Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori. - Related Documents




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588401.0000Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori. BACKGROUND: The drug resistance rate of clinical Helicobacter pylori (H. pylori) isolates has increased. However, the mechanism of drug resistance remains unclear. In this study, drug-resistant H. pylori strains were isolated from different areas and different populations of Chinese for genomic analysis. AIM: To investigate drug-resistant genes in H. pylori and find the genes for the early diagnosis of clarithromycin resistance. METHODS: Three drug-resistant H. pylori strains were isolated from patients with gastritis in Bama County, China. Minimal inhibitory concentrations of clarithromycin, metronidazole, and levofloxacin were determined and complete genome sequencing was performed with annotation. Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction. The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains. The homology of the strains with hp26695 was assessed through complete genome detection and identification. Differences in genome sequences, gene quantity, and gene characteristics were detected amongst the three strains. Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains, which was the same in the artificially induced clarithromycin-resistant bacteria. After gene knockout, the drug sensitivity of the strains was assessed. RESULTS: The strains showing a high degree of homology with hp26695, hp1181, and hp1184 genes were found in these strains; the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance. CONCLUSION: Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance, and they may be the potential target genes for the diagnosis, prevention, and treatment of clarithromycin resistance.202134239272
626410.9993Multi-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
461520.9993Effect 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
596030.999216S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori. Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet(r)) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tet(s) strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tet(r) strain 181, the Tet(s) strain 26695, and four Tet(r) 26695 transformants showed that a single triple-base-pair substitution, AGA(926-928)-->TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet(r) H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet(r) strain 181.200212183259
583640.9992Identification 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
624850.9992Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate. BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.201323349739
598560.9992Alternative 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
626370.9992Gene-Gene Interactions Dictate Ciprofloxacin Resistance in Pseudomonas aeruginosa and Facilitate Prediction of Resistance Phenotype from Genome Sequence Data. Ciprofloxacin is one of the most widely used antibiotics for treating Pseudomonas aeruginosa infections. However, P. aeruginosa acquires mutations that confer ciprofloxacin resistance, making treatment more difficult. Resistance is multifactorial, with mutations in multiple genes influencing the resistance phenotype. However, the contributions of individual mutations and mutation combinations to the amounts of ciprofloxacin that P. aeruginosa can tolerate are not well understood. Engineering P. aeruginosa strain PAO1 to contain mutations in any one of the resistance-associated genes gyrA, nfxB, rnfC, parC, and parE showed that only gyrA mutations increased the MIC for ciprofloxacin. Mutations in parC and parE increased the MIC of a gyrA mutant, making the bacteria ciprofloxacin resistant. Mutations in nfxB and rnfC increased the MIC, conferring resistance, only if both were mutated in a gyrA background. Mutations in all of gyrA, nfxB, rnfC, and parC/E further increased the MIC. These findings reveal an epistatic network of gene-gene interactions in ciprofloxacin resistance. We used this information to predict ciprofloxacin resistance/susceptibility for 274 isolates of P. aeruginosa from their genome sequences. Antibiotic susceptibility profiles were predicted correctly for 84% of the isolates. The majority of isolates for which prediction was unsuccessful were ciprofloxacin resistant, demonstrating the involvement of additional as yet unidentified genes and mutations in resistance. Our data show that gene-gene interactions can play an important role in antibiotic resistance and can be successfully incorporated into models predicting resistance phenotype.202133875431
577080.9992Prevalence of silver resistance genes in bacteria isolated from human and horse wounds. The aim of this study was to investigate the prevalence of silver resistance genes in 172 bacterial strains which had been isolated from both human and equine wounds. PCR screening for 8 currently named genes in 3 silver resistance transcriptional units, silE, silRS and silP, silCBA and silF was performed on total DNA extracted from all clinical isolates. Plasmids were isolated from sil-positive strains to determine if the genes were present on the chromosome. MICs and zone of inhibition assays were utilised to examine phenotypic resistance to silver nitrate and ionic silver. Evidence of silver resistance genes was demonstrated in six strains of Enterobacter cloacae, an organism rarely implicated as a primary pathogen in chronic wounds. MIC data showed that all strains were inhibited at silver nitrate concentrations > or =5mg/L. When tested against a silver-containing absorbent wound dressing all strains showed inhibition of growth after 24h. In MIC and zone of inhibition studies, inhibition was evident but reduced in strains which contained sil genes. Although sil genes were found in six of the wound isolates studied, the genes were consistently associated with a non-pathogenic bacterium. Furthermore, investigation of phenotypic resistance in sil-positive isolates showed that silver continued to be effective.200919362435
588690.9992aac(6')-Iaq, a novel aminoglycoside acetyltransferase gene identified from an animal isolate Brucella intermedia DW0551. BACKGROUND: Bacterial resistance to aminoglycoside antimicrobials is becoming increasingly severe due to their use as commonly prescribed antibiotics. The discovery of new molecular mechanisms of aminoglycoside resistance is critical for the effective treatment of bacterial infections. METHODS: Bacteria in goose feces were isolated by plate streaking. The identification and characterization of a novel resistance gene from the bacterial genome involved various techniques, including molecular cloning, drug susceptibility testing, protein expression and purification, and enzyme kinetic analysis. Additionally, whole-genome sequencing and phylogenetic studies were performed. RESULTS: Brucella intermedia DW0551, isolated from goose feces, was resistant to 35 antibiotics, and the minimum inhibitory concentration (MIC) was particularly high for most aminoglycoside antibiotics. The novel aminoglycoside resistance gene aac(6')-Iaq encoded by B. intermedia DW0551 conferred resistance to netilmicin, sisomicin, amikacin, kanamycin, gentamicin, tobramycin, and ribostamycin. The amino acid sequence of AAC(6')-Iaq shared the highest identity (52.63%) with the functionally characterized aminoglycoside acetyltransferase AAC(6')-If. AAC(6')-Iaq contained all the conserved sites of the acetyltransferase family NAT_SF. The enzyme exhibited strong affinity and catalytic activity toward netilmicin and sisomicin. The mobile genetic element (MGE) was not found in the flanking regions of the aac(6')-Iaq and aac(6')-Iaq-like genes. CONCLUSION: In this work, a novel aminoglycoside acetyltransferase gene, designated aac(6')-Iaq, which conferred resistance to a variety of aminoglycoside antimicrobials, was identified in an animal Brucella intermedia isolate. Identification of new antibiotic resistance mechanisms in bacteria isolated from animals could aid in the treatment of animal and human infectious diseases caused by related bacterial species.202540134786
5768100.9992The resistance mechanism of Escherichia coli induced by ampicillin in laboratory. BACKGROUND: Multi-drug-resistant Escherichia coli poses a great threat to human health, especially resistant to ampicillin (AMP), but the mechanism of drug resistance is not very clear. PURPOSE: To understand the mechanism of resistance of E. coli to beta-lactam antibiotics by inducing drug resistance of sensitive bacteria in laboratory. METHODS: Clinical sensitive E. coli strain was induced into resistance strain by 1/2 minimum inhibitive concentration (MIC) induced trails of AMP. The drug resistance spectrum was measured by modified K-B susceptibility test. Whole-genome sequencing analysis was used to analyze primary sensitive strain, and resequencing was used to analyze induced strains. Protein tertiary structure encoded by the gene containing single nucleotide polymorphism (SNP) was analyzed by bioinformatics. RESULTS: After 315 hrs induced, the MIC value of E. coli 15743 reached to 256 µg/mL, 64 times higher than that of the sensitive bacteria. During the induction process, the bacterial resistance process is divided into two stages. The rate of drug resistance occurs rapidly before reaching the critical concentration of 32 µg/mL, and then the resistance rate slows down. Sequencing of the genome of resistant strain showed that E. coli 15743 drug-resistant strain with the MIC values of 32 and 256 µg/mL contained four and eight non-synonymous SNPs, respectively. These non-synonymous SNPs were distributed in the genes of frdD, ftsI, acrB, OmpD, marR, VgrG, and envZ. CONCLUSION: These studies will improve our understanding of the molecular mechanism of AMP resistance of E. coli, and may provide the basis for prevention and control of multi-drug-resistant bacteria and generation of new antibiotics to treat E. coli infection.201931571941
5838110.9991Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin. Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrB(L208F) mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.202439203486
5766120.9991Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex. Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.202337192202
5765130.9991Expression of Pseudomonas aeruginosa Antibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients. The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.201830201819
6300140.9991Assessing the role of the RND efflux pump in metronidazole resistance of Helicobacter pylori by RT-PCR assay. INTRODUCTION: Metronidazole is a significant antibiotic used for eradication of Helicobacter pylori infections and it is of notice that metronidazole-resistant clinical isolates have been found in high rates worldwide. While the RND family of efflux pumps plays a central role in drug resistance among Gram-negative bacteria, this is questionable for H. pylori. METHODOLOGY: To understand whether TolC homologues of RND pumps contribute to metronidazole resistance in H. pylori isolates, expression of four TolC homologous genes of five resistant clinical isolates exposed to varying concentrations of metronidazole were evaluated by RT-PCR and transcriptional analysis. RESULTS: The results indicate that excess amounts of metronidazole are able to increase the expression level of these genes at the transcriptional stage. CONCLUSIONS: Therefore, it may be hypothesized that use of metronidazole in H. pyori infection can induce metronidazole resistance. Furthermore, the RND family of efflux pumps may contribute to metronidazole resistance in clinical isolates of H. pylori.201121389587
4785150.9991Study of MazEF, sam, and phd-doc putative toxin-antitoxin systems in Staphylococcus epidermidis. Today, to replace the antibacterial targets to overcome antibiotic resistance, toxin-antitoxin (TA) system is noticeable, where the unstable antitoxin neutralizes the stable toxin and protects the bacteria against the toxic effects. The presence and expression of TA genes in clinical and non-clinical strains of Staphylococcus epidermidis were investigated in this study. After identification of three TA pairs (mazEF, sam, and phd-doc) via existing databases (earlier, there has been no information in the case of S. epidermidis isolates), the presence and expression of these pairs were investigated by PCR and q-PCR, respectively. We detected three TA modules in all antibiotic sensitive and resistant isolates. In addition, q-PCR analysis revealed that the transcripts were produced from the three TA modules. This study showed the significant prevalence of these systems in pathogenic bacteria and they were equally found in both oxacillin-resistant and oxacillin-susceptible bacteria. The high prevalence of three systems can make them suitable as potential targets for antibiotic therapy.201829471693
6268160.9991Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.200516048924
5761170.9991The 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
4786180.9991Novel Antimicrobial Target in Acinetobacter Baumannii. BACKGROUND: Resistance to multiple drugs is one of the biggest challenges in managing infectious diseases. Acinetobacter baumannii is considered a nosocomial infection. According to the multiple roles of the toxin-antitoxin system, this system can be considered an antimicrobial target in the presence of bacteria. With the impact on bacterial toxin, it can be used as a new antibacterial target. The purpose of this study was to determine the mazEF genes as a potent antimicrobial target in A. baumannii clinical isolates. METHODS: The functionality of mazEF genes was evaluated by qPCR in fifteen A. baumannii clinical isolates. Then, the mazE locus was targeted by peptide nucleic acid (PNA). RESULTS: The results showed a significant difference in the mean number of copies of mazF gene in normal and stress conditions. Also, we found that at a concentration of 15 µM of PNA the bacteria were killed and confirmed by culture on LB agar. CONCLUSIONS: This research is the first step in introducing mazEF TA loci as a sensitive target in A. baumannii. However, more studies are needed to test the effectiveness in vivo. In addition, the occurrence and potential for activation of the TA system, mazEF in other pathogenic bacteria should be further investigated.202235536074
4960190.9991Bacteriophages and diffusion of genes encoding antimicrobial resistance in cystic fibrosis sputum microbiota. OBJECTIVES: The cystic fibrosis (CF) airway is now considered the site of a complex microbiota, where cross-talking between microbes and lateral gene transfer are believed to contribute to the adaptation of bacteria to this specific environment and to the emergence of multidrug-resistant bacteria. The objective of this study was to retrieve and analyse specific sequences associated with antimicrobial resistance from the CF viromes database. METHODS: Specific sequences from CF metagenomic studies related to the 'antibiotic and toxic compound resistance' dataset were retrieved from the MG-RAST web site, assembled and functionally annotated for identification of the genes. Phylogenetic trees were constructed using a minimum parsimony starting tree topology search strategy. RESULTS: Overall, we found 1031 short sequences in the CF virome putatively encoding resistance to antimicrobials versus only 3 reads in the non-CF virome dataset (P = 0.001). Among them, we could confidently identify 66 efflux pump genes, 15 fluoroquinolone resistance genes and 9 β-lactamase genes. Evolutionary relatedness determined using phylogenetic information demonstrates the different origins of these genes among the CF microbiota. Interestingly, among annotated sequences within CF viromes, we also found matching 16S rDNA sequences from Escherichia, Cyanobacteria and Bacteroidetes. CONCLUSIONS: Our results suggest that phages in the CF sputum microbiota represent a reservoir of mobilizable genes associated with antimicrobial resistance that may spread in this specific niche. This phenomenon could explain the fantastic adaptation of CF strains to their niche and may represent a new potential therapeutic target to prevent the emergence of multidrug-resistant bacteria, which are responsible for most of the deaths in CF.201121816767