scholarly journals Two step PCR method to exchange the resistance cassette of a vector

2021 ◽  
Author(s):  
Nils Cremer ◽  
Anne Diehl
Keyword(s):  
Antibiotic Resistance ◽  
Agar Plate ◽  
Easy Method ◽  
Antibiotic Selection ◽  
E Coli ◽  
Target Vector ◽  
Pcr Product ◽  
Plasmid Preparation ◽  
Pcr Method ◽  
Dna Template

AbstractFor co-transformation of two plasmids, both have to possess different antibiotic selection markers. If that is not the case, normally the gene of interest (GOI) is subcloned into another vector. Here we introduce a fast and easy method to exchange the antibiotic resistance cassette (ARC) in only two PCR steps.Method SummaryTo shuttle the antibiotic resistance cassette (ARC) from one vector to another, one can amplify the ARC of interest and use the resulting PCR-product as a primer pair for the next amplification step. Simply remove parental DNA template by DpnI digestion, transform PCR product directly in E. coli cells, select transformants on an appropriate agar plate and isolate target vector by plasmid preparation.

scholarly journals Detection of biofilm among uropathogenic Escherichia coli and its correlation with antibiotic resistance pattern

2019 ◽  
Vol 11 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Rashmi M. Karigoudar ◽  
Mahesh H. Karigoudar ◽  
Sanjay M. Wavare ◽  
Smita S. Mangalgi
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Agar Plate ◽  
Diffusion Method ◽  
Resistance Pattern ◽  
Susceptibility Pattern ◽  
Sensitivity Testing ◽  
E Coli ◽  
Tract Infections

Abstract BACKGROUND: Escherichia coli accounts for 70%–95% of urinary tract infections (UTIs). UTI is a serious health problem with respect to antibiotic resistance and biofilms formation being the prime cause for the antibiotic resistance. Biofilm can restrict the diffusion of substances and binding of antimicrobials. In this context, the present study is aimed to perform in vitro detection of biofilm formation among E. coli strains isolated from urine and to correlate their susceptibility pattern with biofilm formation. MATERIALS AND METHODS: A total of 100 E. coli strains isolated from patients suffering from UTI were included in the study. The identification of E. coli was performed by colony morphology, Gram staining, and standard biochemical tests. The detection of biofilm was carried out by Congo Red Agar (CRA) method, tube method (TM), and tissue culture plate (TCP) method. Antimicrobial sensitivity testing was performed by Kirby–Bauer disc diffusion method on Muller–Hinton agar plate. RESULTS: Of the 100 E. coli strains, 49 (49%) and 51 (51%) were from catheterized and noncatheterized patients, respectively. Biofilm production was positive by CRA, TM, and TCP method were 49 (49%), 55 (55%), and 69 (69%), respectively. Biofilm producers showed maximum resistance to co-trimoxazole (73.9%), gentamicin (94.2%), and imipenem (11.6%) when compared to nonbiofilm producers. Significant association was seen between resistance to antibiotic and biofilm formation with a P = 0.01 (<0.05). CONCLUSION: A greater understanding of biofilm detection in E. coli will help in the development of newer and more effective treatment. The detection of biofilm formation and antibiotic susceptibility pattern helps in choosing the correct antibiotic therapy.

scholarly journals Phylogenetic Groups ofEscherichia coliStrains from Patients with Urinary Tract Infection in Iran Based on the New Clermont Phylotyping Method

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Darioush Iranpour ◽  
Mojtaba Hassanpour ◽  
Hossein Ansari ◽  
Saeed Tajbakhsh ◽  
Gholamreza Khamisipour ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Cross Sectional Study ◽  
Diffusion Method ◽  
Resistance Pattern ◽  
Multiple Drug ◽  
Phylogenetic Groups ◽  
Disk Diffusion Method ◽  
Cross Sectional ◽  
E Coli ◽  
Pcr Method

Objectives.In 2013, Clermont classifiedE. colistrains into eight phylogenetic groups using a new quadruplex PCR method. The aims of this study were to identify the phylogenetic groups ofE. colibased on this method and to assess their antibiotic resistance patterns in Bushehr, Iran.Methods. In this cross-sectional study, 140E. coliisolates were subjected to phylogenetic typing by a quadruplex PCR method. Antimicrobial susceptibility testing was performed by disk diffusion method.Results. Phylogenetic group B2 was most predominant (39.3%), followed by unknown (27.1%), E (9.3%), C and clade I (each 6.4%), B1 (5%), F and D (each 2.9%), and A (0.7%). The most common antibiotic resistance was related to amoxicillin (82.1%) and the least to meropenem (0.7%). 82.14% of isolates were multiple drug resistant (MDR). Antibiotic resistance was mainly detected in group B2 (50%).Conclusions.Our findings showed the high prevalence of MDRE. coliisolates with dominance of group B2. About 25% ofE. coliisolates belong to the newly described phylogroups C, E, F, and clade I. Such studies need to be done also in other regions to provide greater understanding of the antibiotic resistance pattern and the prevalences of different phylogenetic groups.

scholarly journals Detection of Phylogenetic Groups and Drug Resistance Genes of Escherichia coli Causing Urinary Tract Infection in Southwest Iran

2021 ◽  
Vol 14 (2) ◽  
Author(s):  
Mostafa Boroumand Boroumand ◽  
Mohsen Naghmachi ◽  
Mohammad Amin Ghatee
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Urinary Tract ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Phylogenetic Group ◽  
Phylogenetic Groups ◽  
E Coli ◽  
Pcr Method ◽  
Tract Infections

Background: Many bacteria can cause urinary tract infections (UTIs), among which Escherichia coli is the most common causative agent. E. coli strains are divided into eight phylogenetic groups based on the new Quadroplex-PCR method, which are different in terms of patterns of resistance to antibiotics, virulence, and environmental characteristics. Objectives: This study aimed to determine the phylogenetic groups and the prevalence of drug resistance genes in E. coli strains causing UTIs. Methods: In this descriptive cross-sectional study, 129 E. coli isolates obtained from the culture of patients with UTIs were evaluated for phylogenetic groups using the new method of Clermont et al. The identification of phylogenetic groups and antibiotic resistance genes was performed using the multiplex polymerase chain reaction (PCR) method. Results: In this study, concerning the distribution of phylogenetic groups among E. coli isolates, the phylogenetic group B2 (36.4%) was the most common phylogenetic group, followed by phylogroups C (13.2%), clade I (10.1%), D (9.3%), and A (3.1%) while groups B1 and F were not observed in any of the isolates, and 20.2% had an unknown state. Also, out of 129 E. coli isolates, the total frequency of tetA, tetB, sul1, sul2, CITM, DfrA, and qnr resistance genes was 59.7%, 66.7, 69, 62, 30.2, 23.3, and 20.2%, respectively. In this study, there was a significant relationship between antibiotics (P = 0.026), cefotaxime (P = 0.003), and nalidixic acid (P = 0.044) and E. coli phylogenetic groups. No significant relationship was observed between E. coli phylogenetic groups and antibiotic resistance genes. Conclusions: The results of this study showed that strains belonging to group B2 had the highest prevalence among other phylogroups, and also, the frequency of antibiotic resistance genes and drug-resistant isolates had a higher prevalence in this phylogroup. These results show that phylogroup B2 has a more effective role in causing urinary tract infections compared to other phylogroups, and this phylogroup can be considered a genetic reservoir of antibiotic resistance.

scholarly journals Evaluation of Biofilm Formation and Virulence Genes and Association with Antibiotic Resistance Patterns of Uropathogenic Escherichia coli Strains in Southwestern Iran

2021 ◽  
Vol 14 (9) ◽  
Author(s):  
Mostafa Boroumand ◽  
Asghar Sharifi ◽  
Mohammad Amin Ghatei ◽  
Mohsen Sadrinasab
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Urinary Tract ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Uropathogenic Escherichia Coli ◽  
E Coli ◽  
Resistance Patterns ◽  
Pcr Method ◽  
Antibiotic Resistance Patterns

Background: Uropathogenic Escherichia coli (UPEC) strains, encoding superficial and secretory virulence factors, can lead to colonization and facilitation of bacterial growth in the host urinary tract, causing Urinary Tract Infection (UTI). Objectives: This study determined the ability of biofilm formation by the Congo red agar (CRA) method, the presence of virulence genes using the multiplex polymerase chain reaction (PCR) method, and the relationship between biofilm formation and antibiotic resistance patterns and virulence genes in E. coli clinical isolates in Yasuj. Methods: This cross-sectional study was performed on 144 UPEC isolates collected in 2017. Biofilm formation was detected by the CRA phenotypic assay and virulence factors by the multiplex PCR method. Antibiotic resistance tests were performed by the Kirby-Bauer method. Results: Out of 144 isolates of E. coli, 22 (19.4%) isolates showed to be strong biofilm producers, 27 (23.8%) moderate biofilm producers, and 64 (56.3%) weak biofilm producers. A significant relationship was observed between biofilm-producing strains and resistance to ampicillin (P = 0.020) and cotrimoxazole (P = 0.038). The virulence genes in strong biofilm producers included iutA (95%), FimH (93%), ompT (90%), PAI (90%), and TraT (81%) genes. The phylogroup B2 carried the most virulence genes. A significant correlation was observed between E. coli phylogenetic groups and aer (P = 0.019), iroN (P = 0.042), and ompT (P = 0.032) virulence genes. Conclusions: The results of this study showed a high prevalence of virulence genes, and antibiotic-resistant E. coli strains capable of biofilm formation. The results of this study may help elucidate the pathogenesis of UPEC and facilitate better treatment strategies for patients with UTIs in this geographic area.

Cloning of the High Alkaline Endoglucanase Gene from Bacillus pumilus AC-4 and Expressed in E.coli

2012 ◽  
Vol 249-250 ◽  
pp. 1289-1293
Author(s):  
Yuan Lin ◽  
Xin Zhou Li ◽  
Ji Wei Gao ◽  
Jing Xu ◽  
Chao Du
Keyword(s):  
Gene Sequence ◽  
Bacillus Pumilus ◽  
Gene Fragment ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
E Coli ◽  
Endoglucanase Gene ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Dna Template

The genomic DNA of Bacillus pumilus AC-4 strain, which were separated by our laboratory, was used as the DNA template. The β-1,4-endoglucanase gene fragment was amplified by PCR (Polymerase Chain Reaction). The PCR product was recovered and cloned into plasmid pMD-19T. The DNA sequence analysis showed that the length of the amplified fragment is 1898bp, which has 95% similarities to the β-1,4-endoglucanase gene sequence from other Bacillus strains. Sub-cloned the recombinant plasmid which containing β-1,4-endoglucanase and digested with BamHI and EcoRI. And then linked with the expression plasmid pGEX-4T-1 and transferred into the competent E. coli BL21 for expression. The result of protein electrophoresis showed that there has expressed protein whose molecular weight is about 70 kDa. Measured the enzyme activity of the expression protein of recombinant strains is 0.92 IU/mL, which is 1.52 times as the start strains.

scholarly journals Effects of Selection Pressure and Genetic Association on the Relationship between Antibiotic Resistance and Virulence in Escherichia coli

2015 ◽  
Vol 59 (11) ◽  
pp. 6733-6740 ◽  
Author(s):  
Lixin Zhang ◽  
Karen Levy ◽  
Gabriel Trueba ◽  
William Cevallos ◽  
James Trostle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Virulence Factor ◽  
Selection Pressure ◽  
Antibiotic Use ◽  
Epidemiological Evidence ◽  
Antibiotic Selection ◽  
Content Type ◽  
E Coli ◽  
Antibiotic Selection Pressure

ABSTRACTAntibiotic selection pressure and genetic associations may lead to the cooccurrence of resistance and virulence in individual pathogens. However, there is a lack of rigorous epidemiological evidence that demonstrates the cooccurrence of resistance and virulence at the population level. Using samples from a population-based case-control study in 25 villages in rural Ecuador, we characterized resistance to 12 antibiotics among pathogenic (n= 86) and commensal (n= 761)Escherichia coliisolates, classified by the presence or absence of known diarrheagenic virulence factor genes. The prevalences of resistance to single and multiple antibiotics were significantly higher for pathogenic isolates than for commensal isolates. Using a generalized estimating equation, antibiotic resistance was independently associated with virulence factor carriage, case status, and antibiotic use (for these respective factors: odds ratio [OR] = 3.0, with a 95% confidence interval [CI] of 1.7 to 5.1; OR = 2.0, with a 95% CI of 1.3 to 3.0; and OR = 1.5, with a 95% CI of 0.9 to 2.5). Virulence factor carriage was more strongly related to antibiotic resistance than antibiotic use for all antibiotics examined, with the exception of fluoroquinolones, gentamicin, and cefotaxime. This study provides epidemiological evidence that antibiotic resistance and virulence factor carriage are linked inE. colipopulations in a community setting. Further, these data suggest that while the cooccurrence of resistance and virulence inE. coliis partially due to antibiotic selection pressure, it is also genetically determined. These findings should be considered in developing strategies for treating infections and controlling for antibiotic resistance.

The Order in Which Antibiotics are Administered Affects the Fitness Costs of Adherent-Invasive E. coli (AIEC) Strains

2019 ◽  
Author(s):  
Jordan B Gregg
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Combination Therapy ◽  
Agar Plate ◽  
Sequential Therapy ◽  
Negative Control ◽  
Clinical Settings ◽  
Fitness Costs ◽  
Inflammatory Gene Expression ◽  
E Coli

AIEC-LF82 is a strain of bacteria that is surmised to have a role in causing IBD and Crohn’s disease by activating pro-inflammatory gene expression in organisms. Using antibiotics via combination therapy has been a technique used in clinical settings in an attempt to treat the strains, however, the attempts have not been that effective nor efficient in terms of completely halting the growth and colonization of AIEC to treat IBD and Crohn's disease patients. Research has shown that regarding hindering or preventing the colonization bacterial colonies, sequential therapy tends to be more effective and time-efficient than combination therapy, with fewer adverse effects. To test if this is also the case with the AIEC-LF82 strain of bacteria, I first tested AIEC’s response to combination therapy using the Penicillin-Streptomycin, Kanamycin-Chloramphenicol, antimicrobial peptide (AMP), Kanamycin, SPE phase and LB agar plates, all of which were experimental plates other than the LB agar plate that acted as the negative control. I then tested AIEC-LF82’s response to sequential therapy using the LB+ Kan + Spe, LB + AMP + Spe, LB+ Kan/Cam + Spe, LB + P/S + Spe, LB + P/S + Kan and LB + P/S + AMP and one LB agar plate acting as the negative control. The only differences between sets a and b were the order in which antibiotics were administered in the six aforementioned treatment sets. Ultimately, I found that set b of sequential therapy, strong-weak antibiotic treatments, was the most effective treatment but that set a regarding sequential therapy was actually the least effective of all of the treatments. In conclusion, using strong-weak sequential antibiotic therapy treatments appears to be a potentially promising option to treat patients suffering from Crohn's disease and IBD.

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.

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