scholarly journals Efficiency of Single Phage Suspensions and Phage Cocktail in the Inactivation of Escherichia coli and Salmonella Typhimurium: An In Vitro Preliminary Study

2019 ◽  
Vol 7 (4) ◽  
pp. 94 ◽  
Author(s):  
Pedro Costa ◽  
Carla Pereira ◽  
Ana Gomes ◽  
Adelaide Almeida
Keyword(s):  
Escherichia Coli ◽  
Action Spectrum ◽  
E Coli ◽  
Specific Phage ◽  
Salmonella Infections ◽  
Serovar Typhimurium ◽  
Resistant Mutants ◽  
Community Environments ◽  
Enterobacteriaceae Infections

Enterobacteriaceae Escherichia coli and Salmonella enterica serovar Typhimurium strains are among the main pathogens responsible for moderate and serious infections at hospital and community environments, in part because they frequently present resistance to antibiotics. As the treatment of Enterobacteriaceae infections is empiric, using the same antibiotics to treat E. coli and Salmonella infections, the same concept can be applied with phages. The use of different phages combined in cocktails, frequently used to circumvent the development of phage-resistant mutants, also allows for the treatment of multiple pathogens, broadening the phages’ action spectrum. As such, the aim of this study was to evaluate the efficiency of a cocktail of two phages (ELY-1, produced on E. coli and phSE-5, produced on S. Typhimurium) to control E. coli and S. Typhimurium. Phages ELY-1 and phSE-5 were effective against E. coli (maximum reductions of 4.5 and 3.8 log CFU/mL, respectively), S. Typhimurium (maximum reductions of 2.2 and 2.6 log CFU/mL, respectively), and the mixture of both bacteria (maximum reductions of 2.2 and 2.0 log CFU/mL, respectively). The cocktail ELY-1/phSE-5 was more effective against S. Typhimurium and the mixture of both bacteria (maximum reduction of 3.2 log CFU/mL for both) than the single phage suspensions and as effective against E. coli as its specific phage ELY-1 (maximum reductions of 4.5 log CFU/mL). The use of both the phage cocktails, as well as the single-phage suspensions, however, did not prevent the occurrence of phage-resistant mutants. Overall, the results indicate that the application of the phages in the form of a cocktail show their potential to be used presumptively, that is, prior to the identification of the pathogens, paving its use to control E. coli or S. Typhimurium.

scholarly journals In VitroEffect ofqnrA1,qnrB1, andqnrS1Genes on Fluoroquinolone Activity against IsogenicEscherichia coliIsolates with Mutations ingyrAandparC

2010 ◽  
Vol 55 (3) ◽  
pp. 1266-1269 ◽  
Author(s):  
A. Briales ◽  
J. M. Rodríguez-Martínez ◽  
C. Velasco ◽  
P. Díaz de Alba ◽  
J. Domínguez-Herrera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
E Coli ◽  
Mutant Prevention Concentration ◽  
One Step ◽  
Resistant Mutants

ABSTRACTThis article provides an analysis of thein vitroeffect ofqnrA1,qnrB1, andqnrS1genes, combined with quinolone-resistant Ser83Leu substitutions in GyrA and/or Ser80Arg in ParC, on fluoroquinolone (FQ) resistance in isogenicEscherichia colistrains. The association of Ser83Leu substitution in GyrA, Ser80Arg substitution in ParC, andqnrgene expression increased the MIC of ciprofloxacin to 2 μg/ml.qnrgenes present inE. colithat harbored a Ser83Leu substitution in GyrA increased mutant prevention concentration (MPC) values to 8 to 32 μg/ml.qnrgene expression inE. colimay play an important role in selecting for one-step FQ-resistant mutants.

scholarly journals Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

2004 ◽  
Vol 186 (9) ◽  
pp. 2829-2840 ◽  
Author(s):  
Heather D. Carter ◽  
Vladimir Svetlov ◽  
Irina Artsimovitch
Keyword(s):  
Escherichia Coli ◽  
Expression Vectors ◽  
Divergent Evolution ◽  
Transcriptional Enhancer ◽  
Elongation Complex ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Transcript Elongation

ABSTRACT The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.

scholarly journals ArgR-Independent Induction and ArgR-Dependent Superinduction of the astCADBE Operon in Escherichia coli

2002 ◽  
Vol 184 (11) ◽  
pp. 2940-2950 ◽  
Author(s):  
Alexandros K. Kiupakis ◽  
Larry Reitzer
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Nitrogen Source ◽  
Nitrogen Limitation ◽  
Nitrogen Sources ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Nitrogen Regulator I

ABSTRACT For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ54 and nitrogen regulator I (NRI, also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a σ54-dependent promoter utilized in exponential phase during nitrogen limitation and a σS-dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NRI, and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ54-dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NRI-dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression.

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Investigation of Salmonella Phage–Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1261
Author(s):  
Khatuna Makalatia ◽  
Elene Kakabadze ◽  
Nata Bakuradze ◽  
Nino Grdzelishvili ◽  
Ben Stamp ◽  
...  
Keyword(s):  
Host Range ◽  
Salmonella Enterica ◽  
Target Range ◽  
Nested Subsets ◽  
Specific Phage ◽  
Salmonella Infections ◽  
Specialist Species ◽  
Host Infection ◽  
Salmonella Phage

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus–host infection networks have been used to characterize the “predator–prey” interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage–bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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