Antibiotic resistance markers are a necessary tool in many clinical areas

SUMMARYOf 28 multi-resistant isolates ofStaphylococcus aureuscollected during 1986 from hospitals in major cities around Australia, 27 were found to contain the same prophage (denoted phage 604). Hospital isolates carrying three or fewer resistance markers, and community isolates carrying one or no resistance markers, did not carry this prophage. Phage 604 does not confer antibiotic resistance on its lysogens, nor does it increase virulence in chick embryo assays. Phage 604 appears to be a correlate of antibiotic multi-resistance inS. aureusin Australia, and may provide a molecular marker for incipiently epidemic strains of this bacterium in Australian hospitals.

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TnFLX: a third-generation mariner-based transposon system for Bacillus subtilis

10.1101/825950 ◽

2019 ◽

Author(s):

Felix Dempwolff ◽

Sandra Sanchez ◽

Daniel B. Kearns

Keyword(s):

Antibiotic Resistance ◽

Number System ◽

Cellular Biology ◽

Host Organism ◽

E Coli ◽

Genetic Tool ◽

Translational Activity ◽

Vector Construction ◽

Low Copy Number ◽

Resistance Markers

AbstractRandom transposon mutagenesis is a powerful genetic tool to answer fundamental biological questions in an unbiased approach. Here, we introduce an improved mariner-based transposon system with higher stability, and with versatile applications. We take advantage of the lower frequency of unintended recombination during vector construction and propagation in a low copy number system in E. coli to improve construct integrity. We generated a variety of transposons allowing for gene disruption or artificial overexpression each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance flexibility of future transposon modification and application in Bacillus and other organisms.ImportanceThe optimization of transposase encoding vectors in terms of stability during cloning and propagation is crucial for the reliable application of this system in any host organism. With an increased number of antibiotic resistance markers and the possibility to detect translational activity, the TnFLX transposon system will significantly help the implication of forward genetic methods in the field of cellular biology.

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A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

Applied and Environmental Microbiology ◽

10.1128/aem.02519-20 ◽

2020 ◽

Author(s):

Constantin N. Takacs ◽

Molly Scott ◽

Yunjie Chang ◽

Zachary A. Kloos ◽

Irnov Irnov ◽

...

Keyword(s):

Gene Expression ◽

Antibiotic Resistance ◽

Borrelia Burgdorferi ◽

Gene Function ◽

Electron Tomography ◽

Ease Of Use ◽

Fast Method ◽

Cellular Motility ◽

Crispr Interference ◽

Resistance Markers

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

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Prevalence of Mycoplasma genitalium fluoroquinolone-resistance markers, and dual-class-resistance markers, in asymptomatic men who have sex with men

Journal of Medical Microbiology ◽

10.1099/jmm.0.001429 ◽

2021 ◽

Vol 70 (9) ◽

Author(s):

Teck-Phui Chua ◽

Kaveesha Bodiyabadu ◽

Dorothy A. Machalek ◽

Suzanne M. Garland ◽

Catriona S. Bradshaw ◽

...

Keyword(s):

Antibiotic Resistance ◽

Type Species ◽

Health Centre ◽

Mycoplasma Genitalium ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Link Type ◽

Resistance Markers ◽

Sex With Men ◽

Dual Class

Introduction. Failure of fluoroquinolones, the principal treatment option for macrolide-resistant Mycoplasma genitalium infections, has recently emerged. This is of particular concern for men who have sex with men (MSM), who have high proportions of macrolide-resistant M. genitalium infections. Treatment failure with moxifloxacin is likely the result of single nucleotide polymorphisms (SNPs) in parC, whilst concurrent gyrA mutations may play a role. Gap Statement. The levels of fluoroquinolone resistance and dual-class (i.e. macrolide and fluoroquinolone) resistance in M. genitalium among asymptomatic MSM is unknown. Aim. To (i) determine the proportion of fluoroquinolone resistance and dual-class resistance in M. genitalium infections among asymptomatic MSM, (ii) explore any clinical and behavioural associations with fluoroquinolone resistance, and (iii) determine the distribution of antibiotic resistance among M. genitalium mgpB sequence types (STs). Methodology. M. genitalium positive samples (N=94) were obtained from 1001 asymptomatic MSM enrolled in a study at Melbourne Sexual Health Centre (Carlton, Australia) between August 2016 and September 2017. Sanger sequencing was performed to determine the proportion of M. genitalium infections with SNPs in parC that have previously been associated with failure of moxifloxacin (corresponding to amino changes S83I, D83R, D87Y and D87N) and in gyrA (corresponding to amino acid changes M95I, D99N, D99Y and D99G). Associations between clinical/behavioural factors and parC SNPs were examined. Strain typing was performed by sequencing a portion of the mgpB gene. Results. The proportion of MSM with infections harbouring parC and gyrA SNPs was 13.0 % [95 % confidence interval (CI): 6.8–23.2 %] and 4.7 % (95 % CI: 1.1–13.4 %), respectively; dual-class resistance was 13.0 %. No significant clinical/behavioural associations were found. Antibiotic resistance was not restricted to specific mgpB STs. Conclusion. One in eight (13 %) of asymptomatic MSM with M. genitalium had an infection with dual-class-resistance mutations. Typing by mgpB sequence suggested fluoroquinolone resistance is arising from independent mutation events. This study illustrates that asymptomatic MSM may act as a reservoir for antibiotic-resistant M. genitalium .

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Phenotypic assortment in Tetrahymena thermophila: assortment kinetics of antibiotic-resistance markers, tsA, death, and the highly amplified rDNA locus.

Genetics ◽

10.1093/genetics/120.2.389 ◽

1988 ◽

Vol 120 (2) ◽

pp. 389-395

Author(s):

E V Merriam ◽

P J Bruns

Keyword(s):

Antibiotic Resistance ◽

Random Distribution ◽

Tetrahymena Thermophila ◽

Single Gene ◽

Rdna Locus ◽

Phenotypic Assortment ◽

Resistance Markers ◽

Kinetics Of ◽

Input Ratio ◽

As If

Abstract Phenotypic assortment in Tetrahymena thermophila results from random distribution of alleles during amitotic division of the macronucleus. The rate of assortment is dependent on input ratio and the number of assorting units. The assortment of the antibiotic resistance markers Chx, Mpr and gal was determined and is consistent for each with the model of 45 assorting chromosomes. The gene tsA (previously ts-1) shows normal assortment, in contrast to previous reports. A mutation in the highly amplified ribosomal locus (rdnA2) assorts as if present at only 45 copies. Death of clones occurred at a rate consistent with assortment for a single gene.

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