Name: conjunctival commensal isolation and identification in mice
Uploaded: 2021-05-01T13:00:00
Description: Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Funding from&#160;P30 DK034854&#160;supported VY, LB and studies in the Massachusetts Host-Microbiome Center and funding from NIH/NEI&#160;R01 EY022054 supported MG.

All procedures involving mice follow the Institutional Animal Care and Use Committee guidelines. Follow laboratory safety guidelines (as directed by your Institutional Environmental Health and Safety department) when working with microorganisms and potentially contaminated materials. Use appropriate waste receptacles and decontamination procedures prior to disposal of potentially biohazard contaminated materials.
1. Eye swab preparation, work field set-up, mouse eye swabbing and sample enrichmen...

<ol>
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E., Van Gelder, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+understanding+the+ocular+surface+microbiome.">Considerations in understanding the ocular surface microbiome.</a> American Journal of Opthalmology. 158 (3), 420-422 (2014).</li><li>Fleiszig, S. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-enriched+metagenomic+sequencing+enables+in-depth+profiling+of+the+cystic+fibrosis+lung+microbiota.">Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.</a> Nature Microbiology. 5 (2), 379-390 (2020).</li><li>Raymond, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-enriched+human+gut+microbiomes+reveal+core+and+accessory+resistance+genes.">Culture-enriched human gut microbiomes reveal core and accessory resistance genes.</a> Microbiome. 7, 56 (2019).</li><li>Peto, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+culture+enrichment+and+sequencing+of+feces+to+enhance+detection+of+antimicrobial+resistance+genes+in+third-generation+cephalosporin+resistant+Enterobacteriaceae.">Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.</a> PLoS One. 14 (11), 0222831 (2019).</li><li>Lauer, B. A., Masters, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+effect+of+calcium+alginate+swabs+on+Neisseria+gonorrhoeae.">Toxic effect of calcium alginate swabs on Neisseria gonorrhoeae.</a> Journal of Clinical Microbiology. 26 (1), 54-56 (1988).</li><li>Dubois, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performances+of+the+Vitek+MS+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+system+for+rapid+identification+of+bacteria+in+routine+clinical+microbiology.">Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology.</a> Journal of Clinical Microbiology. 50 (8), 2568-2576 (2012).</li><li>Kawakita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=double-blind+study+of+the+safety+and+Efficacy+of+1%D-3-Hydroxybutyrate+eye+drops+for+Dry+Eye+Disease.">double-blind study of the safety and Efficacy of 1%D-3-Hydroxybutyrate eye drops for Dry Eye Disease.</a> Scientific Reports. 6, 20855 (2016).</li><li>Lee, H. 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A review of evidence.</a> Human Genomics. 14 (1), 11 (2020).</li><li>Stevenson, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+disease:+an+immune-mediated+ocular+surface+disorder.">Dry eye disease: an immune-mediated ocular surface disorder.</a> Archives of Ophthalmology. 130 (1), 90-100 (2012).</li></ol>

Due to the paucibacterial state of the ocular surface, many laboratories have had difficulty isolating ocular commensals7,20, resulting in low number of samples with growth, low abundance and low diversity8. This method significantly improves upon standard culture practices4,21 by the addition of an enrichment step, as well as a redesigned eye swab and identification by MALDI-TOF M...

The aim of this protocol is to enhance specific isolation of viable and rare aerobic and facultative anaerobic microbes from the ocular conjunctiva to characterize the ocular microbiome. Extensive studies have profiled commensal mucosal communities on the skin, gut, respiratory and genital tracts and show that these communities influence the development of the immune system and response1,2,3. Ocular commensal communities have been shown to change during certain disease pathologies, such as Dry eye disease4, Sjogren&#8217;s syndrome5 and diabetes6. Yet, the ability to define a typical ocular surface commensal community is hampered by their relatively low abundance compared to the other mucosal sites6,7,8. This prompts a controversy over whether there is a resident ocular microbiome and if it exists, whether it differs from the skin microbiome and consequently, its local effect on the innate immune system development and response. This protocol can help resolve this question.
Generally, approaches to define the ocular commensal niche are based on sequencing and culture-based techniques4,7,9. 16 S rDNA sequencing and BRISK analysis7 show a broader diversity than culture-based techniques, but are unable to differentiate between live and dead microbes. Since the ocular surface is hostile to many microbes due to tear film&#8217;s anti-microbial properties4 generating a large array of DNA fragments, DNA based approaches will detect these artifacts which may skew the data toward identification of dead bacteria as resident commensals rather than contaminants. This results in aberrant commensal identification and characterization of the ocular space as being higher in microbe abundance and diversity10. This makes it difficult to define the resident ocular microbiome via DNA based methods. Whereas, standard culture-based techniques are unable to detect commensals because the load is too low11. Our method improves upon standard practices by using a thin swab that can target the conjunctiva, thus avoiding contamination from neighboring skin, as well as the concept that viable organisms can be enriched by brief culture in nutrient dense media with the goal of resuscitating viable but non-culturable, as well as, enriching for rare viable microbes.
The results, relative abundance of ocular commensals per eye swab, characterize the conjunctiva resident microbiome and are important for comparative purposes. Our data shows that there is a difference between skin and conjunctival microbiota, as well as greater diversity with increased age and a sex specific difference in abundance. Furthermore, this approach has reproducibly found commensal differences in knock-out mice12. This protocol can be applied to describe the ocular microbiome which may vary due to caging practices, geography, or disease state, as well as the local effects of commensal metabolites and products on immune system development and response.

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Representative results for an eye swab plate demonstrating different methods for plating are pictured in Figure 3A showing morphologically diverse isolates from C57BL/6 mouse. For each distinct isolate, the colonies were counted in the strip and the relative abundance, unique Colony Forming Units (CFUs) per eye swab, calculated and plotted for comparison purposes. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate (c...

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Conjunctival Commensal Isolation and Identification in Mice

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

This method identifies viable ocular conjunctival microorganisms, which is challenging due to the eyes hostile environment. It can help answer whether an ocular microbiome exists and how bacterial presence changes in eye disease. In contrast to DNA sequencing, which identifies both viable and non-viable microorganisms, this method identifies only viable microorganisms, resulting in a clearer understanding of the ocular commensal community.Studies suggest that difficult to diagnose eye diseases such as non autoimmune and autoimmune dry eye have unique ocular bacterial presence. This method could be used to diagnose eye diseases with distinctive microbial signatures. Begin by autoclaving the appropriate amount of cotton batting and toothpicks for the number of mice to be swabbed, then pinch off 1/2 a centimeter long piece of cotton batting and tease it out by pulling on the edges to form a flat single porous layer stopping just before the batting falls apart.Swirl the batting around one of the sharp ends of the toothpick by lightly holding the stretched out piece on the toothpick tip as it is twisted. The finished eye swab will have a very thin layer of cotton stretched over the tip extending approximately 1/2 to one centimeter away from the tip. Insert the swabs into a small beaker, swab side down then cover and autoclave them.Clean the work area with a disinfectant to minimize contamination and aliquot 0.5 milliliters of sterile brain heart infusion, or BHI media into labeled 1.5 milliliter sterile micro centrifuge tubes. Cap the tubes in a rack and set the rack on ice. Set up workflow from left to right starting with the mouse anesthetizing station, which contains the cage with the experimental mice, empty sterile cage, room temperature anesthesia, 25 gauge needle and a one milliliter syringe.Next, prepare an eye swabbing station that contains the aliquoted BHI on ice, sterilized eye swabs, lubricating eyedrops, biohazard container, clean paper towels, and 70%isopropanol spray. Finally, set up the plating station with room temperature blood agar plates, a 10 microliter pipette, 10 microliter sterile disposable tips, and a biohazard waste container. Make sure that the mouse is properly anesthetized by squeezing a hind foot pad.Only proceed if there is no movement. Assign one hand to handle anesthetized mice and the other hands to handle the eye swab and the culture. Remove the mouse from the cage and place it on top of the work surface positioned on its side with the left eye exposed.Spray gloved hands with isopropanol and dry them with a paper towel. Uncap the labeled BHI micro centrifuge tube with the dedicated media handling hand and place the two back in the rack. Dip the cotton coated tip of the eye swab in the BHI, then withdraw the swab from the tube while swirling the tip twice against the inner tube to remove excess liquid and remove it.With the mouse handling hand, gently hold the mouse by the scruff of the neck. With the other hand, place the tip of the eye swab against the medial conjunctival region of the left eye. Lightly depress the eyeball and move the swab in a window washing motion between the lower eyelid and eye 10 times, maintaining constant pressure.Without touching the fur, gently remove the tip of the swab perpendicular to where it was inserted. Place the swab cotton side down, directly into a labeled micro centrifuge tube with BHI media. Apply an eye drop to the swabbed eye.If desired, acquire skin or fur swabs for control samples, sterilizing gloves appropriately between each swab. When finished, return the mouse to the cage. Let the swab stand for 10 to 15 minutes on ice, then sterilize gloved hands and remove the swab while mixing the tip in the media for 10 rotations.Withdraw the swab by swirling the tip against the inner wall of the tube for five rotations and dispose of it in a biohazard container. Repeat the process for each mouse. Enrich the sample by incubating the tube statically for one hour at 37 degrees Celsius.During incubation, label one room temperature TSA plate per mouse eye swab or control swab and divide it in half. Remove the enriched samples from the incubator and place them on ice. Briefly vortex the samples to mix, then aliquot 10 microliters of the sample onto the TSA plate and tilt the plate to form a strip.Repeat this twice. On the other side of the plates dividing line, create 10 dots with 10 microliters of sample each. Incubate the plates at 37 degrees Celsius for 18 hours, two days and four days in a clean chamber that prevents agar plates from drying.Count the colonies in the strips. Note morphology and calculate colony forming units per swab for morphologically similar isolates. Look at the dots where unique organisms are not captured in the strips.To facilitate the visualization, we plated bacteria densely in the middle and right plates. Typically, we observed much fewer bacteria in eye swab plates. Note that on occasion, the eye swabs may not yield recoverable bacteria.A representative eye swab plate with morphologically diverse isolates from a C57 black six mouse is shown here. For each distinct isolate, the colonies were counted in the strip and the relative abundance was calculated and plotted. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate.When growth appeared, additional tests were run to characterize or identify the microbes. The master plate was used to provide enough inoculum to expand the respective isolates. To identify isolates, a TSA plate was streaked and incubated overnight, and MALDI-TOF MS was performed.Examples of isolates shown here were identified as Streptococcus acidominimus and Aerococcus viridans. Significantly different levels of commensal organisms were recovered from male and female C57 black six mice. Streptococcus acidominimus, Aerococcus viridans coagulates negative Staphylococci and E.coli were isolated from C57 black six N mice.When attempting this protocol, keep in mind that the best outcome will be achieved by coating the swab thinly and taking time during the eye swab step. If access to MALDI-TOF MS is limited, then the isolates may be identified by microbiological and biochemical testing.

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