scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

scholarly journals Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli

2015 ◽  
Vol 197 (23) ◽  
pp. 3658-3665 ◽  
Author(s):  
Jia Hu ◽  
Brittany N. Ross ◽  
Roberto J. Cieza ◽  
Alfredo G. Torres
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Colonization Factor ◽  
Initial Adhesion ◽  
Intestinal Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is a human pathogen that requires initial adhesion to the intestine in order to cause disease. Multiple adhesion factors have been identified inE. colistrains, among them the long polar fimbriae (Lpf), a colonization factor associated with intestinal adhesion. The conditions of Lpf expression are well understood in enterohemorrhagicE. coli(EHEC); however, the expression of EPEClpfhas been found to be repressed under anyin vitrocondition tested. Therefore, we decided to identify those factors silencing expression of EPEClpf. Because histone-like nucleoid structuring protein (H-NS) is a known repressor of EHEClpf, we tested it and found that H-NS is a repressor of EPEClpf. We also found that the adhesion of the EPEC Δhnsstrain was significantly enhanced compared to the wild-type strain. Becauselpfexpression was modestly increased in thehnsmutant, transposon mutagenesis was performed to find a strain displaying higherlpfexpression than EPEC Δhns. One Tn5insertion was identified within theyhjXgene, and furtherin vitrocharacterization revealed increasedlpfexpression and adhesion to Caco-2 cells compared with EPEC Δhns. However, in a murine model of intestinal infection, the EPEC Δhnsand EPEC ΔhnsTn5mutants had only a slight change in colonization pattern compared to the wild-type strain. Our data showed that EPEC Lpf is transcribed, but its role in EPEC intestinal colonization requires further analysis.IMPORTANCEData are presented demonstrating that the long polar fimbriae (lpf) operon in enteropathogenicE. coli(EPEC) is highly regulated; however, derepression occurs by mutagenizing two proteins associated with its control. The study demonstrates that the EPEClpfoperon can be expressed and, therefore, participates in the EPEC adherence phenotype.

scholarly journals Borrelia burgdorferi vlsE Antigenic Variation Is Not Mediated by RecA

2008 ◽  
Vol 76 (9) ◽  
pp. 4009-4018 ◽  
Author(s):  
Dionysios Liveris ◽  
Vishwaroop Mulay ◽  
Sabina Sandigursky ◽  
Ira Schwartz
Keyword(s):  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Antigenic Variation ◽  
Genetic Recombination ◽  
Joint Infection ◽  
Functional Characterization ◽  
Wild Type ◽  
Reca Mutant

ABSTRACT RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Construction and Characterization of aMycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF

2000 ◽  
Vol 68 (10) ◽  
pp. 5575-5580 ◽  
Author(s):  
Ping Chen ◽  
Rafael E. Ruiz ◽  
Qing Li ◽  
Richard F. Silver ◽  
William R. Bishai
Keyword(s):  
Stationary Phase ◽  
Sigma Factor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Axenic Culture ◽  
Wild Type ◽  
Intracellular Growth ◽  
Factor Gene ◽  
Time To Death

ABSTRACT The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

2000 ◽  
Vol 182 (23) ◽  
pp. 6630-6637 ◽  
Author(s):  
Chin Li ◽  
Yi Ping Tao ◽  
Lee D. Simon
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Transcription Termination ◽  
Carbon Starvation ◽  
Lower Percentage ◽  
Wild Type ◽  
Lower Efficiency ◽  
E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

scholarly journals A genetic analysis of resistance to nystatin inSaccharomyces cerevisiae

1967 ◽  
Vol 9 (2) ◽  
pp. 179-193 ◽  
Author(s):  
K. A. Ahmed ◽  
R. A. Woods
Keyword(s):  
Genetic Analysis ◽  
Resistance Genes ◽  
Type Strain ◽  
Wild Type Strain ◽  
Second Step ◽  
Wild Type ◽  
Recessive Resistance ◽  
Low Concentrations ◽  
Effects Of Temperature ◽  
Resistant Mutants

1. A number of stable nystatin-resistant mutants of the yeastSaccharomyces cerevisiaehave been isolated from platings of a sensitive wild-type strain on low concentrations of the antibiotic.2. These mutants were found to be resistant to 10, 15 or 60 units of drug/ml.3. Analysis of meiotic segregants from crosses of these mutants to wild-type indicate that resistance is determined by two types of genes; resistance genes and modifiers.4. Functional analysis of the mutants demonstrated the existence of three recessive resistance genes,nys-l,nys-2 andnys-3 and thatnys-1 andnys-2 were linked.5. Genetic analysis showed thatnys-1 was affected by two modifiers,Mnys-1 andMnys-2, but that onlyMnys-2 affectednys-2 andnys-3.6. The modifiersMnys-1 andMnys-2 are dominant.7. An investigation of the effects of temperature and medium on resistance demonstrated marked interactions between genotype and environment for both the resistance genes and the modifiers.8. Second-step mutants have been isolated by plating first-step mutants on higher concentrations of the drug. Some of these are resistant to 800 units/ml.9. Some possible mechanisms of nystatin resistance are discussed.

scholarly journals A Bile Salt Hydrolase of Brucella abortus Contributes to the Establishment of a Successful Infection through the Oral Route in Mice

2006 ◽  
Vol 75 (1) ◽  
pp. 299-305 ◽  
Author(s):  
M. Victoria Delpino ◽  
María I. Marchesini ◽  
Silvia M. Estein ◽  
Diego J. Comerci ◽  
Juliana Cassataro ◽  
...  
Keyword(s):  
Bile Salt ◽  
Brucella Abortus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Interleukin 2 ◽  
Oral Route ◽  
Bile Salt Hydrolase ◽  
Wild Type ◽  
Successful Infection

ABSTRACT Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of Brucella species. We obtained the Brucella CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate. Brucella abortus 2308 (wild type) and its isogenic Δcgh deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Δcgh mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Δcgh mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live B. abortus 2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on Brucella the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of Brucella to infect the host through the oral route.

scholarly journals Lipooligosaccharide Structure Contributes to Multiple Steps in the Virulence of Neisseria meningitidis

2006 ◽  
Vol 74 (2) ◽  
pp. 1360-1367 ◽  
Author(s):  
Laura Plant ◽  
Johanna Sundqvist ◽  
Susu Zughaier ◽  
Lena Lövkvist ◽  
David S. Stephens ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Neisseria Meningitidis ◽  
Type Strain ◽  
Lipid A ◽  
Wild Type Strain ◽  
Inner Core ◽  
Wild Type ◽  
Proinflammatory Response

ABSTRACT Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

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