scholarly journals The effect of some factors on expression of gene encoding endoglucanase from dna metagenome of Binh Chau hot spring in Escherichia coli

2019 ◽  
Vol 41 (2) ◽  
Author(s):  
Tran Thanh Thuy ◽  
Lai Thi Hong Nhung ◽  
Tran Dinh Man ◽  
Le Thi Thanh Xuan ◽  
Nguyen Kim Thoa
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Hot Springs ◽  
Hot Spring ◽  
Gene Encoding ◽  
Culture Volume ◽  
E Coli ◽  
Cell Densities ◽  
High Level ◽  
Expression Time

Expression of microbial target genes in Escherichia coli is broadly used due to its advantages namely: well established system, easy to manipulate, a huge biomass, high level productivity, safe and inexpensive to grow. Metagenomic technique has been applying in Vietnam recently for effective mining of uncultured gene resources, especially in endemic mini-ecologies such as hot springs where the cell densities are low. DNA metagenome of Binh Chau hot spring was isolated and sequenced by Illumia HiseqTM. Based on analyses of databases of cellulase-encoded genes, denovogenes 18736 gene sequence for thermal endoglucanase was selected for expression in E. coli. In this paper, some factors for expression of endoglucanase have been investigated. The results show that appropriate gene expression conditions are:  Expression performed in E. coli C43 (DE3) on TB medium at 30oC with 0.25 mM of IPTG as inducer, the culture volume of 20% compared with the bottle volume and the expression time is 42–48 hours. In this condition, the biomass production and soluble enzyme activity can reached up to 5.54–5.58 g /L and  1.92–1.98 U/mL, respectively. Our results show the prospect of exploiting microbial genes without culture.

scholarly journals Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabriela Gregova ◽  
Vladimir Kmet
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Virulence Genes ◽  
Gene Encoding ◽  
Antibiotic Resistant ◽  
Beta Lactamases ◽  
E Coli ◽  
Animal Wastes ◽  
Extended Spectrum Beta Lactamases ◽  
High Level

Abstract Processing of animal carcasses and other animal wastes in rendering plants is a significant source of antibiotic resistant microorganisms. The main goal of this study was to investigate the resistance to 18 antibacterial agents including β-lactams, fluoroquinolones, colistin and virulence factors (iss, tsh, cvaC, iutA, papC, kps and ibeA genes) in 88 Escherichia coli strains isolated from a rendering plant over 1 year period. ESBL (Extended-spectrum beta-lactamases) and plasmid-mediated Amp were screened by interpretative reading of MIC. ESBL phenotype was detected in 20.4% of samples and high level of resistance to fluoroquinolone was found in 27.2% of strains. Cephalosporinase CTX-M1, cephamycinase CMY-2, integrase 1 and transposon 3 genes were detected by PCR. Furthermore, there were found three CMY-2 producing E. coli with O25b-ST131, resistant to the high level of enrofloxacin and containing the gene encoding the ferric aerobactin receptor (iutA). One enrofloxacin resistant E. coli strain possessed iss, ibeA, kps and papC virulence genes also with CMY-2, integrase1 and Tn3. ST131 E. coli with CMY-2 has a zoonotic potential and presents a serious health risk to humans.

scholarly journals Expression of Alcaligenes eutrophusFlavohemoprotein and Engineered VitreoscillaHemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth ofEscherichia coli

2000 ◽  
Vol 66 (1) ◽  
pp. 98-104 ◽  
Author(s):  
Alexander D. Frey ◽  
James E. Bailey ◽  
Pauli T. Kallio
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Product Formation ◽  
Gene Encoding ◽  
Final Cell Density ◽  
E Coli ◽  
Amino Terminal ◽  
Positive Effects ◽  
Cell Densities ◽  
Carboxy Terminal

ABSTRACT Expression of the vhb gene encoding hemoglobin fromVitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant β-lactamase relative to the VHb-expressing control.

A Polyphasic Approach To Detect Enterotoxigenic Staphylococcus aureus and Diarrheagenic Escherichia coli in Raw Milk Italian Cheeses by Multiplex PCR

2010 ◽  
Vol 73 (12) ◽  
pp. 2281-2284 ◽  
Author(s):  
VALENTINA BERNINI ◽  
ELISA SGARBI ◽  
CLAUDIO GIORGIO BOVE ◽  
MONICA GATTI ◽  
ERASMO NEVIANI
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Raw Milk ◽  
Polyphasic Approach ◽  
Potential Hazard ◽  
Gene Encoding ◽  
Lombardy Region ◽  
E Coli ◽  
Culture Independent ◽  
Encoding Genes

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)–encoding genes (sea, sec, sed, seg, seh, sei, sej, sel) and the Escherichia coli genes most commonly associated with virulence factors (eae, elt, ipaH, stx) in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture-independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community (“bulk”). Genes associated with enteroinvasive E. coli, ipaH, and Shiga toxin–producing E. coli, stx, were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (sea in all three samples, associated with sed and sej in two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel, and sei. No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.

scholarly journals Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV.

1997 ◽  
Vol 41 (1) ◽  
pp. 175-179 ◽  
Author(s):  
D M Breines ◽  
S Ouabdesselam ◽  
E Y Ng ◽  
J Tankovic ◽  
S Shah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Allele ◽  
Quinolone Resistance ◽  
Temperature Sensitive ◽  
Resistance Locus ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Resistance Phenotype ◽  
E Coli ◽  
High Level

The locus nfxD, which contributes to high-level quinolone resistance in Escherichia coli KF111b (gyrAr nfxB nfxD), is only expressed in the presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (gyrAr nfxD). DNA sequence analysis of DH161 revealed no changes in the topoisomerase IV parC quinolone resistance-determining region but did identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-length cloned parE+ partially complemented the resistance phenotype in KF111b and DH161, but did not complement the resistance phenotype in strain KF130 (gyrAr). No complementation was seen with cloned, truncated parE+. To confirm these findings, gyrAr was first outcrossed from KF130 into E. coli W3110parE10 [parE temperature sensitive(Ts)] and KL16. The transduced strains KL16 and W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfloxacin resistance in the parE(Ts) background twofold at 30 degrees C and fourfold at 42 degrees C compared to those for cloned parE from KL16. The same experiment with a non-Ts background revealed a twofold increase in the norfloxacin MIC at both 30 and 42 degrees C. These data identify the nfxD conditional resistance locus as a mutant allele of parE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norfloxacin in E. coli.

scholarly journals Molecular survey of mcr1 and mcr2 plasmid mediated colistin resistance genes in Escherichia coli isolates of animal origin in Iran

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kayhan Ilbeigi ◽  
Mahdi Askari Badouei ◽  
Hossein Vaezi ◽  
Hassan Zaheri ◽  
Sina Aghasharif ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Companion Animals ◽  
Multidrug Resistant ◽  
Animal Origin ◽  
Colistin Resistance ◽  
E Coli ◽  
High Level ◽  
Farming Industry

Abstract Objectives The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is one of the major public health concerns as colistin is the last-resort antibiotic for treating infections caused by multidrug-resistant Gram-negative bacteria. We aimed to determine the prevalence of the prototype widespread colistin resistance genes (mcr-1 and mcr-2) among commensal and pathogenic Escherichia coli strains isolated from food-producing and companion animals in Iran. Results A total of 607 E. coli isolates which were previously collected from different animal sources between 2008 and 2016 used to uncover the possible presence of plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) by PCR. Overall, our results could not confirm the presence of any mcr-1 or mcr-2 positive E. coli among the studied isolates. It is concluded that despite the important role of food-producing animals in transferring the antibiotic resistance, they were not the main source for carriage of mcr-1 and mcr-2 in Iran until 2016. This study suggests that the other mcr variants (mcr-3 to mcr-9) might be responsible for conferring colistin resistance in animal isolates in Iran. The possible linkage between pig farming industry and high level of mcr carriage in some countries needs to be clarified in future prospective studies.

Fate of Escherichia coli O157:H7 and Other Coliforms in Commercial Mayonnaise and Refrigerated Salad Dressing

1995 ◽  
Vol 58 (1) ◽  
pp. 13-18 ◽  
Author(s):  
ERROL V. RAGHUBEER ◽  
JIM S. KE ◽  
MICHAEL L. CAMPBELL ◽  
RICHARD S. MEYER
Keyword(s):  
Escherichia Coli ◽  
Storage Temperature ◽  
Enterobacter Aerogenes ◽  
Antimicrobial Effect ◽  
Coliform Bacteria ◽  
Escherichia Coli O157 ◽  
Salad Dressing ◽  
E Coli ◽  
Survival Pattern ◽  
High Level

Commercial mayonnaise and refrigerated ranch salad dressing were inoculated at two levels with two strains of Escherichia coli O157:H7, a non-pathogenic E. coli, and the non-fecal coliform Enterobacter aerogenes. Results showed that at the high inoculation level (>106 colony forming units [CFU]/g) in mayonnaise stored at room temperature (ca. 22°C) both strains of O157:H7 were undetected at 96 h. At the high inoculation level, all strains of coliform bacteria tested survived longer in salad dressing stored at 4°C than in mayonnaise stored at 22°C. The O157:H7 strains were still present at low levels after 17 days. The survival time in the low-level inoculum (104CFU/g) study decreased, but the survival pattern in the two products was similar to that observed in the high-level inoculum study. Slight differences in survival among strains were observed. The greater antimicrobial effect of mayonnaise may be attributable to differences in pH, water activity (aw), nutrients, storage temperature, and the presence of lysozyme in the whole eggs used in the production of commercial mayonnaise. Coliform bacteria survived longer in refrigerated salad dressing than in mayonnaise particularly at the high-level inoculum. Both mayonnaise (pH 3.91) and salad dressing (pH 4.51) did not support the growth of any of the microorganisms even though survival was observed.

scholarly journals Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

1996 ◽  
Vol 40 (10) ◽  
pp. 2380-2386 ◽  
Author(s):  
M J Everett ◽  
Y F Jin ◽  
V Ricci ◽  
L J Piddock
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Single Mutation ◽  
Wild Type ◽  
Single Strand Conformational Polymorphism ◽  
E Coli ◽  
High Level

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces†

2016 ◽  
Vol 79 (1) ◽  
pp. 66-74 ◽  
Author(s):  
P. B. SHRIDHAR ◽  
L. W. NOLL ◽  
X. SHI ◽  
B. AN ◽  
N. CERNICCHIARO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Virulence Genes ◽  
Target Genes ◽  
Culture Methods ◽  
Fecal Samples ◽  
E Coli ◽  
Cattle Feces ◽  
Before And After

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture–spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P < 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

scholarly journals High Levels of Antibiotic Resistance in Isolates From Diseased Livestock

2021 ◽  
Vol 8 ◽  
Author(s):  
Nurul Asyiqin Haulisah ◽  
Latiffah Hassan ◽  
Siti Khairani Bejo ◽  
Saleh Mohammed Jajere ◽  
Nur Indah Ahmad
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Sensitivity ◽  
Clinical Isolates ◽  
Situational Analysis ◽  
Sensitivity Testing ◽  
Resistance Level ◽  
Diagnostic Laboratory ◽  
E Coli ◽  
Livestock Health ◽  
High Level

Overuse of antimicrobials in livestock health and production beyond therapeutic needs has been highlighted in recent years as one of the major risk factors for the acceleration of antimicrobial resistance (AMR) of bacteria in both humans and animals. While there is an abundance of reports on AMR in clinical isolates from humans, information regarding the patterns of resistance in clinical isolates from animals is scarce. Hence, a situational analysis of AMR based on clinical isolates from a veterinary diagnostic laboratory was performed to examine the extent and patterns of resistance demonstrated by isolates from diseased food animals. Between 2015 and 2017, 241 cases of diseased livestock were received. Clinical specimens from ruminants (cattle, goats and sheep), and non-ruminants (pigs and chicken) were received for culture and sensitivity testing. A total of 701 isolates were recovered from these specimens. From ruminants, Escherichia coli (n = 77, 19.3%) predominated, followed by Staphylococcus aureus (n = 73, 18.3%). Antibiotic sensitivity testing (AST) revealed that E. coli resistance was highest for penicillin, streptomycin, and neomycin (77–93%). In addition, S. aureus was highly resistant to neomycin, followed by streptomycin and ampicillin (68–82%). More than 67% of E. coli isolates were multi-drug resistant (MDR) and only 2.6% were susceptible to all the tested antibiotics. Similarly, 65.6% of S. aureus isolates were MDR and only 5.5% were susceptible to all tested antibiotics. From non-ruminants, a total of 301 isolates were recovered. Escherichia coli (n = 108, 35.9%) and Staphylococcus spp. (n = 27, 9%) were the most frequent isolates obtained. For E. coli, the highest resistance was against amoxicillin, erythromycin, tetracycline, and neomycin (95–100%). Staphylococcus spp. had a high level of resistance to streptomycin, trimethoprim/sulfamethoxazole, tetracycline and gentamicin (80–100%). The MDR levels of E. coli and Staphylococcus spp. isolates from non-ruminants were 72.2 and 74.1%, respectively. Significantly higher resistance level were observed among isolates from non-ruminants compared to ruminants for tetracycline, amoxicillin, enrofloxacin, and trimethoprim/sulfamethoxazole.

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