Enterococcus sp. Streptococcus thermophiles, Micrococcus luteus, and Melissococcus plutonius isolated from contact lens storage.
Introduction
There are about 125 million contact lens wearers worldwide. (Kay JE, 2007) Aside from the people with one-day use only lenses, most contact lens wearers are required to have contact storage to keep their lenses moisturized and protected from potential contaminants. With the help of cleaning solution, the lens itself can be easily washed and disinfected, however the cases are often easily disregarded and left untreated. According to the American Academy of Ophthalmology (AAO), and the American Optometric Association (AOA), it is recommended to replace the storage case at least every three months as microbes from fingers and surrounding area will eventually build up along the bottom and sides of the cases. Also, since many people keep their cases in the bathroom, there are further chances of having contamination that could eventually be transferred to our eyes.
This lab project focused on isolating microbes from a contact lens case that has been used for about 2 months and deciphering whether the found microbes can potentially cause eye infection. To investigate what types of bacteria may have been building up, inoculated samples went through isolation to pure culture. The pure culture was then sequenced to obtain a partial genome via shotgun metagenomics. Identification of my isolated helped to decipher whether it has potential to cause microbial corneal infection, which is a serious complication of contact lens wear.
The environment which housed my contact lens case in this study was a bathroom shared by 4 people. Myself and 2 more people wear contact lenses and keep the storage case extremely near the bathroom sink or inside the cabinet. The bathroom, although a convenient place to keep a contact lens case, is a very hospitable environment for microbes. Microbes can be introduced by potential splashes of droplets while washing hands near the sin, drying hair or shaving.
From this lab project, Enterococcus was found to be the majority portion growing on my contact lens case. Enterococcus in general can cause from minor infection that can be treated with ease to serious infections such as Endocarditis that requires numerous antibiotic treatment as Enterococcus possess intrinsic nature to be resistant to antibiotic drugs and thus is currently the second leading cause of healthcare associated bacteremia. (Agudelo, N, et al. 2014)
Methods
Sample collection and isolation
A sterile swab was used to collect samples from the sides and bottom surfaces of a contact lens case. Since the surface of the case was dry, the swab was first dipped into sterile water then was used to streak the sample surfaces and then the tryptic soy agar (TSA). After the inoculation, the plates were left in room temperature for growth.
Once visible colonies formed on the TSA plate, a single colony was inoculated using the aseptic technique to a new TSA plate. The new TSA plate was then incubated at 37 °C quadrant streak method several times until the microbes on the plate were isolated and pure.
Identification and characterization
When the isolated colonies were cultured, physical morphology was first recorded by observing uniformity of physical characteristics such as color, size and shape. Then Gram staining technique was used to stain the isolated culture and was placed under a microscope to see if the culture was either Gram positive, negative or Gram variable. Morphology of the culture was noted again, as the 100X magnification provided clearer images of the culture’s physical properties.
Further detailed physiological tests were performed such as fluid thiogylcollate test to determine oxygen class, oxidase test to determine if the isolated sample has cytochrome c oxidase, catalase test to see if the strain has the enzyme catalase and lastly API 20E test which consists of 21 miniaturized physiological tests.
For detailed genomic identification, a tryptic soy broth was inoculated with my isolate using aseptic technique. The DNA was then extracted using a Powersoil DNA kit. Once a pure solution of DNA in buffer was made, it went through DNA Core lab for genome sequencing using the Illumina MiSeq DNA sequencer. The results were analyzed on BaseSpace through SPAdes Genome Assembler (to assemble the genome), Kraken metagenomics (for identification of isolate down to genus and species level) and Prokka Genome Annotation (to annotate genes and determine the function).
Antibiotic susceptibility test
In preparation for the antibiotic susceptibility test, my isolate was transferred to a broth culture then incubated. With fresh isolate the culture was spread evenly throughout the two TSA plates. Using the spring-loaded antibiotic disk dispenser, 8 different antibiotics: piperacillin, cefazolin, oxacillin, cefotaxime, tobramycin, amikacin, clindamycin and trimethoprim were placed on the plates. Once the cultures were grown with antibiotic disks, diameter of the zones of inhibition were measured to determine the susceptibility.
Results
The Gram stain revealed that the bacterium was Gram variable and under the microscope and was generally cocci shaped and clustered together. Taxonomic results generated from Kraken Metagenomics revealed that my isolate was Enterococcus and these attributes fitted well with the physical description of Enterococcus from numerous literatures.
Physiological tests included fluid thioglycolate test, oxidase test, catalase test and API 20E strip test. The results for fluid thioglycolate test indicated that my bacterium is a facultative anaerobe as it grew throughout the tube but heavily on oxidized area. For the oxidase and catalase tests, my bacterium showed positive results meaning it can use oxygen as a terminal electron acceptor in respiration and will produce catalase, protecting it from the toxic by-products of oxygen metabolism.
From the API 20E test, my bacterium showed 5 positive results. CIT was positive, indicating the utilization of citrate as the only carbon source. The URE test was positive which tested for the enzyme urease. The TDA test was positive which indicates that the enzyme tryptophan deaminase was detected. The VP test was positive meaning that acetoin or 2,3-butanediol were formed from fermentation. Lastly, a positive GEL test indicated that my bacterium is capable of producing gelatinase enzyme which liquefies gelatin.
From BaseSpace Sequence Hub, the taxonomic results generated from Kraken Metagenomics application yielded 95% reads up to genus level. (Figure 1) From running nucleotide BLAST, different genuses that incorporated the remaining 5% were also found which were: Streptococcus thermophiles, Micrococcus luteus, and Melissococcus plutonius. Along with numbers of different genus, there were also numerous species of Enterococcus present as well.
Figure 1. Classification Statistics generated from Kraken Metagenomics
Upon analysis of my bacterium’s antibiotic susceptiblity, 8 different antibiotics were usded and later analyzed for its inhibition zone diameter (Figure 2)
| Antibiotics | Measured Inhibition Zone Diameter | Susceptibility |
| Piperacillian | 48mm | Susceptible |
| Cefazolin | 50mm | Susceptible |
| Oxacillin | 18mm | Susceptible |
| Cefatoxime | 52m | Susceptible |
| Tobramycin | 14mm | Intermediate |
| Amikacin | 24mm | Susceptible |
| Trimethoprim | 14mm | Intermediate |
| Clindamycin | 20mm | Intermediate |
Figure 2. Potential susceptibility of 8 different antibiotics tested on Enterococcus
Discussion
There were numerous differences between the taxonomic identification and associated literature and results from Gram staining and physiological tests for this microbe. The genus Enterococcus is a Gram-positive, and oxidase and catalase negative cocci. (Castillo-Rojas et al, 2013) This disagrees with majority of the results I acquired from physiological testing as my isolate showed both catalase and oxidase positive.
There are few explanation for this phenomenon. First, my isolated sample, after the bioinformatics analysis using Kraken Metagenomics turned out to be impure. Although the microscopic morphology (all cocci-shaped) looked identical from a TSA plate, there existed numerous species of Enterococcus. In addition to my analyses using Kraken, further taxonomic identification provided via BLAST analysis, revealed there were also species from different genera such as Micrococcus, Streptococcus, and Melissococcus. Thus, it is probable that the numerous contaminants are likely what skewed the physiological test. All the identified species but Micrococcus have both oxidase and catalase negative and Gram-positive and Micrococcus was the only species with both catalase and oxidase positive result. Considering my physiological result, it is probable that Micrococcus leutus heavily influenced the data.
For antibiotic susceptibility, Enterococci are intrinsically resistant to many commonly used antimicrobial agents and show decreased susceptibility to penicillin and ampicillin. Also, they are intrinsically resistant to clindamycin (Miller et al. 2014) which was used during the lab. However, the result disagreed with the literature as my isolate showed susceptibility to almost every antibiotic that was tested. Clindamycin was analyzed as intermediate by observing the zone of inhibition which somewhat fits with the literature.
The reason why antibiotic susceptibility test disagreed with the literature could be because my microbe had numerous Enterococcus species. It is common for strains of species not showing resistance when another strains do because different strains could have adapted or mutated differently to their fitting situations.
The bacteria that were identified through bioinformatics seemed reasonable as I could see why each of them were present on the surfaces of the contact storage. Enterococcus, which inhabits in GI tract or contaminated water, could have been transferred from water droplets from washing hands from myself and 3 other people as I do keep my contact storage extremely close to the sink. Streptococcus thermophiles are microbes that reside in dairy product such as yogurt and milk. I do consume lots of dairy products daily. Micrococcus luetus are found often on mammalian skin and mouth. Considering the location of where my contact storage is, I wasn’t surprised that this microbe was also present. The most unusual microbe was the Melissococcus plutonius which is a pathogenic agent of European foulbrood (Okumura et al. 2012) killing honeybee larvae.
Out of these identified microbes, only Enterococcus faecalis can cause endophthalmitis which is an inflammation to an eye that can potentially cause permeant eye sight damage. However, literatures have mentioned that the cases of endopthalmitis were only occurring to patients who recently underwent eye-related surgery with Enterococcus faecalis contaminated surgical equipment. (Rishi et al. 2009) From this study, Enterococcus that was found on my contact storage cannot cause eye inflammation and so do the other microbes that were also found.
In conclusion, the lab project was successful as isolation and identification of microbes from contact storage revealed multiple species of Enterococcus and different genus. However, further research needs to be done to completely isolate and culture microbes as catalase and oxidase test for my culture did not agree with the literature results. Future researches about possible microbes living inside of contact storage would be interesting as this portion is not exposed to the environment and usually is filled up with cleaning solution that most claims to kill 99% of the bacteria.
Literature Cited
Agudelo, N. I., M. M. Huycke, M. S. Gilmore, D. B. Clewell, Y. Ike, and N. Shankar. “Enterococcal Disease, Epidemiology, and Implications for Treatment.” National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 28 Apr. 2017.
Castillo-Rojas, Gonzalo, Marisa Mazari-HirÃart, Sergio Ponce De León, Rosa I. Amieva-Fernández, Raúl A. Agis-Juárez, Johannes Huebner, and Yolanda López-Vidal. “Comparison of Enterococcus Faecium and Enterococcus Faecalis Strains Isolated from Water and Clinical Samples: Antimicrobial Susceptibility and Genetic Relationships.”PLOS ONE. Public Library of Science, 1 Apr. 2013.
Kay, JE. “Development of Contact Lenses and Their Worldwide Use.” PubMed.gov. NCBI, Nov. 2007.
Miller William, Munita M Jose, and Arias A Cesar “Mechanisms of Antibiotic Resistance in Enterococci’ NCBI 15 May. 2015.
Okumuraab, Kayo, Rie Araicd, Masatoshi Okurae, Teruo Kirikaeb, Daisuke Takamatsuce, and Makoto Osakie* And. “Kayo Okumura.” Journal of Bacteriology. N.p., 01 June 2012.
Rishi, E., P. Rishi, K. Nandi, D. Shroff, and KL Therese. “Endophthalmitis Caused by Enterococcus Faecalis: A Case Series.” NCBI. Retina, 9 Feb. 2009.