Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants inStreptococcus mutans

ABSTRACTBroad spectrum antimicrobials, both in dental products and within the clinic, have been used in the suppression of cariogenic bacteria such asStreptococcus mutansfor over 40 years. One such antimicrobial is chlorhexidine (CHX), and serves as a standard in dental research against which other antimicrobial therapies are compared against for their efficacy. However, very little is known about the mode of action for CHX against Streptococci and whether tolerance can be developed from repeated exposures. Here, we begin to answer such questions by passagingS. mutanswith increasing concentrations of CHX and isolating spontaneously-arising tolerant variants (CTVs) from separate lineages. We find that these CTVs display a higher minimal inhibitory concentration (MIC) against CHX than the wild-type strain and have altered virulence properties such as acid tolerance and biofilm formation. We record higher MICs for the variants against both daptomycin and bacitracin, but find increased sensitivity to triclosan and sodium fluoride. Measurements of antagonistic capabilities against other health-associated oral streptococci show decreased bacteriocin production compared to wild-type and increased sensitivity to hydrogen peroxide. Finally whole genome sequencing of the CTVs show common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, altering LTA accumulation and potentially lipid profile of the cell wall. Together, these findings confirm that streptococci may develop tolerance to antimicrobial agents such as CHX but in the case ofS. mutans,increased tolerance may come at a fitness cost for survival within oral biofilms that keeps variants suppressed within the population.

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Spontaneously ArisingStreptococcus mutansVariants with Reduced Susceptibility to Chlorhexidine Display Genetic Defects and Diminished Fitness

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00161-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 4

Author(s):

Justin R. Kaspar ◽

Matthew J. Godwin ◽

Irina M. Velsko ◽

Vincent P. Richards ◽

Robert A. Burne

Keyword(s):

Ex Vivo ◽

Bacterial Species ◽

Oral Streptococci ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Content Type ◽

Glycolipid Synthesis ◽

Acid Tolerant ◽

Increased Sensitivity ◽

Acidic Conditions

ABSTRACTChlorhexidine (CHX) has been used to control dental caries caused by acid-tolerant bacteria such asStreptococcus mutanssince the 1970s. Repeat CHX exposure for other bacterial species results in the development of variants with reduced susceptibility that also become more resistant to other antimicrobials. It has not been tested if such variants arise when streptococci are exposed to CHX. Here, we passagedS. mutansin increasing concentrations of CHX and isolated spontaneously arisingreducedsusceptibilityvariants (RSVs) from separate lineages that have MICs that are up to 3-fold greater than the parental strain. The RSVs have increased growth rates at neutral pH and under acidic conditions in the presence of CHX but accumulate less biomass in biofilms. RSVs display higher MICs for daptomycin and clindamycin but increased sensitivity to dental-relevant antimicrobials triclosan and sodium fluoride. Plate-based assays for competition with health-associated oral streptococci revealed decreased bacteriocin production by the RSVs, increased sensitivity to hydrogen peroxide, and diminished competitive fitness in a human-derivedex vivobiofilm consortium. Whole-genome sequencing identified common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, which could alter the accumulation of lipoteichoic acids and other envelope constituents, as well as a variety of mutations in other genes. Collectively, these findings confirm thatS. mutansand likely other streptococci can develop tolerance to CHX but that increased tolerance comes at a fitness cost, such that CHX-induced variants that spontaneously arise in the human oral cavity may not persist.

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Significant Associations of lncRNA H19 Genotypes with Susceptibility to Childhood Leukemia in Taiwan

Pharmaceuticals ◽

10.3390/ph14030235 ◽

2021 ◽

Vol 14 (3) ◽

pp. 235

Author(s):

Jen-Sheng Pei ◽

Chao-Chun Chen ◽

Wen-Shin Chang ◽

Yun-Chi Wang ◽

Jaw-Chyun Chen ◽

...

Keyword(s):

Confidence Interval ◽

Childhood Leukemia ◽

Healthy Controls ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Genotypic Distribution ◽

Heterozygous Variant ◽

Significant Difference ◽

The Difference

The purpose of our study was to investigate whether genetic variations in lncRNA H19 were associated with susceptibility to childhood leukemia. Two hundred and sixty-six childhood leukemia patients and 266 healthy controls were enrolled in Taiwan, and two single nucleotide polymorphisms (SNPs), rs2839698 and rs217727, in H19 were genotyped and analyzed. There was a significant difference in the genotypic distribution of rs2839698 between patients and healthy controls (p = 0.0277). Compared to the wild-type CC genotype, the heterozygous variant CT and homozygous variant TT genotypes were associated with significantly increased risks of childhood leukemia with an adjusted odd ratio (OR) of 1.46 (95% confidence interval (CI), 1.08–2.14, p = 0.0429) and 1.94 (95%CI, 1.15–3.31, p = 0.0169), respectively (pfor tread = 0.0277). The difference in allelic frequencies between childhood leukemia patients and controls was also significant (T versus C, adjusted OR = 1.53, 95%CI, 1.13–1.79, p = 0.0077). There were no significant differences in the genotypic and allelic distributions of rs217727 between cases and controls. Interestingly, the average level of H19 rs2839698 was statistically significantly higher for patients with CT and TT genotypes than from those with the CC genotype (p < 0.0001). Our results indicate that H19 SNP rs2839698, but not rs217727, may serve as a novel susceptibility marker for childhood leukemia.

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Characterization of Enterococcus faecium mutants resistant to mundticin KS, a class IIa bacteriocin

Microbiology ◽

10.1099/mic.0.26435-0 ◽

2003 ◽

Vol 149 (10) ◽

pp. 2901-2908 ◽

Cited By ~ 26

Author(s):

Youko Sakayori ◽

Mizuho Muramatsu ◽

Satoshi Hanada ◽

Yoichi Kamagata ◽

Shinichi Kawamoto ◽

...

Keyword(s):

Enterococcus Faecium ◽

Antimicrobial Agents ◽

Wild Type Strain ◽

Unsaturated Fatty Acids ◽

Class I ◽

Wild Type ◽

Food Preservatives ◽

In The Wild ◽

Resistant Mutants

The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. To determine the physiological characteristics of resistant mutants, mutants of Enterococcus faecium resistant to mundticin KS, a class IIa bacteriocin, were isolated. Two types of mutant were found that had different sensitivities to other antimicrobial agents such as nisin (class I) and kanamycin. Both mutants were resistant to mundticin KS even in the absence of Mg2+ ions. The composition of unsaturated fatty acids in the resistant mutants was significantly increased in the presence of mundticin KS. The composition of the phospholipids in the two resistant mutants also differed from those in the wild-type strain. The putative zwitterionic amino-containing phospholipid in both mutants significantly increased, whereas amounts of phosphatidylglycerol and cardiolipin decreased. These changes in membrane structure may influence resistance of enterococci to class IIa and class I bacteriocins.

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Associations between varied susceptibilities of PfATP4 inhibitors and genotypes in Ugandan Plasmodium falciparum isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00771-21 ◽

2021 ◽

Author(s):

Oriana Kreutzfeld ◽

Stephanie A. Rasmussen ◽

Aarti A. Ramanathan ◽

Patrick K. Tumwebaze ◽

Oswald Byaruhanga ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Single Nucleotide Polymorphisms ◽

Ex Vivo ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Field Isolates ◽

Frequent Mutations ◽

Mixed Field ◽

P Type

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda from 2016-2019. Median IC 50 s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many non-synonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%) and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92 and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

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From raw reads to trees: Whole genome SNP phylogenetics across the tree of life

10.1101/032250 ◽

2015 ◽

Cited By ~ 10

Author(s):

Sanaa Afroz Ahmed ◽

Chien-Chi Lo ◽

Po-E Li ◽

Karen W Davenport ◽

Patrick S.G. Chain

Keyword(s):

Ad Hoc ◽

Phylogenetic Reconstruction ◽

Clinical Samples ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Complex Samples ◽

Phylogenetic Characterization ◽

Genome Wide ◽

Genome Assemblies

Next-generation sequencing is increasingly being used to examine closely related organisms. However, while genome-wide single nucleotide polymorphisms (SNPs) provide an excellent resource for phylogenetic reconstruction, to date evolutionary analyses have been performed using different ad hoc methods that are not often widely applicable across different projects. To facilitate the construction of robust phylogenies, we have developed a method for genome-wide identification/characterization of SNPs from sequencing reads and genome assemblies. Our phylogenetic and molecular evolutionary (PhaME) analysis software is unique in its ability to take reads and draft/complete genome(s) as input, derive core genome alignments, identify SNPs, construct phylogenies and perform evolutionary analyses. Several examples using genomes and read datasets for bacterial, eukaryotic and viral linages demonstrate the broad and robust functionality of PhaME. Furthermore, the ability to incorporate raw metagenomic reads from clinical samples with suspected infectious agents shows promise for the rapid phylogenetic characterization of pathogens within complex samples.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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Characterisation of the melanocortin-1 receptor gene in alpaca and identification of possible markers associated with phenotypic variations in colour

Animal Production Science ◽

10.1071/an09005 ◽

2009 ◽

Vol 49 (8) ◽

pp. 675 ◽

Cited By ~ 21

Author(s):

N. L. Feeley ◽

K. A. Munyard

Keyword(s):

Mus Musculus ◽

Bos Taurus ◽

Receptor Gene ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Melanocortin 1 Receptor ◽

Mc1r Gene ◽

Pcr Products ◽

Phenotypic Variations

The aim of this study was to determine if any correlation exists between melanocortin-1 receptor (MC1R) polymorphisms and skin and fibre colour in alpacas. Primers capable of amplifying the entire alpaca MC1R gene were designed from a comparative alignment of Bos taurus and Mus musculus MC1R gene sequences. The complete MC1R gene of 41 alpacas exhibiting a range of fibre colours, and which were sourced from farms across Australia, was sequenced from PCR products. Twenty-one single nucleotide polymorphisms were identified within MC1R. Two of these polymorphisms (A82G and C901T) have the potential to reduce eumelanin production by disrupting the activity of MC1R. No agreement was observed between fibre colour alone and MC1R genotype in the 41 animals in this study. However, when the animals were assigned to groups based on the presence or absence of eumelanin in their fibre and skin, only animals that had at least one allele with the A82/C901 combination expressed eumelanin. We propose that A82/C901 is the wild-type dominant ‘E’ MC1R allele, while alpacas with either G82/T901 or G82/Y901 are homozygous for the recessive ‘e’ MC1R allele and are therefore unable to produce eumelanin.

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Prevalence and Genomic Characterization of Brucella canis Strains Isolated from Kennels, Household, and Stray Dogs in Chile

Animals ◽

10.3390/ani10112073 ◽

2020 ◽

Vol 10 (11) ◽

pp. 2073

Author(s):

Nicolás Galarce ◽

Beatriz Escobar ◽

Eduard Martínez ◽

Natalia Alvarado ◽

Gabriela Peralta ◽

...

Keyword(s):

Public Health ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Stray Dogs ◽

Control Programs ◽

Official Control ◽

Brucella Canis ◽

Genomic Characterization ◽

Canine Brucellosis

Canine brucellosis caused by Brucella canis is a zoonotic disease that causes reproductive alterations in dogs, such as infertility, abortion, and epididymitis. This pathogen is especially prevalent in South America, and due to the lack of official control programs and the growing trend of adopting dogs it constitutes a public health risk that must be addressed. The aim of this study was to determine the prevalence of B. canis infection in kennel, shelter, and household dogs and to characterize the genomic properties of circulating strains, including ure and virB operons and omp25/31 genes. Samples from 771 dogs were obtained, and the infection was detected by blood culture and/or serology in 7.0% of the animals. The complete ure and virB operons and the omp25/31 genes were detected. Interestingly, we found different single-nucleotide polymorphisms (SNPs) in some of the analyzed genes, which could mean a change in the fitness or virulence of these strains. This study provides further evidence about dogs as a source of B. canis strains that can infect people. This also highlights the need to implement official control programs, including the mandatory testing of dogs, especially stray dogs, before adoption.

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