Bevyn Cover
March 17th, 2019
Microbiology
Lab Report
A Look Into an Under Researched Dietzia
Introduction
Skin is the largest organ of the human body and is teeming with a diverse array of microorganisms. “The adult human is covered with approximately 2m^2 of skin, with surface area supporting about 10^12 bacterial cells/person’(Morubagal et al. 2017). This hotspot, which acts as the protective barrier for the human body, is habitat for commensalistic bacteria, like Staphylococcus epidermidis, the microbe most commonly found on the skin, or opportunistic pathogens that can cause infection or even death such as Staphylococcus aureus and Corynebacterium diphtheriae (Cogen & Nizet & Gallo, 2009).
These colonies on the skin come from a lifetime of being inoculated, or touched by people, objects, or even breezes. The recent uptick in human’s access to cell phones brings a new form of inoculation to human skin. Cell phones are often touched with unwashed hands and are rarely stanitized so the possibility of cross transfer is high. After looking into studies of cross contamination in the healthcare worker field, “Ansari and colleagues 149,150 studied rotavirus, human parainfluenza virus 3, and rhinovirus 14 survival on hands and potential for cross-transfer. Survival percentages for rotavirus at 20 minutes and 60 minutes after inoculation were 16.1% and 1.8%, respectively … Harrison and colleagues 157 showed that contaminated hands could contaminate a clean paper towel dispenser and vice versa. The transfer rates ranged from 0.01% to 0.64% and 12.4% to 13.1%, respectively.’(World Health Organization, 2009). These findings show that it is extremely easy for microbes to be transferred from the hand and to the skin. When a phone call is taken, there runs the chance that a potential pathogen present on the phone can be transferred onto the face and into the mouth, nose, or eye. These mucus membranes of the face make it easier for the microbes that might be present on the skin to make their way into the body and potentially infect the host.
The objective of this study was to collect, isolate, and then test both genetically and physiologically bacteria from my phone screen in order to identify it and find out if my phone carried microbes capable of making me sick. Given that I sought to isolate a bacterium from my cell phone, I initially hypothesized it will be that I would identify a microbe that is commonly found on human skin and that it lives aerobically. In an experiment, it was found that, “Microbes on owner’s hands play an important role in the contamination of mobile phone surfaces. Still the count of bacteria on mobile phones was lower than that previously found in skin touch samples where the median colony count was 480 per cm^2’ (Kõljalg et al. 2017). Given this, whatever microbe found on my cell phone should likely be one that is found on my hands as well.
Methods
Sample Collection
In order to get the most accurate swab of the microflora of my phone, I did not in anyway clean the screen. I swabbed the screen with a moistened sterile cotton swab and streaked the swabs on three different kinds of media, Tryptic Soy Agar (TSA), Reasoner’s 2 Agar (R2A), and Sabouraud’s Agar (SA) (Lab Handout 1.) The plates were placed in the 37oC incubator for four days. The colony selected was one of the last ones to show up and was chosen because it was small and the most unique in color of all the colonies on the plate (Figure 1). Once I chose the bacterial colony I wanted to study, I used a sterilized metal loop to inoculate a new, sterile TSA plate with the quadrant method as directed in Lab Handout 2 to begin to isolate a pure culture from the small pink colony (Figure 1.) After two streaks I was confident in the pureness of the sample, but four streaks were performed to increase confidence.
Figure 1. Isolate of pure culture obtained from a cell phone. The bright coral/pink colonies thrived in the 37oC incubator
Genomics
With the Qiagen PowerSoil DNA Isolation Kit, I extracted DNA from my isolate using the protocol found in Lab Handout 5. The gDNAl was sent to to be sequenced in the Genomics Core Lab at UAF using the Illumina MiSeq DNA sequencer.
To analyze the sequence data and gain information on the genome of my isolate , following Lab Handout 7, I used two different web-based bioinformatics pipelines: KAIJU and PATRIC. PATRIC as used to assemble the genome of my isolate and annotate it’s genome via metagenome binning. Kaiju was used to assign taxonomy to the assembled genome and to compare results with PATRIC results.
Physiological
To determine the morphological traits and Gram status of the isolate, the isolate was Gram stained and observed under the microscope. The Gram stain was conducted following the Lab Handout 4 protocol in which I stained my isolate alongside Gram-positive and -negative controls to validate results. This process of Gram staining made it possible to see the shape, size, colony organization of the isolate, and the Gram status of the isolate.
In order to further support the Gram staining results and discern whether my isolate could ferment certain sugars, I streaked my isolate on by MacConkey (MAC) and Eosin Methylene Blue (EMB) agars following Lab Handout 6 instructions. Both MAC and EMB agars are selective for Gram-negative bacteria and reveal whether the bacteria can ferment lactose and sucrose respectively.
Several physiological tests were outlined and performed following the Lab Handout 8 protocol. First, a catalase test was performed by dropping hydrogen peroxide on the bacteria in order to find out if the bacteria produces the catalase enzyme.. An oxidase test using a teststrip was also performed to determine if cytochrome c oxidase was present. Based upon Gram staining results and the physiological test previously mentioned, the Lastly API Coryne physiological test strip, which tests for 21 metabolic processes, was determined to be the best fit for the isolate. The API test strip was inoculated with the isolate, incubated for 36 hours, after which a battery of follow-up tests were performed as highlighted in the API Coryne instruction manual. .
In order to determine the oxygen class of the isolate, a fluid thioglycollate test was performed. A sterile loop was used to inoculate a soft agar to determine if the isolate required oxygen to grow or not.
Lastly, antibiotic test was carried out by suspending the isolate in sterile TSB and then swabbing it onto a Mueller-Hinton agar plate with a lawn streak (Lab Handout 9). Eight different antibiotics; tobramycin, piperacillin, amikacin, gentamicin, cefazolin, cefoperazone, oxacillin, and erythromycin were placed in different quadrants of the plates and allowed to diffuse for several days. After several days of growth, any rings that formed around the antibiotic rings were measured and recorded.
Results
Morphological
The most obvious trait of the isolate was its presentation on the TSA plate. From a macroscopic point of view, the isolate grew in shiny round colonies with jagged edges. During early growth the colonies were yellowish in appearance and turned bright pink after a few days of incubating in 37oC. Under the microscope, Gram staining revealed the isolate was Gram-positive presenting as deep purple (Figure 2.) The Gram-staining also made it possible to see that the bacteria were either elongated cocci or short bacillus, likely coccobacillus in shape. The cells grew in small groups, end-to-end with an almost zigzag type pattern.
Figure 2. The isolate under the microscope after Gram staining. Dark purple indicating this is a Gram-positive bacteria and shows both the cell’s size and shape.
Physiological
When the isolate was streaked on MAC and EMB agars, no sign of growth was present. Because both of these growth media select only for Gram-negative bacteria, this confirms that the isolate is Gram-positive.
The fluid thioglycollate test revealed that the isolate is strictly aerobic as only the the first few millimeters below the surface exhibited growth. During the the catalase test, many bubbles were formed indicating that catalase was present in the isolate. The oxidase test came back with less spectacular results in that nothing happened, meaning the test was negative for the isolate having cytochrome c oxidase.
API Coryne test strip was determined to be the optimal strip based upon aforementioned morphological and physiological test results. The results of that test showed that my ioslate only tested positive for two enzymes, ALkaline Phosphatase and GLUcosidase. The API strip results allowed me to input the data into the API database to get a few possible species it could possibly be. My results brought about six possible matches, and one of these matches was Dietzia timorensis.
The antibiotic testing failed for my isolate as the colony had either died or gone dormant by the time I conducted the antibiotic resistance experiment.. Research through PATRIC using the genetic information indicated that of the eight antibiotics tested (amikacin, cefazolin, cefoperazone, erythromycin, gentamicin, oxacillin, piperacillin, and tobramycin) Erythromycin was the only antibiotic my isolate was resistant to.
Genomic
The metagenome binning results provided by PATRIC indicated that the bacterium I had been isolating and characterizing is Dietzia UCD- THP. The Krona chart (Figure 3) resulting from the KAIJU analysis supports the PATRIC identity of my isolate, Dietzia UCD- THP, and indicated
that 83% of my DNA sequences were Dietzia UCD- THP, the remaining 7% from the Dietzia genus and some minor contamination.
(Figure 3) The Krona Chart of the isolate. The outer ring showing 83% of the DNA belongs to Dietzia UCD- THP.
Discussion:
After learning that my isolate only had one bin in PATRIC and was 83% Dietzia UCD- THP, I was confident that this was in fact the bacteria I had and it was a fairly pure sample. The later tests simply reaffirmed the genetic testing. “Dietziae are aerobic, Gram-positive, non-acid-alcohol fast, non-sporing, catalase-positive actinomycetes that form cocci that germinate into short rods or rod-shaped cells, which exhibit snapping division and produce V-shaped forms. Circular, raised or convex, glistening, orange to coral red colonies with entire edges are formed on agar media’(Koerner et al. 2005). That being said, this particular strain of Dietzia is not well researched, to the point that it still doesn’t have an official name. Most of my research had to be done on the whole genus as there just wasn’t enough literature for this particular species. Dietzia is found mostly in humans and animals, but is not very common. In an article published by FEMS Immunology & Medical Microbiology, it goes on to say the Dietzia is found in the intestinal tracts of carp and halibut, in soil enriched with alkanes, plant tissue, along with sometimes being the culprit for some deep tissue infections in humans meaning they can be a resident of the skin microbiome (Roland et al. 2009). Along with these places there are a few others, “including Korean food (1), a soda lake (2), and a swab sample from a human patient (3)’(Diep et al. 2013). Because they are found in such diverse places, the fact that one was found on a phone is not out of the ordinary.
My API test lead to finding one match that was similar to my isolate. However after doing further research, though it is similar and helps to solidify that I do indeed have a Dietzia, Dietzia timorensis is definitely not a possible candidate for my isolate’s identity as it is forms sandy yellow colonies and has only been found in soil in Southeast Asia (Yamamura et al. 2009).
Seeing as this bacteria was collected from a highly aerobic environment, the fluid thioglycollate test pointed that this was indeed the case in that it only grew on that very top layer. I was not surprised when this bacteria came out being susceptible to many of the antibiotics as this particular isolate is not regarded as a human pathogen and is not generally treated with antibiotics so there is no selective pressure to have those genes.
As a whole, these tests mostly aligned with traits commonly associated with Dietzia, but not exactly like any of the well researched strains. I can confidently say that my strain was a part of the genus Dietzia as many of the results aligned closely with those found within the known species, there has been research on this exact strain, and after genetic testing they also came up with the same conclusion, “Dietzia sp. strain UCD-THP falls within a poorly resolved paraphyletic clade containing 7 species of Dietzia (https://dx.doi.org/10.6084/m9.figshare.646178). Because the 16S rRNA gene sequence of Dietzia sp. strain UCD-THP has >99% identity to homologs from several species of cultured isolates, and the phylogenetic relationships among those species are unclear, we have been unable to assign a species name to this isolate’(Diep et al. 2013). This explains why many of my testings gave results that matched different species within this genus but not exactly with any of them. These findings solidify the fact that the genus Dietzia is still in it’s exploratory stage, finding new strains and researching their similarities and what they might do to both the environment around them and their potential when coming in contact with a human’s cell phone.
Work Cited
Cogen, A L et al. “Skin microbiota: a source of disease or defence?’ British journal of dermatology vol. 158,3 (2008): 442-455.
Morubagal, R. R., Shivappa, S. G., Mahale, R. P., & Neelambike, S. M. (2017). Study of bacterial flora associated with mobile phones of healthcare workers and non-healthcare workers. Iranian journal of microbiology, 9(3), 143—151.
Roland J. Koerner, Michael Goodfellow, Amanda L. Jones. (April 2009). The genus Dietzia: a new home for some known and emerging opportunist pathogens, FEMS Immunology & Medical Microbiology, Volume 55, Issue 3, Pages 296—305
https://doi.org/10.1111/j.1574-695X.2008.00513.x
WHO Guidelines on Hand Hygiene in Health Care: First Global Patient Safety Challenge Clean Care Is Safer Care. Geneva: World Health Organization; 2009. 7, Transmission of pathogens by hands.
https://www.ncbi.nlm.nih.gov/books/NBK144014/
Diep, A. L., Lang, J. M., Darling, A. E., Eisen, J. A., & Coil, D. A. (2013). Draft Genome Sequence of Dietzia sp. Strain UCD-THP (Phylum Actinobacteria). Genome announcements, 1(3),
Yamamura, H., Lisdiyanti, P., Ridwan, R., Ratnakomala, S., Sarawati, R., Lestari, Y., . . . Ando, K. (2009). Dietzia timorensis sp. nov., isolated from soil. International Journal Of Systematic And Evolutionary Microbiology, 60(2), 451-454.
Kõljalg, S., Mändar, R., Sõber, T., Rööp, T., & Mändar, R. (2017). High level bacterial contamination of secondary school students’ mobile phones. Germs, 7(2), 73—77.
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