Clinical Microbiology - Science topic

For genomic DNA, any standard condition, 15-20 min, 5000 g would work, however, good to use at 4 °C if possible. Time can vary based on your sample density.

Why should they be mutually exclusive. The automated methods are generally based on classic methods, so understanding the old methods is important, but knowing how to apply modern technology is equally important.

I support the views raised by Prof.Phil Geis.

You can ,as you say, pcr amplify overlapping 800 base amplimers ( you get no useful sequence under the sequencing primer or for the next 30 bases). If you can amplify the whole 6KB in one amplimer then you just need overlapping sequencing primers at 800 base intervals along the whole sequence but just the one template dna. If you have a friend with a minion sequencer or NGS then new sequencing technology is good and quick but not cheap or easy to analyse the results

This is the procedure I use for E. coli stocks:

- Take a freshly saturated culture in rich medium (2YT or LB) and spin 2 ml in a 2-ml microfuge tube for 2 min in a microcentrifuge.

- Discard 1 ml of the supernatant and use the remaining 1 ml to resuspend the pellet

- To the concentrated suspension, add 0.5 ml 60% (v/v) sterile glycerol and mix thoroughly by pipetting up and down.

-Transfer the mixture to a cryotube and store at -80 °C

I have kept such viable stocks for decades. When needed, scratch the surface of the frozen stock with a toothipck and use the scratched material to inoculate a fresh culture, without thawing the rest of the stock, which should be returned immediately to the -80°C freezer.

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Dear Saravanan Arumugam thanks for sharing this very interesting technical question with the RG community. Personally I'm not an exert in this field of reserach (we are inorganic / organometallic chemists) and I was not even aware of the fact that hetero-atom-doped carbon dots have antibacterial properties. However, there are various reports in the literature in which this effect has been demonstrated. For example, please have a look at the following potentially useful articles which migth help you in your analysis:

and

The first article is freely available as public full text, while the second has been published Open Access (please see attached pdf file). Apparently it might be worth a try using the hetero-atom-doped carbon dots in combination with copper or silver nanoparticles for enhanced antibacterial activity.

Good luck with your work!

Ido From

The best way of end-point amplification of target region using RNA directly extracted from tracheal/cloacal swabs is making complementary DNA (Step 1) and amplify target region from complementary DNA (Step 2).

One-step RT-PCR system can lead to lower yields or efficiency, more complicated to troubleshoot, and does not provide a stock of cDNA for future use. Since RNAs are unstable so, you can reverse transcribe them immediately and perform PCR later (Two-step PCR System).

Still if you want to use One-Step RT-PCR system, you may use following kits;

1. QIAGEN OneStep RT-PCR Kit, https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/pcr-enzymes-and-kits/one-step-rt-pcr/qiagen-onestep-rt-pcr-kit/

2. SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (you mentioned).

The World Health Organisation recommends the use of disinfectants on hard surfaces and hand sanitizers. The formulations that offer the best results are alcohol-based disinfectants. We use Ethanol and Isopropanol (IPA) in our hand sanitizer formulations. These two alcohols are equally effective when it comes to killing bacteria, but to understand the differences between them kindly check:

I have run into a similar identification problem with a gram-negative bacillus that results as Salmonella typhi, S. gallinarum or sometimes as Shigella spp. in biochemical assay (usually using Remel RapID ONE in my case), but consistently produces an oxidase positive reaction (from growth on SBA, MH and others). In addition to this contradiction, the isolate has yet to be H2S positive on any medium (without significant acid production from sugar fermentation/utilization that could hide H2S positivity - good times for any of you out there that run into lac+ Salmonella isolates), and presents significantly enhanced growth under increased CO2 with difficulty at first isolation in aerobic subculture from blood culture vials or thioglycolate broth.

To complicate the identification further, MALDI-TOF protein biotyping always results the isolate with low confidence as Salmonella - oxidase positive genera have never broken the top 10 matched patterns by rank of score value. We test a lot of isolates which cannot be matched in currently available MALDI-TOF spectra libraries so I have worked under the assumption that these low confidence ID's were "overly confident." The isolate obviously needs to be sequenced at this point, but I unfortunately will not have the opportunity to do this and am unsure anyone will pick this work up behind me. As such, I am left only with the contribution of a comment to this thread in case we are encountering the same problem and someone here can solve it.

After repeated testing following countless passages in subculture across various media, my best guesses are as follows:

1. Aggregatibacter actinomycetemcomitans

2. non-motile, L-form Salmonella typhi or S. gallinarum with unknown interference mechanism resulting in false-positive oxidase test result for cytochrome C oxidase activity (thinking along the lines of something like manganese oxide interfering with oxidase test reagent)

Best of luck solving your mystery should you not be able to sequence your isolate...

In my lab, we mostly store our microbial cultures either in soft agar covered with liquid sterile paraffin(molecular grade heavy/light) kept at 10°C to 12°C temperature or use glycerol stocks (70:30 or 80:20 ratio of LB broth & molecular grade glycerol) in 2 ml autoclaved cryovials & preserve it in -20°C. The advantage of storing culture in soft agar is culture can withstand if temperature much fluctuates also & It is durable for long time preservation if the surface of the agar properly covered with paraffin.

sure, I'd like to see the pictures - philageis@aol.com

Hi, good day! Nontuberculous mycobacteria are a diverse group of mycobacterial species that causes different clinical infections in children and adults. According to Ingen (2013), 25 species have been strongly associated with NTM out of the 140 known NTM species in the literature/studies. Isolation of M. kansasii and M. malmoense from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae , M. simiae, or M. chelonae are said to be contaminants rather than causative agents of true disease. Mycobacterium avium complex (MAC), M. xenopi, and M. abscessus form an intermediate category between these two extremes (Ingen,2013). NTM species differ in their clinical relevance therefore, the need for correct species identification is very important. As per your question, to prevent the chance of overdiagnosis and undertreatment of NTM, it is crucial to identify the particular causative agent that caused the condition/illness as well as its clinical relevance. to make a firm diagnosis, a culture of the clinical specimens, and a histological examination of tissue biopsy specimens are needed. Diagnosing NTM disease is complex therefore a good communication between clinicians, radiologists, and microbiologists may help in optimizing the culture conditions in line with the particularities of the patients.

if you want to know more about the diagnosis of NMT, this article might help you:

Ingen, J. (2013). Diagnosis of Nontuberculous Mycobacterial Infections. Diagnosis of Nontuberculous Mycobacterial Infections. DOI:10.1055/s-0033-1333569

The Bile Esculin Azide Agar is a good and recommended selective medium for the isolation and identification of Enterococci. The presence of the bile salt in this agar acts by inhibiting the growth of mostly gram-positive organisms while esculin is hydrolyzed by Enterococci providing the means for selective isolation and presumptively identification of the Group D Streptococi and Enterococci like the Enterococcus faecalis and Enterococcus faecium.

Please go through the following RG links.

It was nice to see this discussion both because there was a clear question, but even more importantly the answers were knowledgeable with helpful information. When possible references should always be given, and several of the answers would have been more helpful if there was a citation that could be checked out.

I do not think that it makes any difference whether you use chloride or sulphate. The important part is the magnesium ions

i agree with Jay Siddharth

Please take a look at this link.

Article Methicillin Resistant Staphylococcus aureus (MRSA) Nasal Car...

Article Comparison of chromogenic agar medium and diffusion disk tes...

Article Detection of methicillin resistant Staphylococcus aureus (MR...

the API system is a good one, Klebsiella sp, the (probably) easiest way would be by the Indol test. K. pneumoniae is indol-negative, whereas K. oxytoca and K. ornitolytica are indol positive. But be aware that e.g. Enterobacter spp are also indol-negative.sugar fermentation

Establishing the relationship of Double J stent Catheters with the episodes of CAUTI and CRBSI and subsequently link its prolonged exposure leading to bacterial translocation as a risk factor for the development of the sepsis.

Please go through the following RG links.

I know this reply is really belated but I was wondering if portable UV-C devices abundantly available on line will do the job. Here is an example of what can be found on ebay: https://www.ebay.com/itm/Nail-Sterilizer-Box-Disinfection-UV-LED-Pedicure-Brush-Cleaner-Tool-HL/123814321847?hash=item1cd3e89eb7:g:2t0AAOSw4Vpb0ofU

I am really curious if anybody used them in research. So far could not find any reviews of such products by scientists at Amazon :)

Thanks for all the tips! This was extremely helpful

I add file.

Yes, I am interested @Giovanni

Hello Kevin. My team compared the biofilms on different brands of 96-wellplates (TC-treated, non-TC-treated), and found that some non-TC-treated plates (super cheap) had the lowest biofilm of the same bacteria. We then concluded that non-biofilm experiments can be used on certain non-TC-treated plates, while biofilm experiments should be used on TC-treated plates.

You can not. The only available test for this purpose is coagulase test.

Other tests or agar media are unreliable.

Regards

It's well-known that most Gram-negative bacteria are intrinsically resistant to vancomycin because their outer membranes are impermeable to large glycopeptide molecules (with the exception of some non-gonococcal Neisseria species).

Regards

Following

I also use 0.5 Mcfarland to determine number of bacteria cell after incubation but in most articles they use Uv/visible spectrophotometer at 600 nm to determine cell concentration. Whic way is more reliable?

At least we would be very happy to test the antiviral activity of ZnO tetrapods, let me know if someone is interested, I am hopeful that it will work.

It is so interesting, unfortunately I will not be able to be there.

My best wishes

Can you provide additional information e.g. Gram stain, colony morphology in other culture medium?

Next to molecular biology ( and perhaps even more promising) you should focus on mass spec techniques.

Read as an introduction:

If I understood your question right, you want to know if other HPV proteins (different from E6/E7) can induce cervical cancer. Besides E6 and E7, E5 has been showed to be an oncoprotein as well. One well explored mechanism of E5 is regarding the evasion of immune system. The high‐risk E5 protein interferes with classical MHC class 1 processing, but also has many other functions related to proliferation.

E5 as an oncogene has been largely investigated in bovine papillomavirus BPV:

Check the review from Dr. Doorbar below:

Hey, for a real time update on classification of beta-lactamases in general, you got to always check this online database

From the website you have all the known abd emerging beta-lactamases list and classifications

Cheers

Hi,

It is believed that brucellosis causes fewer spontaneous

abortions in humans than it does in animals because of

the absence of erythritol in thehumanplacentaandfetus

You can use Galleria for compound toxicity testing, virulence testing, efficacy of novel compounds, evaluation of pathogenicity between strains of bacteria/fungi. In addition, you can now purchase research grade Galleria from https://biosystemstechnology.com/ which are grown free of any hormones or antibiotics and are genome sequenced

Chocolate Mueller Hinton Agar has been developed for fastidious organism susceptibility testing , originally for Haemophilus species and A. pleuropneumoniae and H. somni (both veterinary pathogens).

Currently the CLSI suggests the use of Haemophilus test medium for H. influenza and H. parainfluenzae species.

If you are testing streptococci I would suggest that it is not appropriate as it has X and V factors which were added to this media specifically to allow the organisms above to grow.

CLSI quality control ranges and interpretive criteria for Streptococci, Listeria and N. meningitidis were developed using 5% sheep blood Mueller Hinton agar . So I would not recommend the use of chocolate Mueller Hinton for these species.

I would love to get updated with the results of the survey... How can we stay in touch?

What is the ARMS Plus？

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

Dear Manar, this article will help you:

Semin Respir Crit Care Med. 2003 Feb;24(1):3-22.

Epidemiology, prevalence, and sites of infections in intensive care units.

Richards M1, Thursky K, Buising K.

You will find what you want in the updated version of CLSI 2016 document (attached file).

Regards

Thank you very much for all your help. I really appreciate that.

Becouse of other mechanisms of resistance 

Hi,

Yes, you can use Mueller- Hinton agar or Nutrient agar.

I worked in PhD on Pediocin production and used Nutrient agar in inhibition activity method.

Pa is known to form biofilm and responsible for some of the horrifying nosocomial infection. As pathogen it will grow very slowly outside the host body in absence of body fluids.

Thanks Marion! That answers my question.

I use the CLSI guidelines but that was just a mistake the using standard MHB. For the information, I repeated the procedure with CAMHB and the results didnot change significantly, just for a few samples as only one value of MIC. 

Thanks a lot for your comments, 

All the best

Ezgi

Thank you Dirk Schmidt.

For patients the ct values ranged between 20 and 30.

Is there any guidelines about detection GAS asymptomatic carriers (especially about the bacterial /DNA load)

If the qPCR flags positive at 36 to 40 is it really carrier state positive, since group A streptococcus won't be present as normal flora of upper respiratory tract.

This pdf It may help

The pathogenesis is controversial. According to the most popular hypothesis , dietary tryptophan is converted to indole by gut bacteria, which is further metabolized in the liver to indoxyl sulphate and then excreted in the urine. Constipation favors conversion of tryptophan to indole by gut bacteria.

Once excreted, indoxyl sulphate can be processed by bacteria colonizing the urinary catheter to indoxyl, which is further converted to indigo (blue) and indirubin (red). ( Initially had red tinged urine which became orange as urine flowed)

The most commonly involved

bacteria are Providencia stuartii, Providencia rettgeri, Escherichia coli, Klebsiella pneumoniae,( like our patient) Proteusmirabilis, Morganella morganii, Pseudomonas aeruginosa, and Enterococcus species [4]. These bacteria produce indoxyl phosphatase and sulphatase enzymes.

so it solves our puzzle!!

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:

Yes indeed there is. Well established bacteria have the ability to resist better the effects of agents they are exposed to including antibiotics.  

People interested in an update on predatory journals, following the cessation of Beall's List updates and it's removal, might like to refer to an article in today's Times Higher Education (6 April 2017, pp. 42 & 43), reporting work by Larissa Shamseer and David Moher (link below but access may be restricted to subscribers).

Their study resulted in a list of 13 warning signs:

1. The scope of interest includes non-biomedical subjects alongside biomedical topics, or multiple, wide-ranging and unrelated fields of study are combined

2. The website contains spelling and grammar errors

3, Images are distorted/fuzzy, intended to look like something they are not, or are unauthorised

4.The home page language targets authors rather than readers

5, The Index Copernicus Value is promoted on the website

6.  Description of the manuscript handling process is lacking

7.   Manuscripts are requested to be submitted via email

8.   Rapid publication is promised

9.   There is no retraction policy

10.   Information on whether and how journal content will be digitally preserved Is absent

11. The article processing/publication charge is very low (eg, <$150)

12. Journals claiming to be open access either retain copyright of published research or fail to mention copyright

13.  The contact email address is non-professional and non-journal affiliated (eg, @gmail.com or @yahoo.com)

Couple of thoughts:

Lots of options depending on how many samples you will be processing, how much you're willing to pay and what turnaround time you want. Good luck!

As far as microbiological methods are concern; isolation is not possible from formalized tissue. Acid fast staining, histopathology and immunohistochemistry can be performed. Pcr can be uesd to detect the presence of bacterial DNA.

I'm not aware of defined inhibition zone diameter breakpoints for E. coli and Florfenicol.

In a paper of O Ahmed (2010) [https://ann-clinmicrob.biomedcentral.com/articles/10.1186/1476-0711-9-12] the breakpoint was set to ≤18 mm. Miller et al (2003) [http://jcm.asm.org/content/41/9/4318.full] found E. coli ATCC 25922 (susceptible to florfenicol) showing inhibition zones of 18-33 mm (grown at 28 C!!!) while NCCLS (at that time) approved 20 - 30 mm for that reference strain.

??

You are confused. This question is not for you. This is a general question available for anyone who is a Researchgate member.

We use selective media with amphotericin, vancomycin, cefsulodin and trimethoprim for H. pylori culture and it works very well.

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.

Brain heart infusion broth with 2% glycerol. Then store in -20 °C. This will maintain your culture for 4-6 months.

asymptomatic throat carriers of beta hemolytic streptococci & healthy children should not be treated with antibiotics. 

The classic example of inducible resistance, as you provide, is the D-shaped disk diffusion patterns in gram positives (staph and strep) when you add both erythromycin (ERM) and clindamycin (CLI) disks on the same plate. A strain with inducible clindamycin resistance would appear susceptible if only a CLI disk is used.

This is because clindamycin does not release attenuation of the ermC or similar MLS_B mRNA transcript (so it is never expressed, thus no resistance). However, treatment of infection caused by such an organism with clindamycin monotherapy has a high likelihood of treatment failure due to small subpopulations with mutated ermC sequences able to constitutively express  high-level MLS_B clindamycin resistance. While spontaneous generation of these mutated ermC sequences that are not attenuated is rare, the number of microbes in an average infection is often sufficient to have some of these resistant subpopulations already present. In contrast, even small amounts of erythromycin bind to the ermC or similar MLS_B mRNA transcript, release attenuation and the transcript is expressed resulting in high-level ERY/CLI resistance in the entire population given the presence of erythromycin.

Thus, there will be a blunting of the CLI zone of inhibition where ERY has diffused into the media resulting in a "D" shape because ERY "turns on" expression of ERY/CLI resistance in all members of the population, not just the vanishingly small subpopulation with a mutated (read: constitutively expressed) ermC-like transcript.

Similar inducible resistances can occur in gram negative organisms (e.g. with tetracycline, etc). Hope this helps!

Pathogenicity and detection of virulence genes are necessary for completing the identification criteria for an organism. So PCR test for on of the virulence genes or pathogenicity using experimental animals are two main cases enable us to know wheather the organism is pathogenic or no.

No ...since there are a lot of commensial niesseria which may add to confusion on gram stain. so culture or molecular dtetection is necessary

Thanks for your suggestion

I will check the CLSI, I have 26th edition one, Is it the last version do you have?

Under my personal criterion, it is better to separate both activities, in our laboratory Vero cell cultures are maintained in an area and laminar flow hood separated from the room where we infect those cells with intracellular parasites mainly, the laminar flow hood is also different . In your case one would think Staphylococcus aureus strains are extracellular, but that vision has changed. Attach a reference that might help you.

Curr Protoc Microbiol. 2007 Feb;Chapter 9:Unit 9C.4. doi: 10.1002/9780471729259.mc09c04s4.

Tissue culture assays used to analyze invasion by Staphylococcus aureus.

Cheung AL1, Bayles KW.

Author information

 

Abstract

Many successful pathogens have developed the ability to adhere to and invade animal tissues. Recent experimental evidence suggests that S. aureus, generally perceived as an extracellular pathogen, can also invade and, in some cases, multiply within host cells. As a proxy to infections in animal hosts, the study of S. aureus interactions with tissue culture cells has become an important research tool in many aspects of bacterial pathogenesis. In this unit, we describe two cell culture models, including bovine mammary epithelial cells and human umbilical vein endothelial cells, that investigators have used to study the interactions of S. aureus with host cells.

PMID:

18770623

DOI:

10.1002/9780471729259.mc09c04s4

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.

MBEC is biofilm eradication concentration in which impact of compounds is assessed on pregrown biofilm. MBIC is similar to MIC.

Generally crystal voilet assay is done for quantifying, resazurin assay is also used to assess the viablility.

The following work shows that there are no differences between one or another solvent (DMSO or water)

Abstract

A total of 145 clinical strains of Candida species were tested by the Clinical and Laboratory Standards Institute M27-A3 methodology to determine if replacing water with dimethyl sulfoxide as the solvent for fluconazole and flucytosine impacted the in vitro potency. No significant differences in MIC values were observed with either antifungal between the two solvents against any Candida species, and the essential agreement for each agent between the two solvents was greater than 99%.

Thank you sir