Adenine Deletion Research Articles

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types

Background and purposeOur previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. MethodsA comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. ResultsForty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. ConclusionOur findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

The contribution of a novel PHEX gene mutation to X-linked hypophosphatemic rickets: a case report and an analysis of the gene mutation dosage effect in a rat model.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

Molecular dissection of a rice plant with mutation of the leucine carboxyl methyl transferase gene

Drought is a major environmental factor that negatively affects crop growth and productivity. Rice is a drought-sensitive crop, and its development and yield are greatly affected by stress. Using a forward genetics approach, a drought-insensitive TILLING line, ditl2, induced using gamma-ray irradiation, was selected from 150 rice mutant lines. Under drought treatment conditions, the ditl2 mutant showed a high survival rate, fresh weight, relative water content, soluble sugar content, and low hydrogen peroxide (H2O2) content. Whole-genome re-sequencing of ditl2 showed a single-base adenine deletion of the OsLCMT (LOC_Os03g56370) gene, annotated as leucine carboxyl methyl transferase, resulting in a frameshift (OsLCMTp.Thr163fs) with a short peptide of 163 amino acids. The substrate gene OsSnRK1A of OsLCMT was identified through an experiment using yeast two-hybrid and BiFC assays. The subcellular localization of the two genes was confirmed to be in the cytosol. The methyltransferase activities of OsLCMT and OsLCMT.Thr163fs were examined using the target protein OsSnRK1A in an in vitro assay. Consequently, the methyltransferase activity of OsLCMT.Thr163fs was lower than that of the wild-type gene. High expression of starch hydrolysis pathway genes, such as amylases, amylomaltases, and plastidic sugar transporters, was found in ditl2 compared to wild-type (WT) plants. The ditl2 mutant is useful as a genetic resource for understanding the relationship between protein methylation and drought insensitivity and improving drought tolerance in rice mutant breeding.

Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand.

The increasing rate of antimicrobial-resistant Neisseria gonorrhoeae poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance. We collected 117 N. gonorrhoeae isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and mtrR (a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 μg/ml) were sequenced using Illumina and Nanopore sequencing platforms. The proportions of N. gonorrhoeae isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the mtrR promoter and an H105Y mutation in the mtrR coding region were found in the N. gonorrhoeae isolate with the highest azithromycin MIC value (1 μg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II penA and had mutations in penB and the mtrR coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid. The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of N. gonorrhoeae, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.

Evaluation of Neisseria gonorrhoeae Isolates Susceptibility to Tetracycline Antibiotics from 9 Provinces in China Since 2020.

PurposeThe increasing drug resistance of Neisseria gonorrhoeae has become a serious public health concern. This study investigated N. gonorrhoeae isolates susceptibility to tetracycline antibiotics and the correlation between minimum inhibitory concentrations (MICs) of different antibiotics. The presence of resistance determinants in N. gonorrhoeae strains displaying different levels of tigecycline resistance was also compared.MethodsThe minimum inhibitory concentrations (MICs) of tetracycline, minocycline, tigecycline, eravacycline, omadacycline on 412 N. gonorrhoeae isolates were measured by the agar dilution method. The MICs of ceftriaxone and azithromycin were also measured to determine the correlations between antibiotics by the value of the correlation coefficient R. The presence of resistance determinants was identified through polymerase chain reaction (PCR) and sequencing.ResultsThe MIC90 was 64mg/L for tetracycline, 64mg/L for minocycline, 0.5mg/L for tigecycline, 0.5mg/L for eravacycline, 4mg/L for omadacycline, 0.25 mg/L for ceftriaxone, and 1mg/L for azithromycin. The MIC90 and mode of tigecycline and eravacycline were much lower than those of tetracycline and minocycline. A poor correlation between omadacycline, eravacycline and tetracycline susceptibility was observed. Minocycline has a strong correlation with tetracycline. PorB1 typing, TetM-encoding plasmid, and mtrR promoter adenine deletion were significantly correlated with tigecycline MIC > 0.25mg/L.ConclusionThis study suggested that tigecycline and eravacycline had better in vitro activity and might be alternative antibiotics against resistant N. gonorrhoeae infections. Nevertheless, further in vitro experiments and clinical studies are needed for verification.

Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates.

Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427 motif I; L475Q, 7 aa upstream of the 482SDN484 motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622 motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes.

Mutations of p53 Gene in Canine Sweat Gland Carcinomas Probably Associated with UV Radiation.

IntroductionApocrine sweat gland carcinomas (ASGCs) are rare malignant skin tumours in dogs and humans. The literature published so far focuses mostly on the clinico-epidemiological aspect of these tumours, but little is known about their pathogenesis. In this study we aimed to determine whether the p53 gene is involved in the carcinogenesis of the apocrine sweat gland in dogs and whether ultraviolet radiation (UV) is related to it.Material and MethodsForty canine ASGCs were submitted to laser capture microdissection to isolate neoplastic cells, from which DNA was subsequently extracted. PCR amplification and sequencing of p53 exons 2–8 was then performed, followed by computer analysis of the obtained sequences.ResultsSixteen mutations within the p53 gene were found in 13 tumours. The mutations involved C → T, T → C, G → A, and CC → TT transitions, C → G transversion and adenine deletion, which are gene alteration types known to be related to UV radiation in the process of skin carcinogenesis in humans. Six of the thirteen tumour cases displayed the C → T transitions in the same location in exon 4 and three of the thirteen cases displayed T → C in the same location in exon 5.ConclusionThe results of the present study indicate both the participation of the p53 gene and the influence of UV radiation in the formation of ASGCs in dogs.

Molecular Typing of Neisseria gonorrhoeae Clinical Isolates in Russia, 2018-2019: A Link Between penA Alleles and NG-MAST Types.

This work aimed to study penA gene polymorphisms in clinical isolates of Neisseria gonorrhoeae collected in Russia in 2018–2019 and the contribution of the penA allele type to susceptibility to β-lactam antibiotics. A total of 182 isolates were analyzed. penA allele types were determined by sequencing, and the minimum inhibitory concentrations (MICs) of benzylpenicillin and ceftriaxone were measured. The influence of genetic factors on MICs was evaluated by regression analysis. All isolates were susceptible to ceftriaxone, and 40.1% of isolates were susceptible to penicillin. Eleven penA allele types were identified. The mosaic type XXXIV penA allele and the Gly120Lys substitution in PorB made the greatest contributions to increasing the ceftriaxone MIC; the presence of the blaTEM plasmid, Gly120Asp, Ala121Gly/Asn substitutions in PorB, and the adenine deletion in the promoter region of the mtrR gene caused an increase in the penicillin MIC. Among 61 NG-MAST types identified, the most frequent were types 228, 807, 9486, 1993, and 6226. A link between penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types was established. Resistance to two groups of β-lactam antibiotics was associated with non-identical changes in penA alleles. To prevent the emergence of ceftriaxone resistance in Russia, NG-MAST genotyping must be supplemented with penA allele analysis.

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility–an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)—therefore disrupting the expression of the genes σ54 regulates—coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulence-related loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10–/– mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

Genomic Epidemiology of Carbapenem- and Colistin-Resistant Klebsiella pneumoniae Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid

Klebsiella pneumoniae is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (n = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrBC28S being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the blaOXA–48 genetic background. The blaOXA–48 gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum β-lactamase-encoding gene blaCTX–M–15 and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2∗, K3∗, Q30∗, adenine deletion leading to frameshift and premature termination, IS5-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone.

The fluctuation of APC gene in WNT signaling with adenine deletion of adenomatous polyposis coli, is associated in colorectal cancer

Colorectal cancer is one of the most important malignancies in the classification of gastrointestinal cancers. One of the predisposing factors at molecular level for this cancer is via WNT signaling which is associated with the vast numbers of different genes. Thus, in this study, we aimed to investigate whether Adenomatous Polyposis Coli gene (APC) mutation of rs41115in two locations such as 132.002 and 131.989 acts as a trigger or cause of colorectal cancer. Relatively, 30 blood samples of colorectal cancer patients and 30 normal blood samples as control group after colonoscopy and also confirmation of pathology report at Rohani Hospital in Babol (Iran) were investigated. The primers were designed in order to be included the rs41115 to identify the particular polymorphisms of gene. The polymerase chain reaction (PCR direct sequencing method) was used. Conclusively, deletion of adenine in two specific locations such as 131.989 and 132.002 has been identified, but there was no relationship between rs41115 polymorphisms located in adenomatous polyposis coli gene and colorectal cancer.

Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates from men who have sex with men

ObjectivesTo analyze the susceptibility of Neisseria gonorrhoeae isolates to penicillin (Pen), cefixime (Cfm), ceftriaxone (Cro), tetracycline (Tet), ciprofloxacin (Cip), azithromycin (Azm), and spectinomycin (Spt), and to verify the presence of mutations in resistance genes. MethodsAntibiotic susceptibility testing was performed by Etest method on 30 N. gonorrhoeae isolates collected from the MSM (men who have sex with men) population. PCR and DNA sequencing were performed to identify mutations within the penA, mtrR, gyrA, and parC genes in intermediately resistant and fully resistant isolates. ResultsN. gonorrhoeae isolates showed intermediate or full resistance to Pen (73%), Cfm (3%), Tet (60%), Cip (37%), and Azm (13%). One isolate with resistance to Cfm presented a penicillin-binding protein 2 (PBP2) mosaic XXXIV. All isolates with intermediate or full resistance to Pen (except at PBP2 mosaic) presented a D345a in PBP2. All Cip-resistant isolates had an S91F in the gyrA gene together with mutations in the parC gene. All intermediate or fully resistant isolates to substrates of the MtrCDE efflux pump had an A39T or G45D mutation in the mtrR gene or an adenine deletion within the mtrR promoter. One isolate presented a Neisseria meningitidis-like mtrR promoter sequence. ConclusionsThe results of this study are consistent with the findings of other studies and reinforce the importance of the expedient development of new therapeutic options.

Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis.

ABSTRACTEnterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 −35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.

Ultrasound features and molecular diagnosis testing of thyroid mass

Thyroid gland is one of the most important glands in human body due to the necessary hormones it produces. It can be afffeced by different types of infections and diseases, one of the most risky abnormalities of this gland is thyroid masses. The aim of this study is to find how does thyroid masses start from the molecular stages and is there any relation with it and genetics, for this purpose we scanned more than 273 patients by ultrasound from different ages groups, and genders, thyroid mass detected in 17 of them, 3 with cystic fluid filled mass, and 14 with solid mass . Blood samples from thes patients were analysed and molecular study were performed by taking exon 11 and 15 from Braf gene mution was detected in all patients included frame shift mutaion a deletion of adenine in same position , while no sequence variation was found in the exon 11 Braf gene. In conclusion According to DNA sequence analysis, genomic variation was not found in exon 11 of the BRAF gene in all patients, an exonic alteration was detected in exon 15 in 7 patients was deletion of (A) in coding nucleotide , which the BRAF mutational hot spot region are found. The result of the deletion mutation is conversion of all of codons after deletion

Identification of a new genetic marker in Mycoplasma synoviae vaccine strain MS-H and development of a strategy using polymerase chain reaction and high-resolution melting curve analysis for differentiating MS-H from field strains

Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts+) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs.

Bypassing the Need for the Transcriptional Activator EarA through a Spontaneous Deletion in the BRE Portion of the fla Operon Promoter in Methanococcus maripaludis.

In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

A new mutation at exon 2 of hprt1 locus causing lesch-nyhan syndrome.

Introduction: Lesch-Nyhan síndrome (LNS) is an X-linked recessive inborn error of metabolism, due to deficiency of the enzyme Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; EC.2.4.2.8) resulting in hyperuricemia, neurological and behavioural disturbances. In the present work, we report the results of the study of a Colombian family, where LNS was previously clinically and biochemically diagnosed. Material and Methods: The full HPRT gene, including 9 exons and 8 introns, was amplified on eight separate DNA fragments. Both strands, forward and reverse, of the amplified DNA fragments were analyzed and the obtained sequences were compared with those deposited at National Center for Biotechnology Information. Results and conclusions: Sequence analysis allowed the detection of new LNS causing mutation, an adenine deletion in exon 2 of HPRT1 gene resulting in a frameshift which determines a premature stop codon. This study, besides adding a new mutation to the already large spectrum of disease causing variation at HPRT, allows therefore providing genetic counseling for the family as well as prenatal diagnosis.

Genetic Resistance Determinants for Cefixime and Molecular Analysis of Gonococci Isolated in Italy.

A strictly defined subset of gonococci (n = 65) isolated in Italy from 2011 to 2014 was characterized by antimicrobial susceptibility for cefixime (CFM) and ceftriaxone (CRO) and by sequencing of resistance determinant genes (penA, mtrR, porB1b, ponA) for extended-spectrum cephalosporins and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST). The penA mosaic alleles XXXIV and XXXV were found in all resistant (R) and decreased susceptibility (DS) gonococci to CFM, except for one. They were associated with an adenine deletion in the mtrR promoter plus amino acid substitutions, H105Y or G45D, in the coding region and ponA L421P. The penA mosaic allele XXXIV, and one variant, was found exclusively among genogroup (G) 1407 and its closely related sequence types (STs), as in CFM-DS as well as in CFM-R isolates. Single or combined mutation patterns in penA, mtrR, porB1b, and ponA genes were associated with different CFM susceptibility patterns and NG-MAST STs. Genotyping and antimicrobial resistance (AMR) determinant analyses can be valuable to enhance the gonococcal AMR surveillance.

Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2% had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125μg/mL] and 0.6% to ceftriaxone (MIC > 0.125μg/mL). Azithromycin resistance was documented in 4.3% and ciprofloxacin resistance in 49.2%. Among 48 strains with an MIC ≥ 0.125μg/mL to cefixime, 58.3% showed the penA mosaic pattern XXXIV, 98% a Leu → Pro substitution at position 421 of the ponA gene, 100% amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5% of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125μg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125μg/mL to cefixime, 43.8% belonged to the genogroup G1407 and 27.1% belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125μg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance.

Dent disease: About 4 Adult Observations Monitored in the Department of Nephrology and Organ Transplantation of Toulouse, France

Introduction: The Dent's Disease (Dd) Is An X-Linked Genetic Kidney Disease. We Undertook A Retrospective Study Of 4 Dd Observations Monitored In The Department Of Nephrology And Transplantation In Toulouse To Relate The History Of The Disease. Our Purpose Was To Show The Phenotypic Variability And Therapeutic Difficulties. Case Report Patient 1: Mr. G.O. - The Dd Was Discovered Incidentally Through Proteinuria Of 0.2 G/24 Hours Associated With Polyuria And Polydipsia Syndrome With Impaired Urine Concentrating And Diluting Capacity. A Minus 1 Standard Deviation Was Noted In One Year In Size And Weight, Which Lasted Up To 8 Years. At 7 Years Of Age, Hypercalciuria Of 8.4 Mg/Kg/Day With Nephrocalcinosis Appeared. From 16 Years Onward, Creatinine Degradation Gradually Reached A Peak Of 444 µmol/L Or A Glomerular Filtration Rate (Gfr), Under The Mdrd Formula, At 22 Ml /Minute In June 2014 In The Occurrence Of Epiglottitis. A Point Mutation Of The Chloride Channel Gene (Cncl5) Was Noted. Patient 2: Mr. B.R. The Dd Finding Was Incidental To Proteinuria Of 2.6 G/24 Hours Associated With Polydipsia-Polyuria During A School Visit At The Age Of 6 Without Change In Urinary Diluting Concentration. There Was An Episode Of Renal Colic When He Was 8 Years Old. When He Was 10, Hypercalciuria Of 9 Mg/Kg/Day Was Noticed, Nephrocalcinosis Too. From 14 Years Onward, Creatinine Gradually Deteriorated, Bringing Mr. B. R To Hemodialysis. Genetic Analysis Showed Adenine Deletion At Position 1751, Exon 10. Patient 3: Mr. G.C. The Discovery Of His Kidney Disease Was Fortuitous As Proteinuria Was Detected In Preventive Medicine When He Was 19 Years Old. There Was No Clinical Manifestation. His Gfr Altered To Average At 58 Ml / Min Using Mdrd Formula. Mutation Of Gene Cic.5 Was Noted. There Was Nephrocalcinosis Associated With Renal Cysts. Patient 4: Mr. T.C. Is The Younger Brother Of Mr. G.C. The Genetic Investigation Carried Out In The Family Showed That He Was Bearing The Same Mutation As His Elder Brother. He Had No Symptoms, At Most Proteinuria Of 0.5 G/G With Urinary Calcium/Creatinine Concentration Of 0.6. At The Age 20, His Kidneys Worked Normally. Conclusion: The Therapeutic Resources Were Limited. Several Questions Remained Unanswered, Namely The Impact Of Ace Inhibitors And Arbs In Slowing The Ckd; Also 25-35% Of Cases Of Dd Without Genetic Mutation Was Observed.