Conjunctival Commensal Isolation and Identification in Mice

Abstract

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 "in-lab" 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.

Figure 1:. In-house eye swab.

(A) A thin, approximately 1 cm, piece of pulled cotton batting was held between the sterile toothpick point and index finger and the toothpick was rolled while maintaining slight pressure against the index finger until the cotton was completely rolled onto the toothpick tip. (B) Example of an eye swab. Please click here to view a larger version of this figure.

Figure 2:. Mouse conjunctival eye swab placement.

(A) The BHI wet eye swab tip was inserted, at a 90° angle, into the medial corner of the left eye of an anesthetized mouse, depressing the eyeball. (B) The swab was moved along the conjunctiva while maintaining slight pressure on the eye in a back and forth motion, 10 times. Please click here to view a larger version of this figure.

Figure 3:. Representative results for the eye swab and master plates.

(A) 10 μL of eye swab inoculated enriched media was aliquoted on the right side of the blood agar plate and tilted 30 to 60 degrees to allow the media to form strips and on the left side of the plate, ten 10 μL dots of the sample was dispensed. The photograph of the incubated plate shows individual colonies and morphological diversity. (B) A master plate of isolates was created by picking a colony from the eye swab plate and streaking within one of the squares (grids). Three morphologically similar colonies are streaked in separate grids. Every grid has three streaks of the same colony that was picked from the mouse eye swab plate. After growth appeared on the plates, the isolate was characterized by selective plating and catalase testing. Please click here to view a larger version of this figure.

Figure 4:. Example of MALDI-TOF MS results.

Each isolate was cultured on TSA overnight, applied to MALDI-TOF MS slide and overlaid with MALDI-TOF MS solution, air dried and spotted in duplicate. Results for Streptococcus acidominimus (A) and Aerococcus viridans (B) were positively identified with a confidence value of 99.9. Please click here to view a larger version of this figure.

Figure 5:. Sex-biased commensal conjunctival growth.

Age-matched C57BL6/N mice were compared for relative commensal diversity. Eyes were swabbed and commensal organisms identified. The most predominant species was Streptococcus acidominimus, which was significantly more abundant in male than female mice (2-way ANOVA, p<0.0001). 5 different CNS isolates were identified, Aerococcus viridans and E.coli. While some of the CNS isolates were not present in all mice, the Streptococcus acidominimus, Aerococcus viridans, CNS isolate 1, and E. coli were found in all mice with distinct abundances. Please click here to view a larger version of this figure.