scholarly journals TnFLX: a third-generation mariner-based transposon system for Bacillus subtilis

2019 ◽  
Author(s):  
Felix Dempwolff ◽  
Sandra Sanchez ◽  
Daniel B. Kearns
Keyword(s):  
Antibiotic Resistance ◽  
Number System ◽  
Cellular Biology ◽  
Host Organism ◽  
E Coli ◽  
Genetic Tool ◽  
Translational Activity ◽  
Vector Construction ◽  
Low Copy Number ◽  
Resistance Markers

AbstractRandom transposon mutagenesis is a powerful genetic tool to answer fundamental biological questions in an unbiased approach. Here, we introduce an improved mariner-based transposon system with higher stability, and with versatile applications. We take advantage of the lower frequency of unintended recombination during vector construction and propagation in a low copy number system in E. coli to improve construct integrity. We generated a variety of transposons allowing for gene disruption or artificial overexpression each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance flexibility of future transposon modification and application in Bacillus and other organisms.ImportanceThe optimization of transposase encoding vectors in terms of stability during cloning and propagation is crucial for the reliable application of this system in any host organism. With an increased number of antibiotic resistance markers and the possibility to detect translational activity, the TnFLX transposon system will significantly help the implication of forward genetic methods in the field of cellular biology.

scholarly journals TnFLX: a Third-Generation mariner-Based Transposon System for Bacillus subtilis

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Felix Dempwolff ◽  
Sandra Sanchez ◽  
Daniel B. Kearns
Keyword(s):  
Antibiotic Resistance ◽  
Fluorescent Protein ◽  
Protein Translation ◽  
Delivery Vehicle ◽  
Content Type ◽  
E Coli ◽  
Vector Construction ◽  
Resistance Markers ◽  
The Stability ◽  
Green Fluorescent

ABSTRACT Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms. IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

scholarly journals Antibiotic resistance of E. coli isolated from whiteleg shrimp (Litopenaeus vannamei) collected from wet markets and supermarkets in Ho Chi Minh city and their transferability

2019 ◽  
Vol 02 ◽  
pp. 97-104
Author(s):  
Kha H. N. Nguyen
Keyword(s):  
Antibiotic Resistance ◽  
Multiple Drug Resistance ◽  
Ho Chi Minh City ◽  
Multiple Drug ◽  
Sample Collection ◽  
E Coli ◽  
Risk Sources ◽  
Mar Index ◽  
Resistance Markers ◽  
Successful Transfer

In this study, a collection of 130 E. coli isolated from white-leg shrimp collected from three wet markets and two supermarkets in Ho Chi Minh City was analysed to examine their antibiotic resistance characteristics and the transferability of resistance markers. High levels of resistance to ampicillin, tetracyclines, trimethoprim/sulfamethoxazole, nalidixic acid and chloramphenicol were observed. The percentage of multiple drug resistance (4 to 10 tested antibiotics) was 73.8%. The multiple antibiotic resistance (MAR) index values of 0.4 to 0.73 (of each sample collection site) indicated that these isolates were exposed to high risk sources of contamination where antibiotics were commonly used. Conjugation experiments demonstrated the successful transfer of all or part of the resistance phenotypes of shrimp isolates to the human recipient strains

scholarly journals Diversity of P1 phage-like elements in multidrug resistant Escherichia coli

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carola Venturini ◽  
Tiziana Zingali ◽  
Ethan R. Wyrsch ◽  
Bethany Bowring ◽  
Jonathan Iredell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Multidrug Resistance ◽  
Clinical Isolate ◽  
Insertion Sequence ◽  
Virulence Genes ◽  
Multidrug Resistant ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Resistance Markers

AbstractThe spread of multidrug resistance via mobile genetic elements is a major clinical and veterinary concern. Pathogenic Escherichia coli harbour antibiotic resistance and virulence genes mainly on plasmids, but also bacteriophages and hybrid phage-like plasmids. In this study, the genomes of three E. coli phage-like plasmids, pJIE250-3 from a human E. coli clinical isolate, pSvP1 from a porcine ETEC O157 isolate, and pTZ20_1P from a porcine commensal E. coli, were sequenced (PacBio RSII), annotated and compared. All three elements are coliphage P1 variants, each with unique adaptations. pJIE250-3 is a P1-derivative that has lost lytic functions and contains no accessory genes. In pTZ20_1P and pSvP1, a core P1-like genome is associated with insertion sequence-mediated acquisition of plasmid modules encoding multidrug resistance and virulence, respectively. The transfer ability of pTZ20_1P, carrying antibiotic resistance markers, was also tested and, although this element was not able to transfer by conjugation, it was able to lysogenize a commensal E. coli strain with consequent transfer of resistance. The incidence of P1-like plasmids (~7%) in our E. coli collections correlated well with that in public databases. This study highlights the need to investigate the contribution of phage-like plasmids to the successful spread of antibiotic resistant pathotypes.

scholarly journals The complete sequence, annotation and comparative analysis of the Escherichia coli IncFII family plasmid pMccB17, encoding biosynthetic and immunity functions for the antimicrobial peptide microcin B17

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Mayokun Ajeigbe ◽  
Lewis Bingle
Keyword(s):  
Escherichia Coli ◽  
Pcr Primers ◽  
Complete Sequence ◽  
Conjugative Plasmid ◽  
E Coli ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Resistance Markers ◽  
K 12 ◽  
Microcin B17

Microcin B17 (Mcb17) is a ribosomally synthesized and post-translationally modified peptide (RiPP), produced by Escherichia coli, that inhibits bacterial DNA gyrase in a similar way to quinolones. The Mcb17 operon, consisting of seven genes encoding biosynthetic and immunity/export functions, was originally found on a plasmid, pMccB17. This circular plasmid, previously known as pRYC17, was originally found in Escherichia coli strain LP17, isolated from the intestinal tract of a healthy newborn at Hospital La Paz, Spain and was transferred by conjugation to E. coli K-12 [Baquero et al. (1978) J. Bacteriol. 135: 342]. pMccB17 is a low copy number IncFII plasmid in the same incompatibility group as R100 and R1. Not much is known about this plasmid aside from the facts that it carries the Mcb17 operon, does not possess any conventional antibiotic resistance markers and its size was estimated to be approximately 70 kb. We extracted the plasmid from E. coli K-12 strain RYC1000 [pMccB17] and sequenced it twice using an Illumina short-read method, firstly together with the host bacterial chromosome, then plasmid DNA was purified and sequenced separately. PCR primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete sequence has 83 predicted genes, initially identified by Prokka and subsequently manually reannotated using BLAST. Comparison to other IncFII plasmids shows a large proportion of shared genes, especially in the conjugative plasmid backbone. However, pMccB17 which is a MOBF12 plasmid lacks transposable elements and in addition to the Mcb17 operon, this plasmid carries 25 genes of unknown function.

Relationship of antibiotic resistance to results of treatment in sepsis patients in Hue Central Hospital 2017 – 2018

2019 ◽  
pp. 48-54
Author(s):  
Duy Binh Nguyen ◽  
Trung Tien Phan ◽  
Trong Hanh Hoang ◽  
Van Tuan Mai ◽  
Xuan Chuong Tran
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Infection ◽  
Resistant Bacteria ◽  
Central Hospital ◽  
International Consensus ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Results Of Treatment ◽  
Amoxicillin Clavulanic Acid ◽  
Relationship Of

Sepsis is a serious bacterial infection. The main treatment is using antibiotics. However, the rate of antibiotic resistance is very high and this resistance is related to the outcome of treatment. Objectives: To evaluate the situation of antibiotic resistance of some isolated bacteria in sepsis patients treated at Hue Central Hospital; to evaluate the relationship of antibiotic resistance to the treatment results in patients with sepsis. Subjects and methods: prospective study of 60 sepsis patients diagnosed according to the criteria of the 3rd International Consensus-Sepsis 3 and its susceptibility patterns from April 2017 to August 2018. Results and Conclusions: The current agents of sepsis are mainly S. suis, Burkhoderiae spp. and E. coli. E. coli is resistant to cephalosporins 3rd, 4th generation and quinolone group is over 75%; resistance to imipenem 11.1%; the ESBL rate is 60%. S. suis resistant to ampicilline 11.1%; no resistance has been recorded to ceftriaxone and vancomycine. Resistance of Burkholderiae spp. to cefepime and amoxicillin/clavulanic acid was 42.9% and 55.6%, resistant to imipenem and meropenem is 20%, resistance to ceftazidime was not recorded. The deaths were mostly dued to E. coli and K. pneumoniae. The mortality for patients infected with antibiotic-resistant bacteria are higher than for sensitive groups. Key words: Sepsis, bacterial infection, antibiotics

The Characterization of Antibiotic Resistance of Bacterial Isolates from Intensive Care Unit Patient Samples in a University Affiliated Hospital in Romania

2019 ◽  
Vol 70 (5) ◽  
pp. 1778-1783
Author(s):  
Andreea-Loredana Golli ◽  
Floarea Mimi Nitu ◽  
Maria Balasoiu ◽  
Marina Alina Lungu ◽  
Cristiana Cerasella Dragomirescu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Bacterial Pathogens ◽  
Resistance Rate ◽  
Resistance Pattern ◽  
Bacterial Isolates ◽  
E Coli ◽  
Acinetobacter Spp ◽  
Almost All ◽  
Klebsiella Spp

To determine the resistance pattern of bacterial pathogens involved in infections of the patients aged between 18-64 years, admitted in a ICU from a 1518-bed university-affiliated hospital. A retrospective study of bacterial pathogens was carried out on 351 patients aged between 18-64 years admitted to the ICU, from January to December 2017. In this study there were analysed 469 samples from 351 patients (18-64 years). A total of 566 bacterial isolates were obtained, of which 120 strains of Klebsiella spp. (35.39%%), followed by Nonfermenting Gram negative bacilli, other than Pseudomonas and Acinetobacter (NFB) (75- 22.12%), Acinetobacter spp. (53 - 15.63%), Pseudomonas aeruginosa and Proteus (51 - 15.04%), and Escherichia coli (49 - 14.45%). The most common isolates were from respiratory tract (394 isolates � 69.61%). High rates of MDR were found for Pseudomonas aeruginosa (64.70%), MRSA (62.65%) and Klebsiella spp. (53.33%), while almost all of the isolated NFB strains were MDR (97.33%). There was statistic difference between the drug resistance rate of Klebsiella and E. coli strains to ceftazidime and ceftriaxone (p[0.001), cefuroxime (p[0.01) and to cefepime (p[0.01). The study revealed an alarming pattern of antibiotic resistance in the majority of ICU isolates.

Surveillance of Antibiotic Resistance Among Enterobacteriaceae Strains Isolated in an Infectious Diseases Hospital from Romania

2018 ◽  
Vol 69 (5) ◽  
pp. 1240-1243
Author(s):  
Manuela Arbune ◽  
Mioara Decusara ◽  
Luana Andreea Macovei ◽  
Aurelia Romila ◽  
Alina Viorica Iancu ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Infectious Diseases ◽  
Salmonella Spp ◽  
Resistance Level ◽  
E Coli ◽  
Bacterial Genus ◽  
Antibiotic Resistance Profile ◽  
Carbapenem Resistant ◽  
Invasive Infections ◽  
Klebsiella Spp

The aim of the present study was to characterize the antibiotic resistance profile of enterobacteriaceae strains isolated in Infectious Diseases Hospital Galati, Romania, during 2016, in order to guide the local antibiotic stewardship strategy. There are 597 biological samples with positive cultures for enterobacteriaceae, related to invasive and non-invasive infections. The main bacterial genus were E. coli 62%, Klebsiella spp 15%, Proteus spp 11% and Salmonella spp 6%. Over a half of isolated strains have one or more antibiotic resistance. The resistance level depends on bacterial genus, with highest level found among the rare isolates: Enterobacter spp, Citrobacter spp, Morganella spp and Serratia spp. The rate of MDR was 17.,6% for E. coli, 40.9% for Klebsiella spp and 50.7% for Proteus spp. while the rate of strains producing Extended Spectrum of Beta Lactamase are 7.2% for E. coli, 28.4% for Klebsiella spp and 12.3% for Proteus spp. The carbapenem resistant strains were found in 1.1% cases.

scholarly journals Chromosomal Lesion Suppression and Removal in Escherichia coli via Linear DNA Degradation

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1255-1271 ◽  
Author(s):  
Anabel Miranda ◽  
Andrei Kuzminov
Keyword(s):  
Dna Degradation ◽  
Dna Helicase ◽  
Structural Element ◽  
E Coli ◽  
Linear Dna ◽  
Replication Factory ◽  
Low Copy Number ◽  
Chromosomal Lesion ◽  
Recbcd Enzyme

Abstract RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a ΔrecA ΔrecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of σ-replicating chromosomes could be one of the RecBCD’s roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the ΔrecA ΔrecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.

scholarly journals Antibiotic Resistance of Uropathogens Isolated from Patients Hospitalized in District Hospital in Central Poland in 2020

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 447
Author(s):  
Barbara Kot ◽  
Agata Grużewska ◽  
Piotr Szweda ◽  
Jolanta Wicha ◽  
Urszula Parulska
Keyword(s):  
Antibiotic Resistance ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
Beta Lactamases ◽  
E Coli ◽  
Central Poland ◽  
Resistance Patterns ◽  
Extended Spectrum Beta Lactamases ◽  
Tract Infections

The aim of this study was to determine antibiotic resistance patterns and the prevalence of uropathogenes causing urinary tract infections (UTIs) in patients hospitalized in January–June 2020 in central Poland. Antimicrobial susceptibility testing was performed using the disk-diffusion method. Escherichia coli (52.2%), Klebsiella pneumoniae (13.7%), Enterococcus faecalis (9.3%), E. faecium (6.2%), and Proteus mirabilis (4,3%) were most commonly isolated from urine samples. E. coli was significantly more frequent in women (58.6%) (p = 0.0089) and in the age group 0–18, while K. pneumoniae was more frequent in men (24.4%) (p = 0.0119) and in individuals aged 40–60 and >60. Gram-negative species showed resistance to ampicillin. K. pneumoniae were resistant to amoxicillin plus clavulanic acid (75.0%), piperacillin plus tazobactam (76.2%), cefotaxime (76.2%), cefuroxime (81.0%), ciprofloxacin (81.0%), and trimethoprim plus sulphamethoxazole (81.0%). Carbapenems were effective against all E. coli and P. mirabilis. Some K. pneumoniae (13.6%) produced metallo-β-lactamases (MBLs). E. coli (22.6%), K. pneumoniae (81.8%), and all E. faecium were multidrug-resistant (MDR). Some E. coli (26.2%), K. pneumoniae (63.6%), and P. mirabilis (14.3%) isolates produced extended-spectrum beta-lactamases (ESBL). Vancomycin-resistant E. faecium was also found. This study showed that the possibilities of UTIs therapy using available antibiotics become limited due to the increasing number of antibiotic-resistant uropathogens.

scholarly journals Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved

2021 ◽  
Author(s):  
Cláudia A. Ribeiro ◽  
Luke A. Rahman ◽  
Louis G. Holmes ◽  
Ayrianna M. Woody ◽  
Calum M. Webster ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Antibiotic Resistance ◽  
Antibiotic Susceptibility ◽  
Bacterial Pathogens ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Resistance Mechanisms ◽  
Current Work ◽  
E Coli ◽  
Respiratory Inhibitor

AbstractThe spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

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