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Bacterial Antibiotic Resistance - Science topic

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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.
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Sailee Asolkar You'll still use erythromycin selection like you've been doing. Even in the event of a successful transformation, the overwhelming majority of your cells will remain untransformed and erythromycin sensitive, and will adopt that morphology. Basically I am saying the cell morphology is irrelevant to your failed transformation problem and not to worry about it.
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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.
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The pellets are not actually antibiotics.. They are some kind of dessicants for providing longer shelf life to the discs in the Cartridge
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My project is about root canal and enterococcus faecalis. Can I find vancomycin resistance entrococcus faecalis in root canal samples?
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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 ?
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Since the agar was used with the targeted antibiotic, so the bacteria growing on this agar is resistant (this agar act as a selectable agar for it), to dilute the method is according to the type of bacteria, there are fastidious bacteria that are insulated and shaken by vortex and grown on agar either elective or normal for me I planted it on both and it succeeded. This is called the direct inoculation method...And there are types that need incubation after the inoculation, but in general, the inoculation is with nutritious media. After dilution, it can be planted on selective media or not, according to what is available. Miles and Mazra because they are so accurate. For me, I use the Miles and Mazra method to count the colonies because it is a very accurate method.
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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.
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Good discussion
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Hello
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.
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You don't have to use phosphate buffer, of course, but the buffer is probably not the reason you are having trouble, unless your enzyme is a metallo-beta-lactamase (MBL). Because MBLs require zinc for activity and zinc phosphate has low solubility, it would be better to use a different buffer, such as HEPES. Also, include zinc sulfate in the buffer, and experiment to find the optimal concentration. It may be only a few micromolar.
If your enzyme is a serine beta-lactamase, phosphate buffer is fine.
Make sure your enzyme concentration is consistent. If you are freezing and thawing the solution, make sure you mix it gently but thoroughly after thawing. When refreezing the solution, do it rapidly using dry ice-ethanol, dry ice-acetone, liquid nitrogen, or my favorite, powdered dry ice. Rapid freezing helps reduce separation of solutes as the water freezes. Small volume aliquots (100 µl or less) are preferred because they freeze and thaw quickly.
Titrate the enzyme concentration to get a suitable signal. Monitor the absorbance change continuously, as suggested by Michael J. Benedik
Nitrocefin has a fairly limited solubility in buffer. Try using 100 µM in your reaction. That generally gives a useful absorbance change before the reaction starts to slow down. Later you can titrate the nitrocefin concentration to get the Km.
Not all beta-lactamase enzymes use nitrocefin as a substrate, by the way. Hopefully yours does.
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Hi all,
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!
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Is your strain sequenced ? pCP20 is based on a cI0857-regulated promoter which comes from phage lambda and controls flp induction in your system. Your strain might be a lysogen for a lambdoid prophage that has a similar regulator/promoter which cross-represses flp and prevents resolution of your aphA/KanR cassette. You might want to try other expression systems (ie pBAD) to induce flp.
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Is anybody working on colistin resistance and its relationship with efflux pumps in bacteria?
Best regrades
Mohammed F AL Marjani
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CCCP is not efflux inhibitor, it is a compound deenergizing cells. Efflux pumps are dependent on proton motive force, and, of course, do not work in the presence of CCCP. So, reduction of MIC can be also a result of general deenergization (inhibition of transport, ATP synthesis).
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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.
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@Nikil, if you base any major conclusions in a publication on a protein model, it makes sense to make your specific model available to the reader, either in the supplemental material or in a database. This is all the more important for non-trivial models.
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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!
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They might be mutants, not transformants. This happens with streptomycin (another aminoglycoside); most streptomycin resistant mutants are streptomycin dependent; the strR mutation that is present in most E. coli K12 strains took enormous efforts to isolate for this reason.
On the other hand, if you have checked for the presence of plasmid in your colonies, this explanation is irrelevant.
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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!
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Dear Aditya,
The genotype of E. coli DH5alpha is: F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rKmK+), λ– . If you see it carefully, it contains gyrA96 mutation which provide Nalidixic Acid resistance. So, Tias is right in using Nalidixic Acid while reviving the cells as it would limit growing of contaminants in the plate.
Dear Tias,
I guess your concentration of Nalidixic Acid is sub-optimal. At lower concentrations, Nalidixic acid acts in a bacteriostatic manner (inhibits growth and reproduction of bacteria) while at optimum concentrations it is bactericidal (kills bacteria). Hence, at sub-optimal concentration you may get minute colonies. I generally prepare Nalidixic Acid in NaOH (0.15 M) and use at 25 ug/mL concentration. I am attaching a laboratory exercise protocol which I follow. Hope it will work for you too. Best wishes.
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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?
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Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one ore more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
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Greetings,
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?
Thanks!
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Jhonatan, it may be that you are dealing with a lysogenic phage and thus the presence of antibiotics such as mitomycin in the media may be necessary for plaque formation. Give it a try, or if you can do PCR, use some integrase primers to determine if it is in fact a lysogenic virus. Also, there is always the possibility that the original host has been modified somehow. I would also go back to your original type strain and test it. By the way, let us know how it goes and how you solve the problem.
All the best
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What is the role of extracellular DNA in bacterial antibiotic resistance and how can it be detected in the lab?
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Understanding the mechanisms by which biofilm bacteria develop resistance to antibiotics is paramount to expanding the treatment options available to patients with chronic biofilm infections. ... Extracellular DNA, a known component of biofilms, was found to induce antibiotic resistance
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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).
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Go through this link, which might be helpful: https://doi.org/10.1093/clinids/3.1.45
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Dear All,
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
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Yes, you can.
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Antibiotic sensitivity testing of colistin was done by Kirby Bauer method.I have isolated DNA by boiling method.
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Becouse of other mechanisms of resistance 
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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?
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Check in the literature, MIC stands for Minimum Inhibitory Concentration and is a standard method for analyzing the efficacy of antimicrobials. 
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i need help in explanation of my in vitro results
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Dear, you must knew that chart is old at 1966 but the British  pharmacopoeia is standard 
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please help me
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The specimens in your question, do you mean by bacterial culture? If it is bacterial culture for Antibiotic Susceptibility Tests (AST), the answer is no. Different species of bacteria will have different range/spectrum of antibiotics to test with. As for the recommended culture for AST, please refer to CLSI or BSAC guidelines - the two has difference in media selection for testing. You can check their website for a copy of the AST guideline.
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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?
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This is hardly surprising and was shown multiple times in the past. Keep in mind that most antibiotics were originally produced by natural microflora... Take a look at these papers:
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is there any correlation between biofilm formation and antibiotic resistance in clinical bacterial isolates, if yes what is the mechanisms? 
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Yes indeed there is. Well established bacteria have the ability to resist better the effects of agents they are exposed to including antibiotics.  
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Anyone who shares the supplemental file M27-S4 used as a reference for antifungal susceptibility testing of yeasts?
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??
You are confused. This question is not for you. This is a general question available for anyone who is a Researchgate member.
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Give your views about rapidly developing resistance against many antimicrobial agents.
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Your question comprises a huge, huge topic. 
Antibiotic resistance is a natural and evolutionary event mainly caused by antibiotic misuse and abuse leading to selective pressure.
Emergence and dissemination of multidrug-resistant organisms largely depend on many factors such as the type of bacteria (gram-positive or -negative, and the genus-species), the source (community- vs hospital-acquired), and the geographical area.
Among multidrug-resistant bacteria, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pose the greatest threat to public health generally causing hospital-acquired infections. Other important pathogens with increasing resistance are Mycobacterium tuberculosis, Neisseria gonorrhoeae, and Helicobacter pylori.
Bacterial resistance epidemiology dramatically varies from region to region worldwide and some genetic resistance determinants are endemic to some areas and favored by specific factors. Plasmids and integrative and conjugative elements play a major role for the transmission of resistance mechanisms.
In the last decades, clones of multidrug-resistant bacteria have been described worldwide. These clones carry genetic resistance determinants and virulence factors, and are associated with transmission success.
Here you can take a look of general aspects of multidrug antibiotic resistance and the importance of combined efforts of antibiotic stewardship and infection control to optimize antibiotic use and mitigate antibiotic resistance:
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 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?
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One causes of failure of Disk Diffusion confirmatory test of ESBL production in detecting ESBL-producing enteric bacteria is the over-expression of ESBL and AmpC genes.  Sometimes a bacteria has ESBL and AmpC genes and AmpC enzymes were over-expressed. When this situation occurs, AmpC enzymes is suppress the ESBL confirmatory test. You should check presence of  the bacteria for AmpC genes.
You have to check the susceptibility of your isolate to cefoxitin. 
The attached table may guide you to the suitable answer.
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I'm not able to conclude my result. I am using Acinetobacter species strain to perform Serum bactericidal Assay.
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Thank you so much for your reply sir,
I'm already used as control E.coli as well as Klebsiella pneumoniae as control of serum sensitive but all the ways getting serum resistance. 
I want to measure complement factor in serum could inhibit bacterial growth.
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I am looking for a simple protocol for Quantitative measurement of beta-lactamase without using the Nitrocefin in Gram-negative bacteria.
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Thank you, Mr. Benedik
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Can anyone please suggest me the possible mechanism behind cross resistance of a bacteria to different classes of antibiotic?
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In addition to Oliver Nolte and Mickael J. Benedik
Refer Obolski et al., 2016: Antibiotic cross-resistance in the lab and resistance co-occurrence in the clinic: Discrepancies and implications in E. coli
This manuscript explained all possible mechanism behind antibiotic cross-resistance 
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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? 
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I assume you are using a pure culture of a sensitive E.coli. My answer is speculative and is based on an antimetabolite to cefepime present in the urine together with cefepime (what other medication is the patient on?methotrexate?).The first zone of inhibition (ZOI) closest to the disc is due to the high conc of cefepime acting on  the E.coli. The ring of growth is due to the action of the antimetabolite which has a lower diffusion or conc compared to the cefepime. Therefore the next ZOI is the cefepime acting alone.
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should a MRSA show resistance to methicillin antibiotic disc compulsorily, inspite of them showing resistance to other penicillin antibiotics if not then they are not mrsa? is it like that can anyone help me out
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Thank you sir
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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.
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Dear ARchana
It is depend in what definition of MRSA do yu use; it should be has a mecA gene, means resistant against all beta lactam;
CLSI use OXA and now changed to Cefoxition as better predictor of mecA gene.
CLSI just predictor not absolutely true as MRSA
my experience, res to OXA or FOX is not 100% MRSA, it would be hyper producing beta lactamases.
Thus MRSA should be based on mecA gene detection
Best for you
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Hi
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? 
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Hi Ibrahim
I think this would work, but a nitricefin assay would be more direct, easier and cheaper
Regards, Liam
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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.
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The idea of an adjuvant for antibacterial therapy to permeabilize bacteria is currently the focus of at least one company (Speros). Efflux pump inhibitors for combination with antibacterials have been explored in the past as well (Microcide). It's a challenging approach. Good luck.
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The overlay is with super resistant microbes
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Hello dear.
For detection of antibiotic resistant bacteria, we have enormous methods include of Phenotyping and Genotyping methods. You can find this methods by one simple search or read this attached review 
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.
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TEM-1 is more prevalent in Amp resistant isolate salmonella
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 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  
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Hi Emad:
Please refer to Table 2B-2. Zone Diameter and Minimal Inhibitory Concentration Interpretive Standards for Acinetobacter spp. (in CLSI, 2015, pp. 56), attached below:
Best regards
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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.
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Hi:
You can use CLSI guideline Vet 01-A4 (Susceptibility Tests for Bacteria Isolated From Animals.......). You can purchase it from CLSI bookshop online. However, a sample for this guideline is attached here.
Great regards
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Hi,
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,
Silvia
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Hi:
MIC is defined as the lowest concentration of the agent that inhibits visible growth. The methods are not widely used for routine testing in clinical labs in most countries. Determination of MIC is recommended by CLSI to evaluate rapidly multidrug resistance in the bacterial isolates. 
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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.
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Hi:
Please find attached CLSI, 2015 (Table 6A), pp. 180, which illustrate the Solvents and Diluents for Preparation of Stock Solutions of Antimicrobial Agents
For Nalidix acid, the solvent is 1/2 volume of water, then add 1 mol/L NaOH dropwise to dissolve. etc....
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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?
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I think it depends on the value of "I". If it was closer to the breakpoint of "Sensitive" , it should considered Sensitive, otherwise If it was closer to the breakpoint of "resistant" , it should considered Resistance. However, you have first to re-test your organism three times by DDt before you determine it was "I" or not.
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Is klebsiella oxytoca p2 a sodium azide resistant baceria?
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Certainly No, but in very rare cases mutant strains may be azide-resistant.
Look this article as an example:
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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?
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Dear Kumar. The question of antibiotic resistance is very interesting and it is what we should be trying to find answers to...because the very products of our dear beloved microbes-the antibiotics...that were meant to help mankind heath-wise are now the greatest course of concern..because of the indiscriminate and inappropriate use of antibiotics, the spread of antibiotic resistance is happening fast because as microbes struggle to survive like all other organisms and there are antibiotics left right and center, their only chance of living is to develop resistance..to acquire new genes that code for antibiotic resistance...This is very worrying because most pathogens are also developing resistance and diseases are becoming more and more difficult to treat.
But let me not get carried away and out of the topic. Back to your question..how we can preserve probiotics. when we talk of probiotics we mean the good bacteria in the GIT which can also be wiped out by the antibiotics. This is tough..because unless they evolve to be resistant to the antibiotics, they will be killed. But there is hope.as due to natural selection and adaptation, there is probability that a few species can be resistant naturally and they can spread the resistance by horizontal or vertical gene transfer mechanisms......food for thought. What do you think?
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Hi all,
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..
Thanks
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Dear Silvia,
The last step is executed to avail only those plasmids that contain the resistance gene. I believe that is the whole objective of your experiments - to isolate antibiotic resistance genes in plasmids.
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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.
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MLT test for specific pathogens are necessary to be performed as per the regulatory guidelines. The tests shall be performed as per USP or if any in house developed by you, it shall be validated as per USP method. 
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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.
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formulation of antibiotic policy in hospitals is required so that in inadvertent use of antibiotics can be stopped also strict infection control policies should be implemented at each level of health care. Yes of course since there are no newer drugs in pipeline turning towards our traditional medicines can be an alternative but extensive research is to be done before proven.
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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.
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Please check out the following article.It migth be useful.
Regards
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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
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Hello Gerry,
As far as I Know, there is no specific treatment for infections related to biofilms. Currently, the medical device or site of the body is surgically removed or antibiotics as vancomycin or daptomycin is used. However is frequently the use 10 to 100 times the MIC.
I suggest you read the paper I attached. It helped me a lot. 
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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.
Regards
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The potency of the active substance may be limited by its solubility. Increasing the extract concentration won't increase the active ingredient concentration if the active ingredient has surpassed its limit of solubility.
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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? 
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The CLSI document M45 suggests only MIC testing (i.e. dilution testing or MIC stripes) but not disc testing for Listeria monocytogenes. Three antibiotics are listed: penicillin and ampicillin (≤2 ug/mL susceptible) and trimethoprim-sulfomethoxazole (≤0.5/9.5 ug/mL susceptible). L. monocytogenes should be susceptible to penicillin and ampicillin but is intrinsically resistant to the cephalosporines. The EUCAST, however, provides zone diameter breakpoints for benzylpenicillin (disc Content 1 unit ≥ 13 mm susceptible), ampicillin (2 ug ≥ 16 mm susceptible), meropenem (10 ug ≥ 26 mm susceptible), erythromycin (15 u ≥ 25 mm susceptible) and trimethoprim-sulfomethoxazole (1.25/23.75 ug ≥ 29 mm susceptible). 
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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?
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Very instructive. Thank you very much.
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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
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Geneticin should be toxic to fungi and yeasts at 400 ug/ml concentration, have you tried reducing the geneticin concentration to 250 ug/ml? It might work.
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What is the best method for inhibition of toxin-Antitoxin system in bacteria?
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Dear Mohammed,
The following publications cover the answer to your question:
1-MBio. 2015 May 5;6(3):e00285-15. doi: 10.1128/mBio.00285-15.
New players in the toxin field: polymorphic toxin systems in bacteria.
Jamet A1, Nassif X.
Author information
 
Abstract
Bacteria have evolved numerous strategies to increase their competitiveness and fight against each other. Indeed, a large arsenal of antibacterial weapons is available in order to inhibit the proliferation of competitor cells. Polymorphic toxin systems (PTS), recently identified by bioinformatics in all major bacterial lineages, correspond to such a system primarily involved in conflict between related bacterial strains. They are typically composed of a secreted multidomain toxin, a protective immunity protein, and multiple cassettes encoding alternative toxic domains. The C-terminal domains of polymorphic toxins carry the toxic activity, whereas the N-terminal domains are related to the trafficking mode. In silico analysis of PTS identified over 150 distinct toxin domains, including putative nuclease, deaminase, or peptidase domains. Immunity genes found immediately downstream of the toxin genes encode small proteins that protect bacteria against their own toxins or against toxins secreted by neighboring cells. PTS encompass well-known colicins and pyocins, contact-dependent growth inhibition systems which include CdiA and Rhs toxins and some effectors of type VI secretion systems. We have recently characterized the MafB toxins, a new family of PTS deployed by pathogenic Neisseria spp. Many other putative PTS have been identified by in silico predictions but have yet to be characterized experimentally. However, the high number of these systems suggests that PTS have a fundamental role in bacterial biology that is likely to extend beyond interbacterial competition.
2-Trends Microbiol. 2013 May;21(5):230-7. doi: 10.1016/j.tim.2013.02.003. Epub 2013 Mar 7.
Bacterial contact-dependent growth inhibition.
Ruhe ZC1, Low DA, Hayes CS.
Author information
 
Abstract
Bacteria cooperate to form multicellular communities and compete against one another for environmental resources. Here, we review recent advances in the understanding of bacterial competition mediated by contact-dependent growth inhibition (CDI) systems. Different CDI+ bacteria deploy a variety of toxins to inhibit neighboring cells and protect themselves from autoinhibition by producing specific immunity proteins. The genes encoding CDI toxin-immunity protein pairs appear to be exchanged between cdi loci and are often associated with other toxin-delivery systems in diverse bacterial species. CDI also appears to facilitate cooperative behavior between kin, suggesting that these systems may have other roles beyond competition.
3- The role of contact-dependent growth inhibition toxin systems in bacterial competition and biofilm development
 
 King, Andrew D (2015) The role of contact-dependent growth inhibition toxin systems in bacterial competition and biofilm development. PhD thesis, University of York.
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Abstract
Contact-dependent growth inhibition (CDI) toxins are a recently identified family of polymorphic toxins, initially found in Escherichia coli. CDI toxins are found widely spread in Gram-negative bacterial species, including pathogenic strains, and have been shown to possess a wide range of toxin types which are effective against other bacteria. This research shows that the E. coli EC93 CDI system confers a competitive advantage on bacteria growing in multi strain biofilms with susceptible bacteria. This advantage is due to two factors, firstly the EC93 CDI toxin was shown to be capable of inhibiting the growth of susceptible bacteria in a biofilm and secondly the conserved region of the EC93 CdiA protein was found to increase the rate of biofilm formation. Analysis of the effects of the EC93 and EC869o11 CDI toxins at the single cell level showed that different classes of CDI toxins can act at different rates and with varying degrees of reversibility. Understanding the variable impact of CDI toxins, in concert with CDI’s role in enhancing biofilm formation, aids our understanding of bacterial competition in the natural environment.
4-Bacterial Warfare
UCSB researchers demonstrate how gram-negative bacteria deliver toxins to kill neighboring bacteria
By Jim Logan
It’s bacteria against bacteria, and one of them is going down.
Two UC Santa Barbara graduate students have demonstrated how certain microbes exploit proteins in nearby bacteria to deliver toxins and kill them. The mechanisms behind this bacterial warfare, the researchers suggest, could be harnessed to target pathogenic bacteria. Their findings appear in the Proceedings of the National Academy of Sciences.
Lead authors Julia L.E. Willett and Grant C. Gucinski have detailed how gram-negative bacteria use contact-dependent growth inhibition (CDI) systems to infiltrate and deliver protein toxins into neighboring cells. By studying the bacteria Escherichia coli (E. coli), they were able to document how CDI “translocation domains” can use multiple pathways to transfer those toxins into a cell. By understanding that mechanism, Willett said, it could be possible to use it as a model for pinpoint targeting of bacteria.
“The long-term, real-world potential is that if we know bacteria can deliver their own proteins into other cells, we might be able to use this as a delivery system for antibiotics and other therapeutics,” said Willett, a doctoral student in UCSB’s Department of Molecular, Cellular and Developmental Biology (MCDB). She and Gucinski conducted the work under the direction of faculty adviser and MCDB professor Chris Hayes. Hayes is the second author on the paper.
“If we know the detailed mechanisms of delivery maybe we can target specific groups of bacteria,” Willett continued. “Instead of taking an antibiotic that targets all bacteria, we might be able to deliver one that could specifically target one group of bad bacteria that leaves the good bacteria in your gut alone.”
Gucinski, a graduate student researcher in UCSB’s Biomolecular Science and Engineering Program, began studying E. coli as an undergraduate. Although it has a reputation as a nasty pathogen, that group of bacteria is generic enough to make an ideal research subject.
“E. coli is the easiest system to work with and very representative of the majority of other bacteria,” Gucinski said. “The kind of CDI systems that we study are also found in a lot of different kinds of bacteria. This is the tip of the iceberg in our understanding of what we’ll find in other CDI systems in other bacteria.”
CDI were first described by David Low, professor of MCDB, in 2005. Low, a co-author of the current PNAS paper, reported how a bacterial cell would touch a neighboring cell — one that was competing for resources in the environment — and inject it with a toxin. Willett and Gucinski’s research builds on Low’s work by identifying the multiple ways CDI toxins exploit target cells. The key was in understanding the genetics of those targeted bacteria.
“We know that the cells would have these CDI systems; we know the genetics that are required to make this toxin system, but we were interested in the genetics on the other side, the genetics that are required in the cell that’s being inhibited or the cell that’s receiving this toxin,” Willett explained. “What specifically in that cell is required for the toxin to go from outside the cell to inside the cell?”
Willett and Gucinski found that mutations in the target cells allowed CDI to exploit those cells and inject them with toxins.
“What these CDI systems have done is they’ve actually hijacked machinery that the cells already have,” Willett said. “And so cells when they’re growing need to take in nutrients, and the CDI systems hijack those pre-existing systems to deliver these toxins. It’s not really tricking the target cells, but it’s basically hijacking what’s already there for the inhibitor cell’s own benefit.”
Looking ahead, Willett and Gucinski say potential therapeutic applications are tantalizing but years away. “We’re still trying to understand the routes that we can get different CDI toxins into the cell,” Gucinski said. “One interesting direction would be what other cargo we can deliver with E. coli, how we can manipulate and control the system to target the pathogens.”
Given the rise of drug-resistant bacteria and a dearth of research into new antibiotics, Willett and Gucinski’s research has the potential to open a new front in the fight against pathogenic bacteria.
“We hear on the news that a lot of pathogens are becoming resistant or people can no longer take certain antibiotics,” Willett said. “And so this might be a new way to get around that. Instead of treating everything on a broad spectrum, if we could learn how a natural antibacterial system delivers things that kill other bacteria we might be able to more learn how we can deliver things like specific proteins or specific antibiotics to kill other bacteria.”
Also contributing to the research was UCSB undergraduate Jackson P. Fatheree. The study was supported by a grant from the National Institutes of Health and a National Science Foundation Graduate Research Fellowship.
Hoping this will be helpful,
Rafik
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Papers that contain protocols making use of various techniques and compounds to make a bacteria, preferably a clinical strain, devoid of its antibiotic property.
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Thank you for the very important question.We found effective plasmid elimination in vitro and in vivo in case multidrug resistant infection by combination of plasmid curing Promethazine and antibiotic  gentamycin , Joseph Molnar et al:Synergism between Antiplasmid Promethazine and Antibiotics in Vitro and In Vivo.. Mol. Biol. open access journal. 119.doi:10.4172/2168-9547.1000119 2014, 3:2
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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)
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Thanks for getting back to me Matthaios. We only have a lightcycler though so it would have to be an assay suitable for use on it.
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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?
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In the nature you can find not only one gene on one plasmid,  you can make also different plasmids labellet with diffetrent antibiotics e.g- Km.
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Hi all
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
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Hi Helmi,
if with collateral damage you mean side effects, both antibiotics are quite safe antibiotics. The susceptibility profile you report does not suggest any of the most usual non-fermenter Gram negatives (Pseudomonas, Acinetobacter, Stenotrophomonas), and having accurate identification would help. Anyway, de-escalating antibiotic treatment when beta-lactams other than carbapenems are active is a good measure having in account the current spread of carbapenemase-producing microorganisms.
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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.
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You should not be getting a single crossover with a linear DNA fragment. I would double check your PCR analysis with control strains to show that you have actually deleted the gene and the wild type copy is or is not present. You might have a gene duplication event and just deleted one copy. Also are you certain that deleting ldhA will make the strain non-lactate producing? Is there the possibility of a secondary pathway. Lastly be sure that you have isolated a pure stock and your fermentation run was not with a mixed culture. I would do PCR analysis on the cells from the fermentor to double check.
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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?
Thank you
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What a good question. It depends on how confident is the surgeon. In my present hospital they will give one dose prior to surgery and two doses afterwards if the surgeon is confidence.
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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. 
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I've attached the Clinical and Laboratory Standards Institute's (previously NCCLS)  Antimicrobial Susceptibility Testing Guide, which may be helpful.
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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
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I do not think those are satellite colonies, as this does not happen with aminoglycoside-modifying resistance enzymes. If you really want to find out what they are, do as suggested above: pick a colony, reisolate on same medium and do a PCR:
- If the reisolated colony does not grow or grows very poorly (in more than 36h) on the kanamycin plates, those could be satellite colonies since they would be kanamycin sensitive
- If they grow on the kanamycin plate, you can PCR the marker to see if they carry the kanamycin -resistance marker. My guess is that they will not and that they are spontaneous knamycin-resistant mutants, which happen quite easily if you incubate your plates too long.
Good luck!
Hervé
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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.
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Hello
The most probable is that your isolates will have a fluoroquinolones (FQ) MIC of around 0.25 - 0.5  mg/L , which is considered as susceptible. However, with these MICs values, the isolates usually possess a mutation in gyrA and/or possess any transferable mechanisms of quinolone resistance (TMQR).
I suppose that when you say "phenotypic interpretation revealed it was partially quinolones resistance" you say that your results with disk difussion indicate that your isolates are intermediate resistance. If I am sure in that regarding MIC values this is not surprising, as you are just in the border of the breakpoint, and then the halo diameter will be reduced and may fall within the intermediate interpretation, in special if the inoculum is so high.
Although the current expansión of TMQR may result in the presence of FQ in absence of nalidixic acid resistance, I suggest that use a disk of Nal. If your isolates are Nal-resistant, probably the presence of a mutation in gyrA is the most feasible scenario.
In any case presence of minor discrepances (susceptible / intermediate) using different techniques it is not imposible (J. Clin. Microbiol. September 2011 vol. 49 no. 9 3343-3345) 
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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)
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Here ( http://www.ats.ucla.edu/stat/r/faq/mantel_test.htm) is a worked example in R. If you don't know how to do it in XLSTAT you might be able to do it in R which is free and a lot of people have written code for it to solve various problems. Download R here (http://cran.r-project.org/bin/windows/base/)
Here  is more info on mantel test and R in order to get familiar with the test and with R
LS
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Dear Researcher, I'm currently working on Carbapenem resistance in Acinetobacter. Does anybody have control strains to study efflux pump overexpression and Porin loss ?
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For better results get one from standard culture collection like NCIM, MTCC or NCCS in India.
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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...??? 
thanks
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Hi Morteza,
It depends which bacteria you work with. For example, for Enterococcus species, CLSI guidelines (M100-S25) recommend 120ug gentamicin disk. I suggest to you to check in CLSI guidelines.
Simon
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Hi
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
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Thanks Ramona for your prompt answer and opinion...
they really helped... :)
Thanks again
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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?
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Pathogens of appendicitis includes aerobic and anaerobic microorganism. Gentamicin is primarily used to get rid of aerobic gram-negative bacteria. In the absence of aerobic bacteria anaerobes may fail to survive. However, if anaerobes have established themselves, gentamicin will not be effective. One will have to add an agent with activity against anaerobes such as metronidazole
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Difference between semi resistant and completely resistant colonies
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If you check the MIC of a colony growing in the zone of inhibition, you might get a clue. Often, this kind of behaviour where stray colonies are growing in the zone of inhibition is assiciated with heteroresistance, i.e. resistant subpopulations in the population that you've inoculated onto the plate. So if the MIC of that colony is elevated, you probably have a heteroresistant strain. Or you might just have a contamination...
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If it is, how can I solve this?
Thanks..
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These strains seem to have only mecA. We have found some SCCmec elements that we could not type with the method of Oliveira et al, but could type with the method of Ito et al, but also the opposite. This is more or less normal.
Furthermore, we observed that in some of these cases the quality of the DNA was important. Normally, we used a 0.5 McFarland of the MRSA in water, but after isolation of DNA we could identify the SCCmec elements that were previously not typeable.
We found several strains that had only mecA with method of Oliveira et al, but harboured ccrAB2 of ccrC with method of Ito et al, so that makes them SCCmec IV of V.
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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?
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The central step of biofilm formation is the intercellular adherence between bacteria. Intercellular adherence results in larger cell clusters if the cells are not adherent to a surface. If a substance inhibit intercellular adhesion (and thereby biofilm formation) you will have a higher number of particles even if the total number of bacterial cell is identical (or even slightly reduced). The number of particles affect at the end the optical density of a bacterial culture.
As a control you might use mild ultrasonication to disrupt larger cell clusters and measure your OD prior and after ultrasonication.
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I would like to know anything about Streptococcus antibiogram.
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Please refer to my paper, I have followed CLSI guidelines.
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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?
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RecA is involved primarily in homologous recombination. Deletions are more likely to be caused by non-homologous processes that are independent of RecA. It sounds like your insert is intrinsically unstable in E. coli or alternatively, it, or the variants, are poisonous to the cells. If the latter is the case, you will notice big variations in colony size between whole and deleted inserts. It might help to clone in an expression vector and turn off the expression of the insert, or use a lambda vector that is going to kill the cell anyway. Another advantage of lambda is that it establishes lysogeny at single-copy, so you get less overexpression and less toxicity.
Good luck. Working with unstable inserts can get very complicated. In the worst case you may have to use a different host organism.
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Hi all,
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? 
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Hello,
you should use % 0.9 NACL solution, MHB is unnessesary since you're going to sow the suspension on an Agar Plate as soon as you have misured it, also MHB is "more torbid" by itself (becaused colored in light brown) and would affect the McFarland determination.
Hope to be helpful.
Greetings,
Ramona.
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Dear all 
Is there any reference for carbapenem-resistant Burkholderia psudomallei? 
thanks
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Dear Abdel
There is not too much information about carbapenem-resistant B. pseudomallei. Howevere, there is a recent article:
Aunkham A, Schulte A, Winterhalter M, Suginta W. Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei. PLoS One. 2014 May 1;9(5):e95918. doi: 10.1371/journal.pone.0095918. eCollection 2014.
dealing with this topic.
I attached to this post. I hope would be useful.
Finally, please if you consider this answer appropriate, please upvote it using the green up arrow click. Thanks.
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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.
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Dear William, I believe some concepts are not very clear for you. The MIC50 and MIC90 values are populational scores, employed for determination of a bacterial population against such antimicrobial agent. To do this, MIC of each individual strain must be sorted by value, and MIC of the strain which is the median one represents the MIC50. On the same way, the MIC of the strain which corresponds to percentile 90 indicates the MIC90 value. As already said, the minimal inhibitory concentration must be carried out for determining those parameters, as well as the MIC range (another important information of this methodology). 
Regarding the MAr determination, I recommend this paper: http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2011.03570.x/abstract
If you prefer, please keep in touch for further information.
Best regards!
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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.
Best regards
Sayed S Bukhari MD FRCPath
Consultant in Infection
Midlands, UK
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Dear All,
I see such patients regularly, and indeed, some suffer extensive disease.I diagree with Dr Broxmeyer that antibiotics are first line treatment, as the infectious part is just due due surinfection and not the primary cause. Extensive android development of the sebaceous glands is certainly involved, as you never see the diesese in women without android hallmarks. I Always change their hormonal milieu to a cyproterone dominated milieu. Also ygienic measures, reduction of sugars and animal fat (reducing obesity, often related) is a collateral measure. If a lesion Always re-appears on the same site, it means the glands are destroyed and hide bacteria. This I treat with proper and deep surgical removal. But again, only on the condition it is repeatedly recurent on the same spot.
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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.
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hi
I cant understand the real aim of your question. All micro-organisms have efflux systems, because they need it to survive. If  you want manipulate efflux control in gram negative bacteria you can use molecules as CCCP and motnitoring the effect by the use of staining reagents as EtBR.
kind regards
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