scholarly journals OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC

2020 ◽  
Vol 75 (5) ◽  
pp. 1151-1158
Author(s):  
Corey S Suelter ◽  
Nancy D Hanson
Keyword(s):  
Escherichia Coli ◽  
Half Life ◽  
Selective Advantage ◽  
Resistance Mechanisms ◽  
Northern Blotting ◽  
Rt Pcr ◽  
E Coli ◽  
Protein Levels ◽  
Porin Proteins ◽  
Mrna Half Life

Abstract Background Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic. Objectives To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production. Methods RNA expression was evaluated using real-time RT–PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting. Results Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21–30 min for ST131 isolates compared with <2–23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with −4- to 70-fold for non-ST131 isolates). Conclusions Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.

scholarly journals Reverse Transcription-PCR Differential Display Analysis of Escherichia coli Global Gene Regulation in Response to Heat Shock

1999 ◽  
Vol 65 (12) ◽  
pp. 5386-5393 ◽  
Author(s):  
R. T. Gill ◽  
J. J. Valdes ◽  
W. E. Bentley
Keyword(s):  
Escherichia Coli ◽  
Gene Regulation ◽  
Heat Shock ◽  
Reverse Transcription ◽  
Northern Blotting ◽  
Specific Gene ◽  
Rt Pcr ◽  
Total Rna ◽  
E Coli ◽  
Global Gene Regulation

ABSTRACT A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830–1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495–508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026–2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation inE. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coliglobal gene regulation in response to additional environmental stimuli.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

Antimicrobial Resistance and Association with Class 1 Integrons in Escherichia coli Isolated from Turkey Meat Products

2008 ◽  
Vol 71 (8) ◽  
pp. 1679-1684 ◽  
Author(s):  
M. L. KHAITSA ◽  
J. OLOYA ◽  
D. DOETKOTT ◽  
R. KEGODE
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
Population Attributable Fraction ◽  
Meat Products ◽  
Resistance Mechanisms ◽  
E Coli ◽  
Class 1 Integrons ◽  
Turkey Meat ◽  
Class 1

The objective of this study was to quantify the role of class 1 integrons in antimicrobial resistance in Escherichia coli isolated from turkey meat products purchased from retail outlets in the Midwestern United States. Of 242 E. coli isolates, 41.3% (102 of 242) tested positive for class 1 integrons. A significant association was shown between presence of class 1 integrons in E. coli isolates and the resistance to tetracycline, ampicillin, streptomycin, gentamicin, sulfisoxazole, and trimethoprim-sulfamethoxazole. Attributable risk analysis revealed that for every 100 E. coli isolates carrying class 1 integrons, resistance was demonstrated for ampicillin (22%), gentamycin (48%), streptomycin (29%), sulfisoxazole (40%), trimethoprimsulfamethoxazole (7%), and tetracycline (26%). Non–integron-related antimicrobial resistance was demonstrated for ampicillin (65%), gentamycin (16.9%), streptomycin (42.1%), sulfisoxazole (35.8%), and tetracycline (49.7%). Population-attributable fraction analysis showed that class 1 integrons accounted for the following resistances: gentamycin, 71% (50 of 71), amoxicillin–clavulanic acid, 19.6% (6 of 33), nalidixic acid, 34% (7 of 21), streptomycin, 28% (30 of 107), sulfisoxazole, 38% (40 of 106), and tetracycline, 14%, (26 of 185). In conclusion, although class 1 integrons have been implicated in resistance to antimicrobial agents, other non–integron resistance mechanisms seem to play an important part.

Comparative Evaluation of a Phage Protein Ligand Assay with Real-Time PCR and a Reference Method for the Detection of Escherichia coli O157:H7 in Raw Ground Beef and Trimmings

2011 ◽  
Vol 74 (1) ◽  
pp. 6-12 ◽  
Author(s):  
F. SAVOYE ◽  
P. FENG ◽  
C. ROZAND ◽  
M. BOUVIER ◽  
A. GLEIZAL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Ground Beef ◽  
Reference Method ◽  
Enterohemorrhagic Escherichia Coli ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Rt Pcr ◽  
Raw Meat ◽  
E Coli

Enterohemorrhagic Escherichia coli O157:H7 is an important pathogen associated with infections caused by consumption of undercooked raw meat. Sensitive and rapid detection methods for E. coli O157:H7 are essential for the meat industry to ensure a safe meat supply. This study was conducted to compare the sensitivity of the VIDAS ultraperformance E. coli test (ECPT UP) with a noncommercial real-time (RT) PCR method and the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) reference method for detecting E. coli O157:H7 in raw ground beef. Optimal enrichment times and the efficacy of testing different types of raw meat, either as individual samples (25 g) or as composites (375 g), were examined. For 25-g samples of each type of raw ground beef tested, 6 h of enrichment was sufficient for both the VIDAS ECPT UP and RT-PCR methods, but for 375-g samples, 24 h of enrichment was required. Both the VIDAS ECPT UP and RT-PCR methods produced results similar to those obtained with the USDA-FSIS reference method after 18 to 24 h of enrichment. The primer specificity of the RT-PCR assay and the highly specific phage ligand used in the VIDAS ECPT UP for target recognition enabled the detection of low levels of E. coli O157:H7 in 25 g of various types of raw ground beef. The tests also allowed the detection of E. coli O157:H7 in composite raw ground beef and trimmings in samples of up to 375 g.

scholarly journals Nickel Promotes Biofilm Formation by Escherichia coli K-12 Strains That Produce Curli

2009 ◽  
Vol 75 (6) ◽  
pp. 1723-1733 ◽  
Author(s):  
Claire Perrin ◽  
Romain Briandet ◽  
Gregory Jubelin ◽  
Philippe Lejeune ◽  
Marie-Andrée Mandrand-Berthelot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Selective Advantage ◽  
Bacterial Survival ◽  
Toxic Compounds ◽  
E Coli ◽  
Transcriptional Effect ◽  
K 12 ◽  
Encoding Genes

ABSTRACT The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.

scholarly journals Unexpected Stress-Reducing Effect of PhaP, a Poly(3-Hydroxybutyrate) Granule-Associated Protein, in Escherichia coli

2011 ◽  
Vol 77 (18) ◽  
pp. 6622-6629 ◽  
Author(s):  
Alejandra de Almeida ◽  
Mariela V. Catone ◽  
Virgil A. Rhodius ◽  
Carol A. Gross ◽  
M. Julia Pettinari
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Stress Proteins ◽  
Protective Role ◽  
Pcr Analysis ◽  
Content Type ◽  
E Coli ◽  
Protein Levels ◽  
Stress Related Genes ◽  
Heat Stress Proteins

ABSTRACTPhasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinantEscherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP fromAzotobactersp. strain FA8, on the expression of stress-related genes in PHB-producingE. coli. While PHB accumulation was found to increase the transcription ofdnaKandibpA, the expression of these genes and ofgroES,groEL,rpoH,dps, andyfiDwas reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizingE. coliand linked the effects of the protein to the expression of stress-related genes, especiallyibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect inE. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.

scholarly journals The Streptomycin-Sulfadiazine-Tetracycline Antimicrobial Resistance Element of Calf-Adapted Escherichia coli Is Widely Distributed among Isolates from Washington State Cattle

2007 ◽  
Vol 74 (2) ◽  
pp. 391-395 ◽  
Author(s):  
Artashes R. Khachatryan ◽  
Thomas E. Besser ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Selective Advantage ◽  
Washington State ◽  
Resistance Pattern ◽  
Genetic Element ◽  
Dairy Calf ◽  
Genetic Traits ◽  
E Coli

ABSTRACT Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.

scholarly journals Spanish Multicenter Study of the Epidemiology and Mechanisms of Amoxicillin-Clavulanate Resistance in Escherichia coli

2012 ◽  
Vol 56 (7) ◽  
pp. 3576-3581 ◽  
Author(s):  
Adriana Ortega ◽  
Jesús Oteo ◽  
Maitane Aranzamendi-Zaldumbide ◽  
Rosa M. Bartolomé ◽  
Germán Bou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multicenter Study ◽  
Resistance Mechanisms ◽  
Mechanisms Of Resistance ◽  
Prospective Multicenter Study ◽  
Content Type ◽  
E Coli ◽  
High Prevalence ◽  
Amoxicillin Clavulanate ◽  
High Degree

ABSTRACTWe conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) inEscherichia coli. Up to 44 AMC-resistantE. coliisolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance inE. coliis widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

scholarly journals The Selective Advantage of the lac Operon for Escherichia coli Is Conditional on Diet and Microbiota Composition

2021 ◽  
Vol 12 ◽  
Author(s):  
Catarina Pinto ◽  
Rita Melo-Miranda ◽  
Isabel Gordo ◽  
Ana Sousa
Keyword(s):  
Escherichia Coli ◽  
Selective Advantage ◽  
Lac Operon ◽  
E Coli ◽  
Nutritional Conditions ◽  
Regulatory Circuits ◽  
Primary Habitat ◽  
Gene Regulatory Circuits ◽  
Its Polymorphism ◽  
Mammalian Intestine

The lac operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is nearly ubiquitous in Escherichia coli strains justifying the use of its phenotype, the ability to consume lactose, for species identification. Lactose is the primary sugar found in milk, which is abundant in mammals during the first weeks of life. However, lactose is virtually non-existent after the weaning period, with humans being an exception as many consume dairy products throughout their lives. The absence of lactose during adulthood in most mammals and the rarity of lactose in the environment, means that the selective pressure for maintaining the lac operon could be weak for long periods of time. Despite the ability to metabolize lactose being a hallmark of E. coli’s success when colonizing its primary habitat, the mammalian intestine, the selective value of this trait remains unknown in this ecosystem during adulthood. Here we determine the competitive advantage conferred by the lac operon to a commensal strain of E. coli when colonizing the mouse gut. We find that its benefit, which can be as high as 11%, is contingent on the presence of lactose in the diet and on the presence of other microbiota members in the gut, but the operon is never deleterious. These results help explaining the pervasiveness of the lac operon in E. coli, but also its polymorphism, as lac-negative E. coli strains albeit rare can naturally occur in the gut.

scholarly journals Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

2020 ◽  
Vol 9 (2) ◽  
pp. 448
Author(s):  
Ema Komalasari ◽  
Winiati P. Rahayu ◽  
Siti Nurjanah
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Rt Pcr ◽  
E Coli ◽  
Melting Curves ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Pcr Method

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

Sign in / Sign up

Export Citation Format

Share Document