Anti-Escherichia coliO157:H7 activity of free fatty acids under varying pH

2010 ◽  
Vol 56 (3) ◽  
pp. 263-267 ◽  
Author(s):  
Jinli Yang ◽  
Xianzhi Hou ◽  
Priya S. Mir ◽  
Tim A. McAllister
Keyword(s):  
Fatty Acids ◽  
Bactericidal Activity ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Capric Acid ◽  
Luria Bertani ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Colony Forming Units ◽  
Acid Tolerant

Following screening of 4 strains of Escherichia coli O157:H7 (E32511, E318N, H4420N, and R508N) for acid tolerance, strain H4420N was selected for further study into the influence of pH on bactericidal activity of 6 fatty acids (capric, lauric, palmitic, oleic, linoleic, and linolenic). Strain H4420N was cultured for 6 h in Luria–Bertani broth amended with individual fatty acids at 20 mmol/L, with pH adjusted to 7.0, 4.3, or 2.5. None of the fatty acids exhibited bactericidal activity at pH 7.0 (p >0.05). At pH 4.3, only capric, lauric, and linoleic acids reduced viability of H4420N (p < 0.05). At pH 2.5, oleic (C18:1) and linolenic (C18:3) acids had modest effects on H4420N viability, whereas capric (C10:0), lauric (C12:0), and linoleic (C18:2) acids resulted in a reduction ≥5 log10colony-forming units (CFU)/mL (p < 0.05). Capric and lauric acids were examined further at pH 2.5 over a range of concentrations (0.15–20 mmol/L). After 10 min of exposure, 5 log10 CFU/mL reductions (p < 0.05) were achieved by lauric acid at 2.5 mmol/L and by capric acid at 0.31 mmol/L. Acid stress increased the sensitivity of acid-tolerant E. coli O157:H7 strain H4420N to fatty acids. Including sources of these fatty acids in diets for cattle might impair the ability of this zoonotic pathogen to survive passage through the stomach, possibly reducing the potential for its colonization in the lower gut.

scholarly journals β-Lactams and Florfenicol Antibiotics Remain Bioactive in Soils while Ciprofloxacin, Neomycin, and Tetracycline Are Neutralized

2011 ◽  
Vol 77 (20) ◽  
pp. 7255-7260 ◽  
Author(s):  
Murugan Subbiah ◽  
Shannon M. Mitchell ◽  
Jeffrey L. Ullman ◽  
Douglas R. Call
Keyword(s):  
High Performance ◽  
Selective Pressure ◽  
Luria Bertani ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Adsorption Mechanisms ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Contamination Levels

ABSTRACTIt is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assumption was tested by separately adding 10 different antibiotics (≥200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitiveEscherichia colistrain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicatorE. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dominated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, but sensitive bacteria were still unaffected by tetracycline (P= 0.6). Thus, residual antibiotics in soil do not necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment.

scholarly journals Transcriptome changes and polymyxin resistance of acid-adapted Escherichia coli O157:H7 ATCC 43889

Gut Pathogens ◽  
2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Daekeun Hwang ◽  
Seung Min Kim ◽  
Hyun Jung Kim
Keyword(s):  
Escherichia Coli ◽  
Acid Treatment ◽  
Bacterial Infections ◽  
Acid Tolerance ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Health Concern ◽  
E Coli ◽  
Tolerance Response ◽  
Acid Tolerant

Abstract Background Acid treatment is commonly used for controlling or killing pathogenic microorganisms on medical devices and environments; however, inadequate acid treatment may cause acid tolerance response (ATR) and offer cross-protection against environmental stresses, including antimicrobials. This study aimed to characterise an Escherichia coli strain that can survive in the acidic gastrointestinal environment. Results We developed an acid-tolerant E. coli O157:H7 ATCC 43889 (ATCC 43889) strain that can survive at pH 2.75 via cell adaptation in low pH conditions. We also performed RNA sequencing and qRT-PCR to compare differentially expressed transcripts between acid-adapted and non-adapted cells. Genes related to stress resistance, including kdpA and bshA were upregulated in the acid-adapted ATCC 43889 strain. Furthermore, the polymyxin resistance gene arnA was upregulated in the acid-adapted cells, and resistance against polymyxin B and colistin (polymyxin E) was observed. As polymyxins are important antibiotics, effective against multidrug-resistant gram-negative bacterial infections, the emergence of polymyxin resistance in acid-adapted E. coli is a serious public health concern. Conclusion The transcriptomic and phenotypic changes analysed in this study during the adaptation of E. coli to acid environments can provide useful information for developing intervention technologies and mitigating the risk associated with the emergence and spread of antimicrobial resistance.

scholarly journals Multiple Roles for the sRNA GcvB in the Regulation of Slp Levels in Escherichia coli

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Luria Bertani ◽  
Multiple Roles ◽  
Activation Mechanism ◽  
Mrna Levels ◽  
Rt Pcr ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein

The Escherichia coli gcvB gene encodes a small RNA that regulates many genes involved in the transport of dipeptides, oligopeptides, and amino acids (oppA, dppA, cycA, and sstT). A microarray analysis of RNA isolated from an E. coli wild-type and a ΔgcvB strain grown to midlog phase in Luria-Bertani broth indicated that genes not involved in transport are also regulated by GcvB. One gene identified was slp that encodes an outer membrane lipoprotein of unknown function induced when cells enter stationary phase. The aim of this study was to verify that slp is a new target for GcvB-mediated regulation. In this study we used RT-PCR to show that GcvB regulates slp mRNA levels. GcvB negatively controls slp::lacZ in cells grown in Luria-Bertani broth by preventing an Hfq-mediated activation mechanism for slp::lacZ expression. In contrast, in glucose minimal medium supplemented with glycine, GcvB is required for inhibition of slp::lacZ expression, and Hfq prevents GcvB-mediated repression. Thus, GcvB regulates slp in both LB and in glucose minimal + glycine media and likely by mechanisms different than how it regulates sstT, dppA, cycA, and oppA. Repression of slp by GcvB results in an increase in resistance to chloramphenicol, and overexpression of slp in a ΔgcvB strain results in an increase in sensitivity to chloramphenicol.

scholarly journals Molecular Characterization of the Acid-Inducible asr Gene of Escherichia coli and Its Role in Acid Stress Response

2003 ◽  
Vol 185 (8) ◽  
pp. 2475-2484 ◽  
Author(s):  
Vaida Šeputienė ◽  
Domantas Motiejūnas ◽  
Kęstutis Sužiedėlis ◽  
Henrik Tomenius ◽  
Staffan Normark ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Signal Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Acid Shock

ABSTRACT Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.

Comparative Analysis of Shigella boydii 18 Foodborne Outbreak Isolate and Related Enteric Bacteria: Role of rpoS and adiA in Acid Stress Response

2005 ◽  
Vol 68 (3) ◽  
pp. 521-527 ◽  
Author(s):  
YVONNE C. CHAN ◽  
HANS P. BLASCHEK
Keyword(s):  
Minimal Medium ◽  
Enteric Bacteria ◽  
Arginine Decarboxylase ◽  
Luria Bertani ◽  
Decarboxylase Activity ◽  
E Coli ◽  
Rpos Gene ◽  
Shigella Boydii ◽  
Luria Bertani Broth ◽  
K 12

Shigella boydii CDPH (Chicago Department of Public Health) serotype 18 was implicated in an outbreak of foodborne illness in 1998. The suspected food vehicles were parsley and cilantro imported from Mexico used to prepare bean salad. Previous studies revealed that S. boydii CDPH serotype 18 can survive in bean salad, which contains organic acids and whose pH decreases over time. Acid challenge assays in acidified tryptic soy broth at pH 4.5, acidified Luria-Bertani broth at pH 4.5, and acidified M9 minimal salts medium at pH 2.5 containing amino acids, arginine, or glutamic acid were performed using S. boydii CDPH, S. boydii ATCC 35966, S. flexneri 3136, Escherichia coli O157:H7 dd8872, and E. coli O157:H7 dd642 to compare differences in acid tolerance. Differences in survival of exponential-phase cells were detected in acidified tryptic soy broth and Luria-Bertani broth at pH 4.5. In acidified minimal medium containing arginine, S. boydii strains were able to survive at pH 2.5. The arginine decarboxylase gene (adiA) present in S. boydii is involved in survival at extremely low pH. The discovery of adiA expression in S. boydii serotype 18 by use of an acidified minimal medium challenge and arginine decarboxylase biochemical assay is significant because arginine decarboxylase activity was thought to be unique to E. coli. Sequencing of the rpoS gene from the S. boydii outbreak strain indicates that it is 99% conserved compared with the E. coli K-12 rpoS gene and plays a vital role in survival under acidic conditions.

Detection of viable but nonculturable Escherichia coli O157:H7 using propidium monoazide treatments and qPCR

2013 ◽  
Vol 59 (3) ◽  
pp. 157-163 ◽  
Author(s):  
Xing-long Xiao ◽  
Cong Tian ◽  
Yi-gang Yu ◽  
Hui Wu
Keyword(s):  
Saline Solution ◽  
Tap Water ◽  
Physiological Saline ◽  
Luria Bertani ◽  
Escherichia Coli O157 ◽  
Propidium Monoazide ◽  
Vbnc State ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Physiological Saline Solution

Escherichia coli O157:H7 can enter into a viable but nonculturable (VBNC) state under stress conditions. The aims of the present study were to examine the influences of environmental factors on the survivability and culturability of E. coli O157:H7 and to develop an approach for accurate detection of VBNC E. coli O157:H7. The E. coli O157:H7 strain ATCC 6589 was inoculated into 3 induction microcosm models: (i) Luria–Bertani broth, (ii) sterilized tap water, and (iii) sterilized physiological saline solution. Our results showed that low temperature and nutritional starvation significantly impacted on the survivability of E. coli O157:H7 cells and that the in-vitro-induced VBNC cells were capable of resuscitating under normal temperature and appropriate nutrients. We tested the effectiveness of an approach combining propidium monoazide (PMA) treatment with real-time polymerase chain reaction (PMA–qPCR) for accurate quantification of total, viable, dead, and VBNC cells under different induction microcosm models. Our results indicated different threshold cycle (Ct) values for PMA-treated cells and untreated cells (ΔCt = 4.97, 4.29, and 3.30 for Luria–Bertani broth, sterilized tap water, and sterilized physiological saline solution, respectively). We determined the quantification limit of this PMA–qPCR approach to be 1 × 102 cells·mL–1, providing sufficient sensitivity for detection of VBNC E. coli O157:H7 cells to no less than 100 cells·mL–1. This study clearly demonstrated the feasibility and effectiveness of using PMA–qPCR to accurately quantify E. coli O157:H7 in a VBNC state.

scholarly journals Acid Tolerance of Biofilm Cells of Streptococcus mutans

2007 ◽  
Vol 73 (17) ◽  
pp. 5633-5638 ◽  
Author(s):  
Jessica Welin-Neilands ◽  
Gunnel Svensäter
Keyword(s):  
Streptococcus Mutans ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Planktonic Cells ◽  
Viable Cells ◽  
Tolerance Response ◽  
Acid Tolerant ◽  
Carious Process ◽  
Different Strains ◽  
Viability Stain

ABSTRACT Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.

scholarly journals Production of 7-methylxanthine from Theobromine by Metabolically Engineered E. coli

2020 ◽  
Vol 21 (3) ◽  
pp. 19-27
Author(s):  
Khalid Hussein Rheima Algharrawi ◽  
Mani Subramanian
Keyword(s):  
Potassium Phosphate ◽  
Reaction Medium ◽  
Luria Bertani ◽  
Preparative Chromatography ◽  
Growth Media ◽  
Scale Production ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Product Solution ◽  
Biocatalytic Process

In this work, a novel biocatalytic process for the production of 7-methylxanthines from theobromine, an economic feedstock has been developed. Bench scale production of 7-methlxanthine has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmB/D genes combinations. These modifications enabled specific N7- demethylation of theobromine to 7-methylxanthine. This production process consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for 7-methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure 7-methylxanthine.    Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction of theobromine. Strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. That strain was used to find the optimum amount of cells required to achieve complete conversion of theobromine to 7-methylxanthine within two hours. It was found that the optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. The cell growth of pBD2dDB strain was studied using two different growth media, (Luria-Bertani Broth and Super Broth). Super broth was found to be the best medium to produce the highest amount of cell paste (1.5 g). Subsequently, the process was scaled up in which 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. The reactions was carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). 7-methylxanthines was separated by preparative chromatography with high recovery, and the product solution was collected, purified by drying at 120-140 oC for 4 hours and, recovered (127 mg). Purity of the isolated 7-methylxanthine was comparable to authentic standards with no contaminant peaks, as observed by HPLC, LC-MS, and NMR. 

Effect of Stress on Non-O157 Shiga Toxin–Producing Escherichia coli†

2012 ◽  
Vol 75 (12) ◽  
pp. 2241-2250 ◽  
Author(s):  
JAMES L. SMITH ◽  
PINA M. FRATAMICO
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Stress Responses ◽  
Shiga Toxin ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Arginine Decarboxylase ◽  
E Coli ◽  
Tolerance Response ◽  
Inspection Service

Non-O157 Shiga toxin–producing Escherichia coli (non-O157 STEC) strains have emerged as important foodborne pathogens worldwide. Non-O157 STEC serogroups O26, O45, O103, O111, O121, and O145 have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service. While documentation is limited, treatments including heat and acid that have been shown to inactivate E. coli O157:H7 will likely also destroy non-O157 STEC; however, non-O157 STEC strains show variability in their responses to stress. It has been shown that non-O157 STEC may survive in fermented sausages and cheeses, and treatments such as high pressure may be necessary to eliminate non-O157 STEC from these products. The mechanisms used by non-O157 STEC to resist acid environments are similar to those used by O157:H7 strains and include the acid tolerance response, the oxidative system, and the glutamate and arginine decarboxylase systems. However, one study demonstrated that some non-O157 STEC strains utilize a chaperone-based acid stress response (HdeA and HdeB) to combat acidic conditions, which is lacking in E. coli O157:H7. Genomic studies suggest that while non-O157 STEC can cause diseases similar to those caused by E. coli O157:H7, O157 and non-O157 STECs have different evolutionary histories. Non-O157 STECs are a heterogeneous group of organisms, and there is currently a limited amount of information on their virulence, fitness, and stress responses, rendering it difficult to draw firm conclusions on their behavior when exposed to stress in the environment, in food, and during processing.

scholarly journals Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

2011 ◽  
Vol 77 (10) ◽  
pp. 3327-3334 ◽  
Author(s):  
Thi Mai Huong To ◽  
Cosette Grandvalet ◽  
Raphaëlle Tourdot-Maréchal
Keyword(s):  
Fatty Acids ◽  
Stationary Phase ◽  
Lactococcus Lactis ◽  
Unsaturated Fatty Acids ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Phase Cell ◽  
Wild Type ◽  
Membrane Phospholipids ◽  
Content Type

ABSTRACTCyclopropane fatty acids (CFAs) are synthetizedin situby the transfer of a methylene group fromS-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacteriumLactococcus lactisMG1363. A mutant of thecfagene was constructed by allelic exchange. Thecfagene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of thecfagene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential forL. lactissubsp.cremorissurvival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Sign in / Sign up

Export Citation Format

Share Document