A β-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly

2019 ◽  
Vol 74 (10) ◽  
pp. 2904-2912 ◽  
Author(s):  
Reema Singh ◽  
Sumudu R Perera ◽  
George S Katselis ◽  
Paulos Chumala ◽  
Irene Martin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Neisseria Gonorrhoeae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Start Codon ◽  
Surveillance Program ◽  
Binding Properties ◽  
Antimicrobial Surveillance ◽  
Agar Dilution ◽  
Hydrolysis Of

AbstractBackgroundSeven structurally related β-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903.ObjectivesTo compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 β-lactamases.MethodsN. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. β-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays.ResultsN. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow β-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 β-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours.ConclusionsThis unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 β-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

scholarly journals Drift in Susceptibility of Neisseria gonorrhoeae to Ciprofloxacin and Emergence of Therapeutic Failure

1998 ◽  
Vol 42 (11) ◽  
pp. 2919-2922 ◽  
Author(s):  
Catherine A. Ison ◽  
Patricia J. Woodford ◽  
Helen Madders ◽  
Elizabeth Claydon
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Therapeutic Failure ◽  
Surveillance Program ◽  
Topoisomerase Iv ◽  
First Line Therapy ◽  
Agar Dilution ◽  
High Level ◽  
Emergence Of Resistance ◽  
Level Resistance

ABSTRACT Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary’s Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary’s Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 μg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, ≤0.008 μg of ciprofloxacin/ml), there was a drift toward reduced susceptibility in N. gonorrhoeaeisolated between 1993 and 1996 (P < 0.001). In 1997 this drift was reduced but remained above pre-1993 levels. Isolates from 18 patients were classed as potentially resistant (MIC, >0.12 μg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic againstN. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.

scholarly journals Insertion Mutations in pilEDifferentially Alter Gonococcal Pilin Antigenic Variation

1999 ◽  
Vol 181 (19) ◽  
pp. 6133-6141 ◽  
Author(s):  
Becky Howell-Adams ◽  
H. Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Protein Sequence ◽  
High Frequency ◽  
Antigenic Variation ◽  
Pilin Gene ◽  
Pilin Protein ◽  
Unidirectional Transfer ◽  
Insertion Mutations

ABSTRACT Pilus antigenic variation in Neisseria gonorrhoeaeoccurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of thepilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions inpilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilSand recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.

scholarly journals Summary and Trends of the Russian Gonococcal Antimicrobial Surveillance Programme, 2005 to 2016

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Alexey Kubanov ◽  
Viktoria Solomka ◽  
Xenia Plakhova ◽  
Aleksandr Chestkov ◽  
Natalya Petrova ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Penicillin G ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Surveillance Program ◽  
Surveillance Programme ◽  
Content Type ◽  
Antimicrobial Surveillance ◽  
Agar Dilution ◽  
Chromosomal Mutations

ABSTRACT The Russian Gonococcal Antimicrobial Surveillance Programme (RU-GASP) was established in 2004 and operated continuously during the years from 2005 to 2016. The aims of this study were to summarize the RU-GASP results over this 12-year period and evaluate the trends in Neisseria gonorrhoeae antimicrobial resistance in Russia. In total, 5,038 verified N. gonorrhoeae isolates from 40 participating regions were tested for susceptibility to six antimicrobials via an agar dilution method. DNA loci involved in antimicrobial resistance were identified via minisequencing or DNA microarray techniques. From 2005 to 2016, increasing susceptibility to penicillin G (from 22.6% to 63.0%), tetracycline (from 34.8% to 53.0%), and ciprofloxacin (from 50.6% to 68.6%) was observed, but resistance to these drugs remained high. The proportions of isolates nonsusceptible to azithromycin and spectinomycin peaked in 2011 and decreased thereafter. Of the isolates, only 6 and 23 were identified as nonsusceptible to ceftriaxone according to the CLSI definitions and EUCAST breakpoint (0.57% of the total population), respectively. Comparison of N. gonorrhoeae antimicrobial resistance genetic determinants in 2005 versus those in 2016 showed a significant decrease in the number of isolates carrying chromosomal mutations. The proportion of isolates with wild-type genotypes increased from 11.7% in 2005 to 30.3% in 2016. Thus, the RU-GASP can be considered a successful gonorrhea surveillance program, and the current state of N. gonorrhoeae antimicrobial resistance in Russia is less serious than that in other WHO GASP regions.

Genomic epidemiology of Neisseria gonorrhoeae elucidating the gonococcal antimicrobial resistance and lineages/sublineages across Brazil, 2015–16

2020 ◽  
Vol 75 (11) ◽  
pp. 3163-3172
Author(s):  
Daniel Golparian ◽  
Maria Luiza Bazzo ◽  
Lisléia Golfetto ◽  
Pamela Cristina Gaspar ◽  
Marcos André Schörner ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Genomic Diversity ◽  
Surveillance Program ◽  
Phylogenomic Analysis ◽  
Resistance Mutations ◽  
Genomic Epidemiology ◽  
Antimicrobial Surveillance ◽  
Genomic Studies ◽  
Azithromycin Resistance

Abstract Objectives Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is imperative internationally, but only eight (22.9%) countries in the WHO Region of the Americas reported complete AMR data to the WHO Global Gonococcal Antimicrobial Surveillance Program (WHO GASP) in 2016. Genomic studies are ideal for enhanced understanding of gonococcal populations, including the spread of AMR strains. To elucidate the circulating gonococcal lineages/sublineages, including their AMR determinants, and the baseline genomic diversity among gonococcal strains in Brazil, we conducted WGS on 548 isolates obtained in 2015–16 across all five macroregions in Brazil. Methods A total of 548 gonococcal isolates cultured across Brazil in 2015–16 were genome sequenced. AMR was determined using agar dilution and/or Etest. Genome sequences of isolates from Argentina (n = 158) and the 2016 WHO reference strains (n = 14) were included in the analysis. Results We found 302, 68 and 214 different NG-MAST, MLST and NG-STAR STs, respectively. The phylogenomic analysis identified one main antimicrobial-susceptible lineage and one AMR lineage, which was divided into two sublineages with different AMR profiles. Determination of NG-STAR networks of clonal complexes was shown as a new and valuable molecular epidemiological analysis. Several novel mosaic mtrD (and mtrR and mtrE) variants associated with azithromycin resistance were identified. Conclusions We describe the first genomic baseline data to support the Brazilian GASP. The high prevalence of resistance to ciprofloxacin, tetracycline and benzylpenicillin, and the high number of isolates with mosaic penA and azithromycin resistance mutations, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Brazil.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Studies on Chromatographic Fractioning by Cations Exchangers of a Bovine Hemoglobin Hydrolysate

2018 ◽  
Vol 69 (10) ◽  
pp. 2794-2798
Author(s):  
Alina Diana Panainte ◽  
Ionela Daniela Morariu ◽  
Nela Bibire ◽  
Madalina Vieriu ◽  
Gladiola Tantaru ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Retention Time ◽  
Chromatographic Separation ◽  
Bovine Hemoglobin ◽  
Reverse Phase ◽  
Hplc System ◽  
Fast Flow ◽  
Hydrolysis Of ◽  
Phase Column

A peptidic hydrolysate has been obtained through hydrolysis of bovine hemoglobin using pepsin. The fractioning of the hydrolysate was performed on a column packed with CM-Sepharose Fast Flow. The hydrolysate and each fraction was filtered and then injected into a HPLC system equipped with a Vydak C4 reverse phase column (0.46 x 25 cm), suitable for the chromatographic separation of large peptides with 20 to 30 amino acids. The detection was done using mass spectrometry, and the retention time, size and distribution of the peptides were determined.

scholarly journals DNA thermodynamics shape chromosome organization and topology

2013 ◽  
Vol 41 (2) ◽  
pp. 548-553 ◽  
Author(s):  
Andrew A. Travers ◽  
Georgi Muskhelishvili
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Chromosome Organization ◽  
Topological Properties ◽  
Genetic Organization ◽  
And Topology ◽  
Dna Translocases

How much information is encoded in the DNA sequence of an organism? We argue that the informational, mechanical and topological properties of DNA are interdependent and act together to specify the primary characteristics of genetic organization and chromatin structures. Superhelicity generated in vivo, in part by the action of DNA translocases, can be transmitted to topologically sensitive regions encoded by less stable DNA sequences.

scholarly journals An application of slow feature analysis to the genetic sequences of coronaviruses and influenza viruses

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Anastasios A. Tsonis ◽  
Geli Wang ◽  
Lvyi Zhang ◽  
Wenxu Lu ◽  
Aristotle Kayafas ◽  
...  
Keyword(s):  
Mathematical Method ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Transmission Rate ◽  
Complex Structure ◽  
Mortality Rates ◽  
Influenza Viruses ◽  
Feature Analysis ◽  
Slow Feature Analysis ◽  
Genetic Sequences

Abstract Background Mathematical approaches have been for decades used to probe the structure of DNA sequences. This has led to the development of Bioinformatics. In this exploratory work, a novel mathematical method is applied to probe the DNA structure of two related viral families: those of coronaviruses and those of influenza viruses. The coronaviruses are SARS-CoV-2, SARS-CoV-1, and MERS. The influenza viruses include H1N1-1918, H1N1-2009, H2N2-1957, and H3N2-1968. Methods The mathematical method used is the slow feature analysis (SFA), a rather new but promising method to delineate complex structure in DNA sequences. Results The analysis indicates that the DNA sequences exhibit an elaborate and convoluted structure akin to complex networks. We define a measure of complexity and show that each DNA sequence exhibits a certain degree of complexity within itself, while at the same time there exists complex inter-relationships between the sequences within a family and between the two families. From these relationships, we find evidence, especially for the coronavirus family, that increasing complexity in a sequence is associated with higher transmission rate but with lower mortality. Conclusions The complexity measure defined here may hold a promise and could become a useful tool in the prediction of transmission and mortality rates in future new viral strains.

scholarly journals The Use of MALDI-TOF Mass Spectrometry to Analyze Commensal Oral Yeasts in Nursing Home Residents

2021 ◽  
Vol 9 (1) ◽  
pp. 142
Author(s):  
Jang-Jih Lu ◽  
Hsiu-Jung Lo ◽  
Chih-Hua Lee ◽  
Mei-Jun Chen ◽  
Chih-Chao Lin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Nursing Home ◽  
Dna Sequences ◽  
Nursing Home Residents ◽  
Practical Method ◽  
Elderly Subjects ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Oral Yeasts ◽  
Tof Ms

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and accurate method to identify microorganisms in clinical laboratories. This study isolates yeast-like microorganisms in the oral washes that are collected from non-bedridden nursing home residents, using CHROMagar Candida plates, and identifies them using Bruker MALDI-TOF MS. The ribosomal DNA sequences of the isolates are then examined. Three hundred and twenty yeast isolates are isolated from the oral washes. Candida species form the majority (78.1%), followed by Trichosporon/Cutaneotrichosporon species (8.8%). Bruker MALDI-TOF MS gives a high-level confidence, with a log(score) value of ≥1.8, and identifies 96.9% of the isolates. There are six inconclusive results (1.9%), and those sequences are verified as rare clinical species, including Candida ethanolica, Cutaneotrichosporon jirovecii, Exophiala dermatitidis, and Fereydounia khargensis. Almost all of the isolates have a regular color on the CHROMagar Candida plates. If the colonies are grouped by color on the plates, a specific dominant yeast species is present in each color group, except for purple or orange isolates. In conclusion, MALDI-TOF MS is verified as a fast, accurate and practical method to analyze oral yeasts in elderly subjects.

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