein and R-plasmid among the isolated bacteria. Principal component analysis was applied in determining bacterial resistance to antibiotics and heavy metals. Both lactose-fermenting ( Escherichia coli ) and non-fermenting ( Acinetobacter baumannii and Pseudomonas putida ) Gram-negative bacterial strains were procured, and showed MAR phenotypes with respect to three or more antibiotics, along with resistance to the heavy metals Hg(2+), Cd(2+), Cr(2+) and Cu(2+). The Gram-positive bacteria, Enterococcus faecalis , isolated had 'ampicillin-kanamycin-nalidixic acid' resistance. The bacterial isolates had MAR indices of 0.3-0.9, indicating their ( E. faecalis , E. coli , A. baumannii and P. putida ) origin from niches with high antibiotic pollution and human faecal contamination. The Gram-negative bacteria isolated contained a single plasmid (≈54 kb) conferring multiple antibiotic resistance, which was linked to heavy metal tolerance; the SDS-PAGE analysis demonstrated the expression of heavy metal stress proteins (≈59 and ≈10 kDa) in wastewater bacteria with a Cd(2+) stressor. The study results grant an insight into the co-occurrence of antibiotic resistance and heavy metal tolerance among clinically relevant bacteria in sewage wastewater, prompting an intense health impact over antibiotic usage.202032974572
4739150.9997Indirect resistance to several classes of antibiotics in cocultures with resistant bacteria expressing antibiotic-modifying or -degrading enzymes. OBJECTIVES: Indirect resistance (IR), the ability of an antibiotic-resistant population of bacteria to protect a susceptible population, has been previously observed for β-lactamase-producing bacteria and associated with antimicrobial treatment failures. Here, we determined whether other resistance determinants could cause IR in the presence of five other classes of antibiotics. METHODS: A test was designed to detect IR and 14 antibiotic resistance genes were tested in the presence of 13 antibiotics from six classes. A bioassay was used to measure the ability of resistance-causing enzymes to decrease the concentration of active antibiotics in the medium. RESULTS: We confirmed IR in the presence of β-lactam antibiotics (ampicillin and mecillinam) when TEM-1A was expressed. We found that bacteria expressing antibiotic-modifying or -degrading enzymes Ere(A), Tet(X2) or CatA1 caused IR in the presence of macrolides (erythromycin and clarithromycin), tetracyclines (tetracycline and tigecycline) and chloramphenicol, respectively. IR was not observed with resistance determinants that did not modify or destroy antibiotics or with enzymes modifying aminoglycosides or degrading fosfomycin. IR was dependent on the resistance enzymes decreasing the concentration of active antibiotics in the medium, hence allowing nearby susceptible bacteria to resume growth once the antibiotic concentration fell below their MIC. CONCLUSIONS: IR was not limited to β-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.201626467993
5761160.9997The Effects of Sub-inhibitory Antibiotic Concentrations on Pseudomonas aeruginosa: Reduced Susceptibility Due to Mutations. Pseudomonas aeruginosa chronically infects in the lungs of people with cystic fibrosis and other forms of lung disease. Infections are treated with antibiotics, but over time, the bacteria acquire mutations that reduce their antibiotic susceptibility. The effects of inhibitory amounts of antibiotics in selecting for antibiotic-resistant mutants have been well studied. However, the concentrations of antibiotics that reach infecting bacteria can be sub-inhibitory and but may nonetheless promote emergence of antibiotic-resistant bacteria. Therefore, the aim of this research was to investigate the effects of sub-inhibitory concentrations of antibiotics on the antibiotic susceptibility of P. aeruginosa. Two P. aeruginosa reference strains, PAO1 and PA14, and six isolates from individuals with cystic fibrosis were studied. The bacteria were passaged in the presence of antibiotics (ceftazidime, ciprofloxacin, meropenem or tobramycin) at sub-inhibitory amounts. Fifteen populations of bacteria (up to five per strain) were exposed to each of the four antibiotics. Antibiotic susceptibility was determined following 10 passages on agar supplemented with antibiotic and compared with susceptibility prior to antibiotic exposure. Antibiotic exposure resulted in susceptibility being significantly (>2-fold) reduced for 13 of the 60 populations. Seven samples had reduced susceptibility to ciprofloxacin, three to tobramycin, two to ceftazidime and one to meropenem. Whole-genome sequencing revealed the mutations arising following antibiotic exposure. Mutants with reduced antibiotic susceptibility had mutations in genes known to affect antibiotic resistance, including regulators of efflux pumps (mexR, mexS, mexZ and nalC) and the fusA1 gene that is associated with aminoglycoside resistance. Genes not previously associated with resistance, including gacS, sigX and crfX and two genes with no known function, were also mutated in some isolates with reduced antibiotic susceptibility. Our results show that exposure to sub-inhibitory amounts of antibiotics can select for mutations that reduce the susceptibility of P. aeruginosa to antibiotics and that the profile of mutations is different from that arising during selection with inhibitory antibiotic concentrations. It is likely that exposure to sub-inhibitory amounts of antibiotics during infection contributes to P. aeruginosa becoming antibiotic-resistant.202134987489
3685170.9997Selective concentration for ciprofloxacin resistance in Escherichia coli grown in complex aquatic bacterial biofilms. There is concern that antibiotics in the environment can select for and enrich bacteria carrying acquired antibiotic resistance genes, thus increasing the potential of those genes to emerge in a clinical context. A critical question for understanding and managing such risks is what levels of antibiotics are needed to select for resistance in complex bacterial communities. Here, we address this question by examining the phenotypic and genotypic profiles of aquatic communities exposed to ciprofloxacin, also evaluating the within-species selection of resistant E. coli in complex communities. The taxonomic composition was significantly altered at ciprofloxacin exposure concentrations down to 1 μg/L. Shotgun metagenomic analysis indicated that mobile quinolone resistance determinants (qnrD, qnrS and qnrB) were enriched as a direct consequence of ciprofloxacin exposure from 1 μg/L or higher. Only at 5-10 μg/L resistant E.coli increased relative to their sensitive counterparts. These resistant E. coli predominantly harbored non-transferrable, chromosomal triple mutations (gyrA S83 L, D87N and parC S80I), which confer high-level resistance. In a controlled experimental setup such as this, we interpret effects on taxonomic composition and enrichment of mobile quinolone resistance genes as relevant indicators of risk. Hence, the lowest observed effect concentration for resistance selection in complex communities by ciprofloxacin was 1 μg/L and the corresponding no observed effect concentration 0.1 μg/L. These findings can be used to define and implement discharge or surface water limits to reduce risks for selection of antibiotic resistance in the environment.201829704804
3805180.9997De Novo Characterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli. Sensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in an Escherichia coli mutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level.201627431218
4738190.9997Detection and evaluation of susceptibility to antibiotics in non-hydrogen sulfide-producing antibiotic-resistant soil microbe: Pseudomonas guariconensis. Antimicrobial resistance in bacteria is a global threat that can make antibacterial treatments ineffective. One well-known method of antibiotic resistance and a common defensive mechanism in many harmful bacteria is the synthesis of endogenous hydrogen sulfide (H(2)S) in bacteria. In this study, soil bacteria were screened using the lead acetate agar test and the triple sugar iron test to determine that they were non-endogenous H(2)S producers. This was further validated by full genome analysis of the identified organism against the gene sequences of H(2)S-producing genes. Antibacterial resistance of the bacteria was phenotypically analyzed using the Kirby-Bauer disk diffusion method. Then, the effect of exogenous H(2)S on the antibiotic-resistant bacteria was checked in sodium sulfide, leading to antibiotic re-sensitization.202538767682