Validation of Pefloxacin for detection of fluoroquinolone (FQ) resistance among Salmonella Typhi with special reference to GyrB mutations

2021 ◽  
Vol 70 (8) ◽  
Author(s):  
Shakila Banu Inayath ◽  
Shobha Broor ◽  
Ruchi Gupta ◽  
Priti Agarwal ◽  
Subhradeep Majumder ◽  
...  
Keyword(s):  
Special Reference ◽  
Nalidixic Acid ◽  
Inhibitory Concentration ◽  
Resistance Mechanism ◽  
Salmonella Typhi ◽  
Wild Type ◽  
Mechanisms Of Resistance ◽  
Content Type ◽  
Link Type ◽  
Genetic Mechanisms

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml−1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella . Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species. Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations. Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005–2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30μg), ciprofloxacin (5μg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi. Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml−1) and non-susceptible (≥0.125 µg ml−1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type. Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

scholarly journals Molecular Characterization of the CytochromebGene andIn VitroAtovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

2015 ◽  
Vol 59 (3) ◽  
pp. 1818-1821 ◽  
Author(s):  
Luicer A. Ingasia ◽  
Hoseah M. Akala ◽  
Mabel O. Imbuga ◽  
Benjamin H. Opot ◽  
Fredrick L. Eyase ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Polymorphism ◽  
Molecular Characterization ◽  
Mutant Allele ◽  
Inhibitory Concentration ◽  
Wild Type Allele ◽  
Wild Type ◽  
Western Kenya ◽  
Content Type

ABSTRACTThe prevalence of a genetic polymorphism(s) at codon 268 in the cytochromebgene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227Plasmodium falciparumparasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
Mengting Shi ◽  
Yue Zheng ◽  
Xianghong Wang ◽  
Zhengjia Wang ◽  
Menghua Yang
Keyword(s):  
Mouse Model ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Type Species ◽  
Wild Type ◽  
Expression Library ◽  
Content Type ◽  
Genomic Expression ◽  
Infant Mouse ◽  
Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

scholarly journals Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽  
2020 ◽  
Vol 166 (5) ◽  
pp. 484-497 ◽  
Author(s):  
Alejandra Arteaga Ide ◽  
Victor M. Hernández ◽  
Liliana Medina-Aparicio ◽  
Edson Carcamo-Noriega ◽  
Lourdes Girard ◽  
...  
Keyword(s):  
Type Species ◽  
Sinorhizobium Meliloti ◽  
Arginase Activity ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Link Type ◽  
Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella

Microbiology ◽  
2021 ◽  
Author(s):  
Karine Dufresne ◽  
France Daigle
Keyword(s):  
Wild Type Strain ◽  
Distal Region ◽  
Transcriptional Factor ◽  
Major Influence ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Serovar Typhimurium ◽  
Metabolic Regulators

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella .

scholarly journals ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans

2019 ◽  
Vol 1 (8) ◽  
Author(s):  
Xiao-Lin Tian ◽  
Miao Li ◽  
Zachariah Scinocca ◽  
Heather Rutherford ◽  
Yung-Hua Li
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Critical Role ◽  
Cell Formation ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Viability Assay ◽  
Persister Cell

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

scholarly journals Discerning the role of a functional arsenic-resistance cassette in the evolution and adaptation of a rice pathogen

2021 ◽  
Vol 7 (7) ◽  
Author(s):  
Amandeep Kaur ◽  
Rekha Rana ◽  
Tanu Saroha ◽  
Prabhu B. Patil
Keyword(s):  
Type Species ◽  
Evolutionary Dynamics ◽  
Iron Acquisition ◽  
Arsenic Exposure ◽  
Resistance Mechanism ◽  
Transcriptional Response ◽  
Adaptation Mechanism ◽  
Arsenic Resistance ◽  
Content Type ◽  
Link Type

Arsenic is highly toxic element to all forms of life and is a major environmental contaminant. Understanding acquisition, detoxification and adaptation mechanisms in bacteria that are associated with the host in arsenic-rich conditions can provide novel insights into the evolutionary dynamics of host–microbe–environment interactions. In the present study, we have investigated an arsenic-resistance mechanism acquired during the evolution of a particular lineage in the population of Xanthomonas oryzae pv. oryzae, which is a serious plant pathogen infecting rice. Our study revealed the horizontal acquisition of a novel chromosomal 12 kb ars cassette in X. oryzae pv. oryzae IXO1088 that confers high resistance to arsenate/arsenite. The ars cassette comprises several genes that constitute an operon induced in the presence of arsenate/arsenite. Transfer of the cloned ars cassette to X. oryzae pv. oryzae BXO512, which lacks the cassette, confers an arsenic-resistance phenotype. Furthermore, the transcriptional response of X. oryzae pv. oryzae IXO1088 under arsenate/arsenite exposure was analysed using RNA sequencing. Arsenic detoxification and efflux, oxidative stress, iron acquisition/storage, and damage repair are the main cellular responses to arsenic exposure. Our investigation has provided insights into the existence of a novel detoxification and adaptation mechanism within the X. oryzae pv. oryzae population to deal with high-arsenic conditions outside the rice plant.

scholarly journals Differential susceptibility of airway and ocular surface cell lines to FlhDC-mediated virulence factors PhlA and ShlA from Serratia marcescens

2020 ◽  
Author(s):  
Nicholas A. Stella ◽  
Kimberly M. Brothers ◽  
Robert M. Q. Shanks
Keyword(s):  
Epithelial Cells ◽  
Serratia Marcescens ◽  
Cell Lines ◽  
Ocular Surface ◽  
Type Species ◽  
Wild Type ◽  
Bacterial Killing ◽  
Content Type ◽  
Link Type ◽  
Surface Cell

Introduction. Serratia marcescens is a bacterial pathogen that causes ventilator-associated pneumonia and ocular infections. The FlhD and FlhC proteins complex to form a heteromeric transcription factor whose regulon, in S. marcescens , regulates genes for the production of flagellum, phospholipase A and the cytolysin ShlA. The previously identified mutation, scrp-31, resulted in highly elevated expression of the flhDC operon. The scrp-31 mutant was observed to be more cytotoxic to human airway and ocular surface epithelial cells than the wild-type bacteria and the present study sought to identify the mechanism underlying the increased cytotoxicity phenotype. Hypothesis/Gap Statement. Although FlhC and FlhD have been implicated as virulence determinants, the mechanisms by which these proteins regulate bacterial cytotoxicity to different cell types remains unclear. Aim. This study aimed to evaluate the mechanisms of FlhDC-mediated cytotoxicity to human epithelial cells by S. marcescens . Methodology. Wild-type and mutant bacteria and bacterial secretomes were used to challenge airway and ocular surface cell lines as evaluated by resazurin and calcein AM staining. Pathogenesis was further tested using a Galleria mellonella infection model. Results. The increased cytotoxicity of scrp-31 bacteria and secretomes to both cell lines was eliminated by mutation of flhD and shlA. Mutation of the flagellin gene had no impact on cytotoxicity under any tested condition. Elimination of the phospholipase gene, phlA, had no effect on bacteria-induced cytotoxicity to either cell line, but reduced cytotoxicity caused by secretomes to airway epithelial cells. Mutation of flhD and shlA, but not phlA, reduced bacterial killing of G. mellonella larvae. Conclusion. This study indicates that the S. marcescens FlhDC-regulated secreted proteins PhlA and ShlA, but not flagellin, are cytotoxic to airway and ocular surface cells and demonstrates differences in human epithelial cell susceptibility to PhlA.

scholarly journals Polymyxin Susceptibility in Pseudomonas aeruginosa Linked to the MexXY-OprM Multidrug Efflux System

2015 ◽  
Vol 59 (12) ◽  
pp. 7276-7289 ◽  
Author(s):  
Keith Poole ◽  
Calvin Ho-Fung Lau ◽  
Christie Gilmour ◽  
Youai Hao ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Mechanism ◽  
Polymyxin B ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Polymyxin E ◽  
Increased Susceptibility ◽  
Multidrug Efflux System

ABSTRACTThe ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced themexXYgenes of the MexXY-OprM multidrug efflux system inPseudomonas aeruginosaand increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci,arnBCADTEFand PA4773-74. Consistent with the SPC-promoted reduction inarnand PA4773-74 expression being linked tomexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressingmexXYand enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lackingarnBand/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lackingmexXYwas unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance ‘mechanism’ is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-typeP. aeruginosabut not in the ΔmexXYmutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-inducedmexXYexpression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.

scholarly journals SbcCD-Mediated Processing of Covalent Gyrase-DNA Complex in Escherichia coli

2013 ◽  
Vol 57 (10) ◽  
pp. 5116-5119 ◽  
Author(s):  
Sandra Aedo ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Nalidixic Acid ◽  
Mutant Strain ◽  
Nuclease Activity ◽  
Bactericidal Action ◽  
Wild Type ◽  
Content Type ◽  
Dna Complex ◽  
Covalent Complex

ABSTRACTQuinolones trap the covalent gyrase-DNA complex inEscherichia coli, leading to cell death. Processing activities for trapped covalent complex have not been characterized. A mutant strain lacking SbcCD nuclease activity was examined for both accumulation of gyrase-DNA complex and viability after quinolone treatment. Higher complex levels were found in ΔsbcCDcells than in wild-type cells after incubation with nalidixic acid and ciprofloxacin. However, SbcCD activity protected cells against the bactericidal action of nalidixic acid but not ciprofloxacin.

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