scholarly journals Bactericidal Activities of BMS-284756, a Novel Des-F(6)-Quinolone, against Staphylococcus aureus Strains with Topoisomerase Mutations

2002 ◽  
Vol 46 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Laura E. Lawrence ◽  
MaryBeth Frosco ◽  
Brenda Ryan ◽  
Susan Chaniewski ◽  
Hyekyung Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
In Vivo Efficacy ◽  
Mutant Strains ◽  
Bactericidal Activities ◽  
Time Kill

ABSTRACT The antistaphylococcal activities of BMS-284756 (T-3811ME), levofloxacin, moxifloxacin, and ciprofloxacin were compared against wild-type and grlA and grlA/gyrA mutant strains of Staphylococcus aureus. BMS-284756 was the most active quinolone tested, with MICs and minimal bactericidal concentrations against S. aureus wild-type strain MT5, grlA mutant MT5224c4, and grlA/gyrA mutant EN8 of 0.03 and 0.06, 0.125 and 0.125, and 4 and 4 μg/ml, respectively. In the time-kill studies, BMS-284756 and levofloxacin exhibited rapid killing against all strains. Ciprofloxacin, however, was not bactericidal for the double mutant, EN8. BMS-284756 and levofloxacin were bactericidal (3 log10 decrease in CFU/ml) against the MT5 and MT5224c4 strains at two and four times the MIC within 2 to 4 h. Against EN8, BMS-284756 was bactericidal within 4 h at two and four times the MIC, and levofloxacin achieved similar results within 4 to 6 h. Both the wild-type strain MT5 and grlA mutant MT5224c4 should be considered susceptible to both BMS-284756 and levofloxacin, and both quinolones are predicted to have clinical efficacy. The in vivo efficacy of BMS-284756, levofloxacin, and moxifloxacin against S. aureus strain ISP794 and its single mutant 2C6(1)-1 directly reflected the in vitro activity: increased MICs correlated with decreased in vivo efficacy. The 50% protective doses of BMS-284756 against wild-type and mutant strains were 2.2 and 1.6 mg/kg of body weight/day, respectively, compared to the levofloxacin values of 16 and 71 mg/kg/day and moxifloxacin values of 4.7 and 61.6 mg/kg/day. BMS-284756 was more potent than levofloxacin and equipotent with moxifloxacin against ISP794 both in vitro and in vivo, while BMS-284756 was more potent than levofloxacin and moxifloxacin against 2C6(1)-1.

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 Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals A Single Amino Acid at Residue 188 of Hexon Protein is Responsible for the Pathogenicity of the Emerging Novel Fowl Adenovirus 4

2021 ◽  
Author(s):  
Yu Zhang ◽  
Aijing Liu ◽  
Yanan Wang ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Molecular Basis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Hexon Protein

Since 2015, severe hydropericardium-hepatitis syndrome (HHS) associated with a novel fowl adenovirus 4 (FAdV-4) has emerged in China, representing a new challenge for the poultry industry. Although various highly pathogenic FAdV-4 strains have been isolated, the virulence factor and the pathogenesis of novel FAdV-4 are unclear. In our previous studies, we reported that a large genomic deletion (1966 bp) is not related to increased virulence. In this study, two recombinant chimeric viruses, rHN20 strain and rFB2 strain, were generated from a highly pathogenic FAdV-4 strain by replacing hexon or fiber-2 gene of a non-pathogenic FAdV-4, respectively. Both chimeric strains showed similar titers to the wild type strain in vitro . Notably, rFB2 and the wild type strain induced 100% mortality, while no mortality or clinical signs appeared in chickens inoculated with rHN20, indicating that hexon, but not fiber-2, determines the novel FAdV-4 virulence. Furthermore, an R188I mutation in the hexon protein identified residue 188 as the key amino acid for the reduced pathogenicity. The rR188I mutant strain was significantly neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was able to replicate efficiently. Finally, the immunogenicity of the rescued rR188I was investigated. Non-pathogenic rR188I provided full protection against lethal FAdV-4 challenge. Collectively, these findings provide an in-depth understanding of the molecular basis of novel FAdV-4 pathogenicity and present rR188I as a potential live attenuated vaccine candidate or a novel vaccine vector for HHS vaccines. Importance HHS associated with a novel FAdV-4 infection in chickens has caused huge economic losses to the poultry industry in China since 2015. The molecular basis for the increased virulence remains largely unknown. Here, we demonstrate that the hexon gene is vital for FAdV-4 pathogenicity. Furthermore, we show that the amino acid residue at position 188 of the hexon protein is responsible for pathogenicity. Importantly, the rR188I mutant strain was neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was not. Further, the rR188I mutant strain provided complete protection against FAdV-4 challenge. Our results provide a molecular basis of the increased virulence of novel FAdV-4. We propose that the rR188I mutant is a potential live attenuated vaccine against HHS and a new vaccine vector for HHS-combined vaccines.

scholarly journals Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae

2001 ◽  
Vol 45 (6) ◽  
pp. 1649-1653 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaya Takei ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Methoxy Group ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

scholarly journals Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Monchaya Rattanaprasert ◽  
Jan-Peter van Pijkeren ◽  
Amanda E. Ramer-Tait ◽  
Maria Quintero ◽  
Car Reen Kok ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lactobacillus Reuteri ◽  
Selective Advantage ◽  
Wild Type ◽  
Carbohydrate Source ◽  
Content Type ◽  
Gnotobiotic Mice

ABSTRACT Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo. In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

scholarly journals Bacillus subtilis Phosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-Responsive phoB-PS+V Promoters during Pho Response

2005 ◽  
Vol 187 (15) ◽  
pp. 5166-5178 ◽  
Author(s):  
Wael R. Abdel-Fattah ◽  
Yinghua Chen ◽  
Amr Eldakak ◽  
F. Marion Hulett
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphate Deprivation

ABSTRACT The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-PV. EσE was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σE is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PS in vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

scholarly journals The Role of graRS in Regulating Virulence and Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Le Chen ◽  
Zihui Wang ◽  
Tao Xu ◽  
Hongfei Ge ◽  
Fangyue Zhou ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Bacterial Resistance ◽  
Wild Type Strain ◽  
Lysine Biosynthesis ◽  
Infection Model ◽  
Wild Type ◽  
Methicillin Resistant

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the ΔgraRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “de novo” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (ΔgraRS) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500ΔgraRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS. Meanwhile, an expression of the virulence-associated genes (coa, hla, hlb, agrA, and mgrA) was downregulated in the ΔgraRS mutant. Consistently, the A549 epithelial cells invasion of the ΔgraRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500ΔgraRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo. In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.

Impact of sarA Mutation on Immune System Evasion and Stress Response in Staphylococcus aureus

2020 ◽  
Vol 29 (3) ◽  
pp. 105-112
Author(s):  
Yomna A. Hagag ◽  
Abdelaziz Elgaml ◽  
Ramadan Hassan ◽  
Hany I. Kenawy
Keyword(s):  
Staphylococcus Aureus ◽  
Immune System ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Human Neutrophils ◽  
Bacterial Survival ◽  
Wild Type ◽  
The Impact

Background: Staphylococcus aureus is a major human pathogen responsible for a large number of infections. In S. aureus, SarA is an important global locus responsible for the regulation of virulence factors, as well as biofilm formation. Objectives: The aim of this work is to clarify the impact of SarA on biofilm formation, immune system evasion, as well as the survival of S. aureus under stress conditions. Methodology: A comparative study between S. aureus wild type strain, sarA mutant and complemented strains was established addressing the biofilm formation, opsonization, phagocytosis, as well as ability of the bacterium to survive in stressful environments including acidic pH, hyperosmotic and oxidative stress. The in vitro experiments were confirmed by challenging of mice via intraperitoneal injection with the wild type strain, sarA mutant and complemented strains. Results: Mutation of sarA diminished significantly biofilm formation. Moreover, this mutation resulted in a slight decrease in the deposition of the most important opsonin in complement-mediated immunity, named C3 on S. aureus cells. However, this mutation was associated with a significant enhancement of bacterial phagocytosis and killing by human neutrophils. Furthermore, this mutation altered bacterial survival in stressful conditions. It is also noteworthy that sarA mutation resulted in a significant higher survival rates during the challenging of mice. Conclusion: SarA plays a role as a key regulator of biofilm formation, which in turn has a great impact on immune system evasion through affecting opsonization and phagocytosis. In addition, SarA improves the ability of S. aureus to survive in stressful conditions.

A RIBONUCLEIC ACID FRACTION OF NEUROSPORA CRASSA EVOKING DEVELOPMENT OF A PSEUDO-TRYPTOPHAN SYNTHETASE ACTIVITY BY EXTRACTS OF MUTANTS TD1, TD2, AND TD3

1965 ◽  
Vol 43 (11) ◽  
pp. 1813-1828 ◽  
Author(s):  
S. D. Wainwright ◽  
E. Sandra McFarlane
Keyword(s):  
Neurospora Crassa ◽  
Type Strain ◽  
Wild Type Strain ◽  
Enzymic Activity ◽  
Synthetase Activity ◽  
Acid Fraction ◽  
Wild Type ◽  
Mutant Strains ◽  
Tryptophan Synthetase

"Soluble RNA" fractions isolated from the wild-type strain of Neurospora crassa evoked development of a pseudo-tryptophan synthetase enzyme activity in vitro by extracts of mutant strains lacking ability to produce the enzymic activity. The RNA fractions contained no detectable "template RNA". Some properties of the component eliciting development of pseudo-tryptophan synthetase activity are reported.

scholarly journals Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in Bacillus subtilis

2001 ◽  
Vol 183 (17) ◽  
pp. 4958-4963 ◽  
Author(s):  
Takashi Inaoka ◽  
Koji Kasai ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Type Strain ◽  
Wild Type Strain ◽  
Assay System ◽  
Wild Type ◽  
Translational Accuracy ◽  
Free Translation

ABSTRACT To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in aram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

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