Bacterial Antibiotic Resistance - Science topic
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Questions related to Bacterial Antibiotic Resistance
I have constructed a Bacillus subtilis 168 knock out strain (resistant to chloramphenicol) by homologous recombination. I have been trying to transform my recombinant pMUTIN plasmid into it, but the transformation fails. Also, instead of regular short rod like morphology on my negative and transformant plates (erythromycin 4 ug/ml + chloramphenicol 5 ug/ml), I get long rod like morphology. In all this, the viability control i.e. my host strain shows lawn growth on LB agar plate with pure short rod like morphology.
I am using Vojcic et al, 2012 protocol for Bacillus subtilis transformation. This protocol has previously worked well while transforming pDG1662 into Bacillus subtilis 168 wt strain.
Even if I consider this as contamination, I have applied decontamination measures such as fumigation and other sterility measures. What could be the reason behind this failure of transformation while getting such colonies of unknown morphology. Attaching pictures for better understanding.
How can we determine the concentration of antibiotic pellets available with the antibiotic cartridge? For example, Meropenem concentration from Oxoid is 10 ug per disc, but what would be the concentration of the pellet included in the cartridge.
My project is about root canal and enterococcus faecalis. Can I find vancomycin resistance entrococcus faecalis in root canal samples?
Hello, I first need to select antibiotic resistant bacteria from a bacterial solution so I will use an agar plate with the target antibiotic to do so , then I need to dilute the resistant bacteria to get countable colonies how can I do this? should I inoculate the bacteria then perform serial dilution? Or can I use the selective media directly when performing the serial dilution? is there any other way ?
I have just been looking at:
Hsu, J. (2020). How covid-19 is accelerating the threat of antimicrobial resistance. BMJ, 369.
It sounds worrying. The threat of antibiotic resistance has been increasing year on year and now the added use of broad spectrum antibiotics in patients who are being treated with COVID-19, to stop secondary bacterial infection, is very concerning.
I am working with recombinant β-lactamase enzymes (350-400 aa) with C-terminal his tag and the tag is not cleaved. I know nitrocefin is a chromogenic cephalosporin for β-lactamase activity assay. However, wondering if I could do kinetic analysis with nitrocefin. I performed a couple of test-run with 50 mM phosphate buffer and took absorbance at A490. Every run given me data which was abnormal (very high or very low). Anyone can help me, please?
Thanks in advance.
For a rotation project, I need to flip a Kan cassette out of its site on the shigella genome. I've used FLP before with no issues in e. coli, and the lab uses this method frequently. Recently, it's stopped working. We've made new preps, checked plasmid size, etc. but the cassettes will NOT flip out. We use the temp-sensitive pCP20. The site has been sequenced, so the FRT sites are definitely there.
The method we use is as follows:
-Electroporate plasmid into hypercompetent shigella. Recover at 30C in SOC for 1 hour, plate on TSB+Congo Red+Amp and grow ~24h at 30C. I get 30-50 colonies for this step.
-Restreak on TSB+CR and grow at 37C to induce FLP and cure plasmid.
-Patch onto Kan, Amp, and antibiotic-free to test sensitivity. We usually have no problem curing the plasmid.
I've tried shifting to 42C, which speeds up plasmid loss but doesn't seem to have affected Kan resolution. I might try growing in liquid, since a few scattered protocols suggest that. I'm not the only one in the lab struggling with it. Any suggestions would be appreciated!
Dear research professors and scholars,
I have developed a novel 3D protein structure (mutant DNA Gyrase enzyme of antibiotic-resistant E. coli) by homology modeling technique. Because this type of protein was not deposited in the protein data bank (www.pdb.com). So, I attempted to create mutant DNA Gyrase protein in homology modeling method. I would like to this protein in some online protein bank for future research on antibiotic-resistant related studies. Please suggest some online 3D protein upload website except www.pdb.com.
The answer may be as obvious as there must be something wrong with the plates, but I am still perplexed. I have a plasmid system which has kanamycin resistance on the plasmid vector while expressing chloramphenicol resistance on the antisense strand of RNA produced. I did not expect growth on my plates with CAM or CAM and KAN. However, I did expect growth on LB and on KAN. To my surprise however, the first time I had tried growing my DH5alpha cells from NEB after Gibson cloning of large vector backbones/inserts (around 7000-8000 bp in the final product) nothing had survived. However, after leaving my transformed cells in SOC at four degrees for two days I tried plating again on different plates.
None, however, grew on the LB + Agar plates while I have colonies on my all my new KAN plates. The only noticeable difference between all the plates that I had used and these KAN ones is that they are a much darker yellow, making it seem as though they have a much higher LB content than the other plates. Might this at all be the reason why I have growth on these plates but not the others? Or could something else be the culprit?
Any advice would be appreciated. Thank you!
I'm trying to revive glycerol stock of E. coli DH5alpha by streaking on a Nalidixic acid (NA) containing plate. I have prepared fresh nalidixic acid (15ug/mL) but I'm getting mixed reviews on the optimal concentration of NA to be used. Also, some suggest making it in NaOH and others in water. If it is NaOH, then what dilution should be used to dissolve? When I streak the cells in NA containing plate, even after 12 hours colonies are too small to inoculate. When inoculated in LB with NA then also the growth is very less after 14 hours. Do you think it is the problem with NA or the cells?
Thanks in advance!
I recently bought pVIB plasmid modified e.coli (BL21 strain) and now I want to grow them on LB agar containing ampicillin for selective pressure. I wondered, whether there is a chance due to spontanous mutations that a non plasmid containing e.coli might survive on the amp agar?? Could it ruin my culture?
What can I do to prevent it?
I´m trying to characterize a Klebsiella oxytoca bacteriophage, but always that I'm doing some experiments it stops to form plaques on the media. I mean, I have worked with this phage without any problem during some time. However, after short periods of time ( some weeks) the phage seems to lose infectivity or viability and I stop to obtain the phages plaques after incubation. It has happened around 3 or 4 times, and every time it lasts more than a month without forming plaques. I changed the old bacteria for a new one of the same strain and the media, I tried to decontaminate the lab, I used different bacteria O.D., but anything that I did didn't seem to work. Suddenly, plaques started to appear again after some weeks.
Currently, it has occurred again and the phage has been "lost" for almost two months. I don´t know what to do, I think I have done everything that it´s supposed to do, I have proven distinct strains of the same specie, but again, nothing works.
What do you recommend me to do? what could be the cause of this problem? How can I solve it? Has it happened to another person, with another phage?
Different antibiotic has different tissue penetration abilities. That is why to treat deep tissue infections, need to be chosen such an antibiotic that could penetrate deep into the tissue to reach where pathogens are colonized or have produced infections and to kill them.
For example, Ciprofloxacin is known for its deep tissue penetration (almost 70%) and that is why it is widely used to treat deep infections such as Shigellosis (infected in the very deep of intestine by Shigella dysenteriae).
While working on a project on Antibiotic resistance I was performing Antibiotic Susceptibility Test for E. coli against Colistin as well as for Staphylococcus against Vancomycin. Standards for both the combinations are not there in CLSI. However, quality control limits are given. Can we refer to those quality control limits and compare to see whether our isolate is sensitive or resistant. Kindly help by posting your suggestions.
Thanks in Advance
In my current research, I want to synthesize some compound. Then I want to test it on some resistant bacteria to detect whether it can overcome the resistance or not. How can I detect the particular molecular mechanism or specific gene behind this occurrence of resistance overcoming?
I am working on antimicrobial resistance in bacteria isolated from non anthropogenic environment. Majority of the isolates are non pathogenic, however resistance are there? So is there any guideline like (CLSI guidelines for clinical isolates) to deal with such resistance?
Anyone who shares the supplemental file M27-S4 used as a reference for antifungal susceptibility testing of yeasts?
If one of the species of the Enterobacteriaceae family that is resistant to ceftazidime and cefotaxime, also ceftazidime-clauvunic and cefotaxime -clauvunic and the difference of zone diameter between CAZ and CAZ-CV and also for CTX are less than 5 mm(0 mm).it is ESBL positive or not?
I'm not able to conclude my result. I am using Acinetobacter species strain to perform Serum bactericidal Assay.
After 24h incubation, I observed two ring pattern for one antibiotic,even after repeating of the test.
Please see the attached images.
Whats your idea?
In disk diffusion test if my isolated staphylococcus aureus didnt show resistance to methicillin antibiotic but resistance to all other penicillin class antibiotic, dont it mean that it is not a MRSA
Is it compulsary that MRSA should show resistance to methicillin antibiotic also inspite of it showinfg resistance to other penicillin class antibiotic.
My question is, is it acceptable or valid to detect plasmid mediated antimicrobial resistance genes via Rt-Qpcr. İs there any limitaitons to isolate RNA from plasmid and calculate gene expression?
We have observed some synergy between herbal antimicrobials and antibiotics. It may be due to inhibition of efflux pump by herbal antimicrobials or due to some other kind of interaction.
How good this combination can be exploited clinically.
From my previous screening study, Acinetobacter baumannii was found to be highly resistant to many antibiotics tested. I think it should be some guidelines or documents for microbiologists and doctors about the effective antibiotics against this bacterium. Thanks
I look for published works on direct susceptibility testing especially in poultry. there are several works regarding human practice but I could not find works on animal side.
I am studying the resistance to antibiotics in bacteria and I often happen to read papers where the authors report minimal inhibitory concentration tests performed on these resistant bacteria. I didn't understand why the authors use a wide range of antibiotics for these MIC tests. I mean, I read a few papers in which carbapenem resistant bacteria were tested on carbapenems, as well as on cephalosporins, aminoglycosides..So, I was wondering what s the point to test also cephalosporins or other antibioitcs if the strains are resistant to carbapenems?
Thank you very much in advance,
We'll be adding these to our culture media so we are looking for solvents that won't be toxic to our culture but will still be able to dilute the aforementioned supplements. Thanks.
As you know, antibiogram resulted as susceptible (S), intermediate (I) or resistant(R). i also want to detect gene expression between resistant and susceptibel strains. Can i think intermediates as a resistant?
Increasing incidence of antibiotic resistance are of great concern now a days. How can we safeguard the current probiotics of great concern?
What consequences may arise in future and any solution to the problem?
I am reading different papers about typing of antibiotic-resistant bacteria and I noticed that many studies carry out the plasmid profiles identification followed by transformation experiments in e.coli with the plasmidic extracts and successive re-extraction of plasmids from the transformed strains. I did not understand why one might need to do this last step , that is transformation and re-extraction of plasmids..
i have done verification of microbial limit test for material with strong antibacterial effect, after long time i had excellent recovery for TAMC&TYMC by suitable neutralizers and dilution 1/1000.
during performing specific test (salmonella & E.coli) the used neutralizers weren't compatabile with selective media(change the physical characteristics of the media) and there was no recovery for salmonella or E.coli in 10 gm ,1 gm as USP.
i ask if we replace specific test by adding specific microorganisms like salmonella to microorganisms used in verification of count.
It is an increasingly serious threat to global public health that requires action across all government sectors and society.Antimicrobial resistance is present in all parts of the world. New resistance mechanisms emerge and spread globally.
How can I measure the ROS of bacteria ( E coli) using DCFH-DA? I am confused by reading different methods in papers and no one wrote the method very precisely. Is there any protocol for detecting ROS in bacteria since it's not adherent? Thank you.
In previous research I tested the effectiveness of biosurfactants on the removal of microbial biofilms. Having moved more into proteomics I have not kept abreast of current developments in biosurfactants. I wondered if there were any current medical therapies that used conventional antibiotics + biosurfactants for treating infections with a heavy biofilm component such as leg ulcers.
One hypothesis that has been hanging around in the back of my mind for a few years was that by conjugating an existing hydrophilic antibiotic to a hydrophobic fatty acids tail or some similar hydrophobic chain it might make a better chemotherapeutic agent against biofilms. However I have never got around to synthesizing any of these compounds. I would be very happy to see the field move forward if somebody else was to develop a similar idea if it is feasible. Perhaps this combination therapy has already been achieved or is currently in progress in some lab.
I have read a little about Daptomycin , Dalbavancin and Teicoplanin but I am not sure if these are preferred agents of biofilm removal. I would be keen to find out more on new research and trends in the subject of biofilm removal and biosurfactants if anybody has any good references. thanks
Why when higher concentration of plant extract tested against the bacteria, same results will get as in lower concentration?
Note that Disc diffusion method was used.
Can anybody tell me what are all are the antibiotics disc we can use for Antibiotic sensitivity test against Listeria species? or specifically against Listeria monocytogenus?
1. How to digest plasmid completely, without even one copy of intact circular plasmid?
2. Is it possible to insert this DNA fragment into the genome of TG1 cells randomly? It looks possible from the paper: Nucleic Acids Research, 1999, Vol. 27, No. 5: 1296–1299. Does anybody have experience?
Do you know this one? So, Please let me know the reason.
Recently I tried to do test the possibility of using the geneticin in A. fumigatus in the 400 ug/ml concentration. But it is not working. I am trying to figure out the reasons. Help me please
Papers that contain protocols making use of various techniques and compounds to make a bacteria, preferably a clinical strain, devoid of its antibiotic property.
Current evaluating PCR versus the standard method (which is culture using chromogenic agar) to detect VRE. Literature is pointing towards PCR being more efficient (quicker turnaround times, less expensive and less labour intensive than culture)
I am using the plate KAA (kanamycin azide agar) to find enterococci or staphilococci. But in reality I find a great variety of bacteria such as Weisella Confusa, Lactococcus lactis, Lactob. pentosus, Lacob. plantanum and
Leuc. pseudomesenteroides. So now I am curious how is this possible? I have even used more kanamycin then recommended. Could someone please help me?
May I ask whether there is any data that pits these two against each others in term of collateral damage
Reason for asking: say you are faced with a lactose fermentor bacteremia
and the results than show:
R to ampicillin
S to ceftriaxone and pip tazobactam
And the patient is on carbapenem for 3 days and now better clinically
the source: perhaps lung
What would you do?
Would you downgrade to pip tax/ ceftriaxone or it does not matter?
If it matters, can someone lead me to a specific papers that pit these 2 in term of collateral damage
I have tried to delete the ldhA gene of E. coli K-12 strain W3110. I use a lambda recombination method kit. I successfully transform using a linear fragment containing the homologous region.
Transformed colonies were confirmed in an LB plate containing proper antibiotic. I used colony PCR and confirmed the specific size by primers, but in fermentation lactate was produced in a control strain. Could this be a single crossover? A linear fragment into another region? I confirmed the right size in a PCR experiment.
I don't know why I am seeing this. Please give me advice.
Because of the theoretical immunosuppression in neonates and infants, the trend has been to overuse perioperative antibiotics after surgery in this patient population. Is there any evidence in the literature that justify the use of prophylactic antibiotics for more than 24 hours after clean or clean-contaminated procedures in neonates?
Hi everyone！I got all the information about MIC designed for specific isolates. However, I want to test the antibiotic tolerance of microbial community separated from marine sediment. It will be appreciated if some one could guide me on this.
Recently, I do transformation in Kanamycin resistant plate and I observe a weird phenomenon. After incubation at 37oC for 16-20h of my ligation product, I only observed few colonies. However, the following day (36 hr incubation), I observe a bundle of colonies. It does not look like satellite colonies which I observed in the Ampicillin resistant plate if I incubate too long.
In the Kanamycin plate,the colonies look very normal. Is it satellite colonies or my target clone? I did not pick up the colonies and do sequence. Is there anyone can explain this phenomenon for me? Thanks
I ran an antibiotic susceptibility test using Vitek2 on Klebsiella pneumoniae. The result shown it was sensitive to ciprofloxacin and levofloxacin (quinolones drugs) judging from MIC values. However, phenotypic interpretation revealed it was partially quinolones resistance. How does both results correlated? Please enlighten me. Thank you.
I don t know how to enter my data in mantel test. I want to know if there is a positive correlation between antibiotic (7 antibiotics) resistance and heavy metal (7 heavy metals) resistance in 237 bacteria isolated from 3 areas (agricultural, pasture and mine soils)
Dear Researcher, I'm currently working on Carbapenem resistance in Acinetobacter. Does anybody have control strains to study efflux pump overexpression and Porin loss ?
I work about that high level resistant drug in bacteria. I want to know what is better concentration of Gentamycin useful for disc diffusion detection. I want to say "at least concentration is useful for detection high level gentamycin resistance."
for example: 100 ug ...200 ug...???
May I ask
when a patient is infected by MRSA invasive infection, is there a role of doing nasal swab in the patients as the new NHS guideline released in 2015 (Dumfries and Galloway) did not specifically state whether perianal and nasal swabs need to be taken unlike the recommendation for those with positive wound swab whereby the above needed to done for the patients with wound
As such, do we do it and give chlorhexidine regardless of the result and if the result + then the patient is prescribed mupirocin or we give both chlorhexidine and mupirocin without doing the swab to reduce the cost of nasal swab and use of chromogenic agar
Will this approach then cost us the mupirocin resistance ?
My next question is, if a patient is treated with IV vancomycin , can that eradicate the MRSA colonisation state as the data from clinical study state the AUC/MIC more than 850 needed to be achieved for this in the VAP study. This value is just way too high to be achieved in clinical practice
This bacterium is one of pathogens causing appendicitis and wound infections and some of people use this antibiotic as a treatment to get red of this pathogen as they think.I am asking is this pathogen susceptible to gentamicin?
Difference between semi resistant and completely resistant colonies
I use tryptic soy broth supplemented with 1% glucose for biofilm production.
My experiment flow is that MRSA ATCC 43300 (5*105 CFU) in TSB-glucose with different concentration of drug was incubated at 37°C, 24 h.
(concentration serial twofold dilutions (2x→1x→0.5x→0.25x→0.125xMIC))
Then draw the supernatant and measure OD600 for bacterial viability
After measure OD570 for biofilm mass.
The aim is whether drug can inhibit biofilm formation of MRSA43300 or not
In the 0.5Xmic inhibit the amount of biofilm formation but OD600absorbance increased higher than the positive control
I think it very strange.
Why is there an increase the viability of bacterial?
I have a library with variants of the same gene, and after selection steps (antibiotic resistance) I have observed a large number of variants with deletions. I don't have these deletions in my naive library, they are found only in the selected clones. I use the SNO 301 strain (AMPD1, ampA1, ampC8, pyrB, recA, rpsL) for screenings. Anybody have any suggestions about this issue?
I am working on detecting antibiotic susceptibility of klebsiella pneumoniae with different discs. My question is which solution must be use to determine 0.5 Mcfarland? 0.9% NaCl or Müller Hinton Broth?
I am in the research to analyze the antibiotic resistance bacteria. The method I chose to analyse is Kirby Bauer Method (I will not use PCR because it is so expensive). I want to ask if you have the list for Minimal Inhibition Concentration (especially for Coliform and Enterococci), for any kind of bacteria and the antibiotics. I also confuse how to determine the percent inhibition. Most of the paper I read did't show me how to read it. Just like how come to know if one bacteria have 90% resistance to specific kind of antibiotic ,or only 30% or 50% resistance. Moreover, can someone explain to me how to determine MAR (multiple antibiotic resistance). Thanks before, I appreciate any kind of help you can give.
As you are probably aware hidradenitis suppurativa is a chronic pyogenic and scarring condition involving apocrine glands in the axillary, perineal and genital areas of the body. The aetiology is shrouded in mystery. However the pathophysiology involves an unexplained keratinous plugging of the ducts of the apocrine glands. This leads to dilatation and rupture of the glands with intense inflammation in the surrounding tissues.
The lesions may subsequently become infected with a variety of Gram positive and Gram negative microorganisms including Staphylococcus aureus, non-haemolytic streptococci, E. coli, Proteus species, Pseudomonas aeruginosa, and Bacteroides fragilis.
Chronic extensive disease could be extremely distressing to the patient. In advanced disease there is often poor response to antibiotic therapy (including tetracycline, ciprofloxacin and clindamycin) and surgical intervention may become necessary followed by skin grafting.
I would welcome your thoughts on the management of a patient with chronic and extensive ciccatricial disease. Many thanks in anticipation for your suggestions.
Sayed S Bukhari MD FRCPath
Consultant in Infection
I am looking for microbial controls for an Efflux project. Does anyone have the following efflux control: S. aureus strain SA-1199B - a multidrug-resistant strain that overexpresses the NorA efflux mechanisms with a high level of resistance to certain fluroquinolone. Any Gram-negative efflux-positive strain would also be appreciated.