High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7. - Related Documents




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595901.0000High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7. The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR(+) open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10-100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.201424361397
598110.9997Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.19968723458
360120.9996R 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
595830.9996Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress. Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.202438378700
598540.9996Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae. BACKGROUND: Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. OBJECTIVES: To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. METHODS: Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. RESULTS: By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. CONCLUSIONS: These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.202236124853
565150.9996Class 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
597960.9996Mutations in gyrA, gyrB, parC, and parE in quinolone-resistant strains of Neisseria gonorrhoeae. Mutations in the genes for the subunits GyrA and ParC of the target enzymes DNA gyrase and topoisomerase IV are important mechanisms of resistance in quinolone-resistant bacteria, including Neisseria gonorrhoeae. The target enzymes also consist of the subunits GyrB and ParE, respectively, though their role in quinolone-resistance has not been fully investigated. We sequenced the quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE in 25 ciprofloxacin-resistant strains from Bangladesh (MIC 4-->32 mg/l) and 5 susceptible strains of N. gonorrhoeae. All the resistant strains had three or four mutations. Two of these were at positions 91 and 95 of gyrA. Fourteen strains had an additional mutation in parC at position 91, and 17 strains had an additional mutation in parE in position 439. No alterations were found in gyrB. The five susceptible strains had identical DNA sequences. Data indicate that the mutations detected in the QRDR of gyrA and parC may be important in the development of quinolone resistance. According to transformation experiments we assume that the alteration in parE is not related to a high degree of quinolone resistance. There was no correlation between ciprofloxacin MICs and pattern or number of mutations in the target genes.200212529019
45770.9996Molecular characterization of the genes encoding DNA gyrase and topoisomerase IV of Listeria monocytogenes. The genes encoding subunits A and B of DNA gyrase and subunits C and E of topoisomerase IV of Listeria monocytogenes, gyrA, gyrB, parC and parE, respectively, were cloned and sequenced. Compared with the sequences of quinolone-susceptible bacteria, such as Escherichia coli and Bacillus subtilis, the quinolone resistance-determining region (QRDR) of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed the substitutions Ser-84-->Thr and Asp/Glu-88-->Phe, two amino acid variations at hot spots, commonly associated with resistance to quinolones. No relevant divergences from QRDR consensus sequences were observed in GyrB or both topoisomerase IV subunits. Thus, it could be argued that the amino acid substitutions in GyrA would explain the intrinsic resistance of L. monocytogenes to nalidixic acid. In order to analyse the actual role of the GyrA alterations, a plasmid-encoded gyrA allele was mutated and transformed into L. monocytogenes. However, these heterodiploid strains were not affected in their resistance to nalidixic acid. The effects of the mutant plasmids on ciprofloxacin and sparfloxacin susceptibility were only modest.200212039883
626480.9996Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains. OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.202438041251
42190.9995Effect of pap copy number and receptor specificity on virulence of fimbriated Escherichia coli in a murine urinary tract colonization model. Escherichia coli FN506 containing pap genes that encode two different P fimbriae adherence specificity types were tested for virulence in a murine urinary colonization model. Strains containing adherence genes on either high copy or low copy plasmids were compared. Bacteria that harbored the adherence genes on high copy plasmids colonized mouse kidneys less well than bacteria with the same adherence genes in low copy even though the high copy strains exhibited greater hemagglutination capacity. Bacteria with either type of P fimbriae were able to colonize but pap-2+ bacteria showed increased colonizing capacity when strains containing pap-1 or pap-2 genes on low copy plasmids were compared. Bacteria containing plasmids with both adherence specificities had a similar colonizing capacity as bacteria with either type separately.19947861959
422100.9995Further characterization of complement resistance conferred on Escherichia coli by the plasmid genes traT of R100 and iss of ColV,I-K94. We have shown that the traT gene product was responsible for the complement resistance of the R100 plasmid. We compared this resistance with that specified by the iss gene of the ColV,I-K94 plasmid. The levels of resistance specified by the two genes were similar, and there was no additive effect on resistance when both genes were present together. Under conditions in which traT and iss conferred at least a 50- and 10-fold increase in survival, respectively, the consumption of C6, C7, C8, and C9 was the same for bacteria with and without the plasmid genes. This result indicated that it was the action of the terminal complex, not its formation, which was blocked by traT and iss.19827035371
5961110.9995Characterization of novel antibiotic resistance genes identified by functional metagenomics on soil samples. The soil microbial community is highly complex and contains a high density of antibiotic-producing bacteria, making it a likely source of diverse antibiotic resistance determinants. We used functional metagenomics to search for antibiotic resistance genes in libraries generated from three different soil samples, containing 3.6 Gb of DNA in total. We identified 11 new antibiotic resistance genes: 3 conferring resistance to ampicillin, 2 to gentamicin, 2 to chloramphenicol and 4 to trimethoprim. One of the clones identified was a new trimethoprim resistance gene encoding a 26.8 kDa protein closely resembling unassigned reductases of the dihydrofolate reductase group. This protein, Tm8-3, conferred trimethoprim resistance in Escherichia coli and Sinorhizobium meliloti (γ- and α-proteobacteria respectively). We demonstrated that this gene encoded an enzyme with dihydrofolate reductase activity, with kinetic constants similar to other type I and II dihydrofolate reductases (K(m) of 8.9 µM for NADPH and 3.7 µM for dihydrofolate and IC(50) of 20 µM for trimethoprim). This is the first description of a new type of reductase conferring resistance to trimethoprim. Our results indicate that soil bacteria display a high level of genetic diversity and are a reservoir of antibiotic resistance genes, supporting the use of this approach for the discovery of novel enzymes with unexpected activities unpredictable from their amino acid sequences.201121281423
6255120.9995Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli. Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis.201526014933
4498130.9995A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae. Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.200818024520
5963140.9995Expression of the mphB gene for macrolide 2'-phosphotransferase II from Escherichia coli in Staphylococcus aureus. The genes mphA and mphB encode macrolide 2'-phosphotransferases I and II, respectively, and they confer resistance to macrolide antibiotics in Escherichia coli. To study the expression of these genes in Gram-positive bacteria, we constructed recombinant plasmids that consisted of an mph gene and the pUB110 vector in Bacillus subtilis. When these plasmids were introduced into Staphylococcus aureus, the mphB gene was active and macrolide 2'-phosphotransferase II was produced. The gene endowed S. aureus with high-level resistance to spiramycin, a macrolide antibiotic with a 16-membered ring. Moreover, transcription of the mphB gene in S. aureus began at the promoter that was active in E. coli.19989503630
6259150.9995Evidence of an efflux pump in Serratia marcescens. Spontaneous mutants resistant to fluoroquinolones were obtained by exposing Serratia marcescens NIMA (wild-type strain) to increasing concentrations of ciprofloxacin both in liquid and on solid media. Frequencies of mutation ranged from 10(-7) to 10(-9). Active expulsion of antibiotic was explored as a possible mechanism of resistance in mutants as well as changes in topoisomerase target genes. The role of extrusion mechanisms in determining the emergence of multidrug-resistant bacteria was also examined. Mutants resistant to high concentrations of fluoroquinolones had a single mutation in their gyrA QRDR sequences, whereas the moderate resistance in the rest of mutants was due to extrusion of the drug.200010990265
4496160.9995Phenotypic and genetic barriers to establishment of horizontally transferred genes encoding ribosomal protection proteins. BACKGROUND: Ribosomal protection proteins (RPPs) interact with bacterial ribosomes to prevent inhibition of protein synthesis by tetracycline. RPP genes have evolved from a common ancestor into at least 12 distinct classes and spread by horizontal genetic transfer into a wide range of bacteria. Many bacterial genera host RPP genes from multiple classes but tet(M) is the predominant RPP gene found in Escherichia coli. OBJECTIVES: We asked whether phenotypic barriers (low-level resistance, high fitness cost) might constrain the fixation of other RPP genes in E. coli. METHODS: We expressed a diverse set of six different RPP genes in E. coli, including tet(M), and quantified tetracycline susceptibility and growth phenotypes as a function of expression level, and evolvability to overcome identified phenotypic barriers. RESULTS: The genes tet(M) and tet(Q) conferred high-level tetracycline resistance without reducing fitness; tet(O) and tet(W) conferred high-level resistance but significantly reduced growth fitness; tetB(P) conferred low-level resistance and while mutants conferring high-level resistance were selectable these had reduced growth fitness; otr(A) did not confer resistance and resistant mutants could not be selected. Evolution experiments suggested that codon usage patterns in tet(O) and tet(W), and transcriptional silencing associated with nucleotide composition in tetB(P), accounted for the observed phenotypic barriers. CONCLUSIONS: With the exception of tet(Q), the data reveal significant phenotypic and genetic barriers to the fixation of additional RPP genes in E. coli.202133655294
5763170.9995Development 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
6258180.9995Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.199910103206
4912190.9995Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli. Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.202438396221