Overview of Metabolism of S. Epidermidis and E. Coli on Phenethyl Alcohol, Mannitol Salt Agar, and MacConkey’s Agar

Abstract:

Escherichia coli is a gram-negative bacterium that is found in the human digestive system. Staphylococcus epidermidis is a gram-positive coccus-shaped bacterium commonly found on the human skin. In this study, we looked at the metabolism and physiology of both organisms on differential and selective media. Bacteria were grown and observed on Tryptic Soy Agar (TSA), Phenylethyl Agar (PEA), Mannitol Salt Agar (MSA), and MacConkey’s Agar (MAC). E. coli was able to grow on TSA, MSA, and MAC, and was partially inhibited on the PEA plate. On the MAC plate, the surrounding media was a hot pink color due to lactose fermentation. E. coli grew yellow on the MSA plate, which suggests that it can survive in high salt conditions and ferment mannitol, however, we believe a false positive result is due to inoculation technique error. The growth of E. coli was inhibited on the PEA plate because phenylethyl alcohol inhibits Gram-negative organisms by breaking down the outer membrane of the cell wall. S. epidermidis was able to grow on the TSA, PEA, and MSA plates. No growth was observed on the MAC plate because the bile salts break down the cytoplasmic membrane in Gram-positive organisms. S. epidermidis was able to grow on PEA because Gram-positive organisms do not contain an outer membrane in the cell wall structures, so phenylethyl alcohol cannot interfere with DNA synthesis. S. epidermidis grew pink on the MSA plate because it is a halotolerant organism and cannot ferment mannitol unlike other staphylococci such as S. aureus.

Introduction:

Escherichia coli is a gram-negative rod-shaped bacterium that is between 1.1-6 μmeters long. It is a facultative anaerobe, meaning that it can have a respiratory and fermentative metabolism. It is commonly found in the lower part of the intestines in both humans and other mammals. They are considered an opportunistic pathogen. Certain strains of E. coli are known to cause digestive issues in infants, humans, and animals alike (Bergey 1984). E. coli can ferment glucose and many other carbohydrates, and produce pyruvate as a by-product. This pyruvate can be broken down further into acetic, lactic, and formic acids. E. coli can ferment lactose, and some strains can ferment D-mannitol (Bergey 1984).

The mtlA gene in E. coli encodes for enzyme IIMtl. This enzyme converts D-mannitol to D-mannitol-1-phosphate (Figure 1). D-mannitol-1-phosphate is produced endogenously by E. coli, even if the cells are able to grow on another carbon source. The mtlD gene encodes a dehydrogenase, which converts D-mannitol-1-phosphate to fructose-6-phosphate. mtlR is the repressor gene for the D-mannitol operon. Internalized D-mannitol is the effector. Mutations of MtlA prevent growth of E. coli on D-mannitol. Expression of the MTL operon is dependent on the cAMP-CRP complex (Neidhardt et al. 1987).IIMtl

Dehydrogenase

mtlD
mtlA
Fructose-6-phosphate
D-mannitol-1-phosphate
D-mannitol

Figure 1. Simple depiction of D-mannitol fermentation and mtl operon shown by E. coli. Structures were created using ChemDraw software.

The lac operon in E. coli is well-studied. The lac-operon in E. coli consists of 3 genes: lacZ, lacY, and lacA gene (Figure 2). The lacZ gene encodes for ß-galactosidase, which cleaves lactose into both galactose and glucose. The lacY gene is responsible for encoding the enzyme permease, which transports lactose into E. coli cells. Transport of lactose by the integral cytoplasmic membrane LacY protein depends on the membrane potential of the cell. The LacA gene is the third gene in the lac operon. The LacA gene encodes for transacetylase, which is the enzyme that is responsible for adding an acetyl CoA group to ß-galactosidase. Studies have found that the lacA gene is not necessary for the normal function of the lac operon system (Neidhardt et al. 1987).

If no lactose is present in the cell, the lac repressor is bound to the lac operator (lacO), which prevents RNA polymerase from binding to the lac promoter (lacP). Transcription cannot occur, so no mRNA is created (Figure 2a) (Neidhardt et. al 1987). If lactose is present and there is sufficient cAMP in the cell, the repressor is inactivated, and the lac operon is expressed. In order for lactose to enter the cell, it must be transported by Lac-permease. The presence of lactose causes an allosteric change of the repressor, allowing the repressor to detach from lacO and allow RNA polymerase to bind to lacP. Transcription is initiated 38 bp before the lacZ gene (Neidhardt et. al 1987). Transcription occurs as usual, and eventually, translation of the final mRNA product, which will produce the enzymes ß-galactosidase, permease, and transacetylase from the lacZ, lacY, and lacA genes, respectively (Figure 2b).

Figure 2. Overview of the lac operon in E. coli cells. Photo used from 2011 Pearson Campbell biology textbook.

Staphylococcus epidermidis is a gram-positive bacterium that is roughly 0.5-1.5 μmeter in size. It is a facultative anaerobe and generally non-motile (Bergey 1984). Staphylococci grow in grape-like clusters (Foster 1996). Staphylococci can be distinguished from other coccus-shaped bacteria such as streptococci and micrococci by performing a catalase test. In this test, you take a culture of bacteria either on a plate or a slide and immerse them with hydrogen peroxide. If the culture bubbles, then you have a catalase-positive bacterium. Both streptococcus and micrococcus are catalase-negative.

S. epidermidis is the most common species of staphylococcus found on the human skin (Fey and Olson 2010). It is frequently found on the head, in the nose, and around the armpits. Previous epidemiological studies have shown that humans can carry anywhere from 10 to 24 different strains of S. epidermidis at a time (Kloos and Musselwhite 1975). S. epidermidis can be transferred through common contact.
S. epidermidis is a coagulase-negative staphylococci (CoNS). It lacks the enzyme coagulase, which coagulase-positive staphylococci (such as Staphylococcus aureus) have (Otto 2009). It is now known that some strains of S. aureus are actually coagulase-negative too. There are over 30 species of CoNS, but S. epidermidis is the most common. Coagulase is not an enzyme, but rather an extracellular protein. It binds to prothrombin in the host to form a complex known as staphylothrombin. Thrombin has protease activity, which allows it to convert fibrinogen to fibrin. Thrombin can bind and form clots in blood plasma after it is incubated with an S. aureus broth culture. This is essentially how you test for coagulase in the lab (Foster 1996).
S. epidermidis is known to produce biofilms, especially on foreign objects in the body such as implants and catheters. (4). These biofilms consist of teichoic acid, extracellular DNA, polysaccharide intracellular adhesion (PIA), and proteinaceous factors (Bhp, Aap, and Embp) (Fey and Olson 2010). S. epidermidis does not bind to fibrinogen on foreign objects in the body, but rather to fibronectin (Foster 1996). S. epidermidis can produce “slime” in the body on the objects it attaches to. This slime is primarily composed of teichoic acid, which is commonly found in the cell walls of gram-positive bacteria (Foster 1996).
Unlike S. aureus, S. epidermidis is benign to its host. It does not produce virulence factors, but rather is a skin commensal (Foster 1996, Otto 2009). It is more difficult to diagnose a CoNS infection, due to the lack of virulence factors (Foster 1996). Among CoNS, S. epidermidis is the most common bacteria for skin infections (Otto 2009). S. epidermidis is also known to be resistant to many types of antibiotics, including methicillin (Foster 1996, Otto 2011). Methicillin-resistant-Staphylococcus-epidermidis (MRSE) contain the metA gene, which encodes for a penicillin-binding protein (PBP2a) to decrease its affinity to methicillin (Otto 2011).
S. epidermidis has eight different sodium ion exchangers, and six transport systems for osmoprotectants, which allows the bacterium to handle extreme salt concentration and osmotic pressure (Rogers and Fey 2009). S. epidermidis can produce poly-γ-glutamic acid (PGA) and PNAG/PIA, which are exopolymers that help the bacterium to survive under its host’s immune defense. PGA is a pseudopolymer that originates from genes on the cap locus. PGA allows for S. epidermidis to grow under high salt concentrations. PGA is found in other CoNS but is absent from S. aureus. Many halotolerant bacteria contain PGA (Otto 2009). S. epidermidis does not ferment mannitol, but some other species of CoNS can ferment the sugar (Sah et al. 2018). S. epidermidis can also produce acid from lactose via metabolism of the D-tagatose-6-phosphate pathway (Bergey 1984).

Materials and methods:

S. epidermidis and E. coli were aseptically transferred from slants onto four different plates according to the protocol. All organisms were grown on a TSA plate and then grown on Mannitol Salt Agar, Phenylethyl Alcohol, and MacConkey’s Agar plates. Media was inoculated using aseptic technique according to protocol. The bacteria were spread onto the plates in a zigzag fashion. The plates were sectored with a permanent marker and labeled with a number that was assigned to each organism tested (Table 1). The plates were incubated at 37°C. Observations were made and recorded into a lab notebook at 24 and 48 hours.

M. luteus
S. epidermidis
S. aureus
B. subtilis
E. coli
E. aerogenes
P. aeruginosa

Unknown #14

Unknown #15

M. luteus
S. epidermidis
E. coli
E. aerogenes

Unknown #14

Unknown #15

M. luteus
S. epidermidis
S. aureus
B. subtilis
E. coli

Unknown #14

Unknown #15

S. epidermidis
E. coli
E. aerogenes
P. aeruginosa

Unknown #14

Unknown #15

Table 1. Organisms used for this experiment were labeled onto each plate according to this chart. Unknown assigned organisms were also tested on each plate.

Tryptic Soy Agar (TSA):

Tryptic Soy Agar (TSA) is a complex media that is produced by enzymatic digestion of casein and soybean meal. It contains the basic nutrients needed for most organisms to grow. All organisms that were used in this experiment are able to grow on the TSA media, so it was used as a control plate.

Phenylethyl Alcohol (PEA):

Phenylethyl Alcohol (PEA) is a selective media only. It contains many of the same components that TSA media contains such as casein, soybean meal, sodium chloride, and agar. The only other component the media contains is phenylethyl alcohol. The media partially inhibits the growth of Gram-negative organisms. Phenylethyl alcohol interferes with DNA synthesis and melts the outer membrane of Gram-negative organisms, which selects for Gram-positive organisms to grow. This plate was compared to the TSA plate as a reference.

Mannitol Salt Agar (MSA):

Mannitol Salt Agar (MSA) is a selective and differential media. The media contains 7.5% NaCl, mannitol, phenol red, and peptones. MSA selects for any organism that is able to grow in high salt concentrations (halotolerant organisms). The mannitol salt agar plate is primarily used to distinguish different Staphylococcus species. It differentiates organisms that are able to ferment mannitol. Organisms that are able to ferment mannitol will appear yellow on the plate. This color change is due to the organism producing an acid from fermentation, which changes the phenol red indicator to yellow. Organisms that are able to grow on the media, but do not ferment mannitol appear a pink/red color on the media due to peptone breakdown. Yellow on the plate indicates a pH that is < 6.9, whereas pink indicates a pH > 8.4.

MacConkey’s Agar (MAC):

MacConkey’s Agar (MAC) is a selective and differential media that contains crystal violet, lactose, peptones, bile salts, and neutral red. The media selects for Gram-negative organisms. Crystal violet and bile salts in the media inhibit the growth of any Gram-positive organisms. This media differentiates organisms that are able to ferment lactose. The neutral red dye is a pH indicator that turns red/pink below a pH of 6.8 and remains colorless with a pH above 6.8. Organisms that are able to ferment lactose, will appear a pink/red color on the media. This red color change is due to acid accumulating from lactose fermentation, which changes the neutral red dye indicator to hot pink/red. Non-lactose fermenting organisms that are able to grow on the media will appear colorless.

Observation/Results:

After incubation, observations were made and recorded at both 24 and 48 hours for all plates and organisms tested. A scoring indicator was created to compare growth from the PEA and TSA plates (Table 2).

Figure 4. TSA Plate after 24 hours of incubation at 37°C. Organism #2 is E. coli.

Figure 3. TSA Plate after 24 hours of incubation at 37°C. Organism #5 is S. epidermidis.

Tryptic Soy Agar (TSA) Plate:

After 24 hours, both S. epidermidis and E. coli grew white on the TSA plate. The E. coli grew smooth, round with an entire edge. After 48 hours, E. coli grew more. S. epidermidis did not grow anymore after 48 hours.

Phenylethyl Alcohol (PEA) Plate:

After incubating for 24 hours, S. epidermidis grew white. The bacterium grew a similar amount compared to the TSA plate. E. coli only partially grew white on the plate. There was not as much growth as compared to the TSA plate. After 48 hours, S. epidermidis grew more and E. coli looked the same as the 24-hour observations.

Mannitol Salt Agar (MSA) Plate:

After 24 hours, S. epidermidis grew a faint pinkish color on the MSA plate. E. coli grew yellow. The medium surrounding both S. epidermidis and E. coli was pink in color. After 48 hours, there was significantly more growth for E. coli and S. epidermidis. S. epidermidis was more clearly hot pink, whereas after 24 hours the bacteria were only faintly pink in color. The medium surrounding the E. coli colonies was yellow after 48 hours, whereas it was still a pink color with yellow colonies after 24 hours.

MacConkey’s Agar (MAC) Plate:

After 24 hours, E. coli grew hot pink, and the medium surrounding the colonies was also hot pink in color. S. epidermidis did not grow on the plate. After 48 hours, E. coli grew more and S. epidermidis still did not grow. The E. coli had a pink halo surrounding the growth after 48 hours.

Discussion/conclusions:

Both organisms were able to grow on the TSA plate. This is because tryptic soy agar is a complex media that supplies the basic necessary nutrients that allow most organisms to grow.

S. epidermidis was able to grow on the PEA plate because phenylethyl alcohol is selective for Gram-positive organisms. S. epidermidis is a Gram-positive bacterium. Phenylethyl alcohol interferes with DNA synthesis and breaks down the outer membrane of Gram-negative organisms. Since Gram-positive bacteria do not have an outer membrane layer to their cell wall, just a thick layer of peptidoglycan, PEA does not inhibit their growth. As seen in FIGURE ??? and TABLE S. epidermidis grew just as much on the TSA plate as the PEA plate.

On the Mannitol Salt plate, S. epidermidis was able to grow because it is a halotolerant bacterium. S. epidermidis can produce PGA, which allows it to grow under high salt conditions (Otto 2009). This is why we see growth of S. epidermidis on the MSA plate (FIGURE ). S. epidermidis does not ferment mannitol, unlike other staphylococci such as S. aureus, which is why it grew pink on the MSA plate. If it were able to ferment the mannitol, the organism would produce an acid which would change the phenol red indicator dye to a yellow color. As a reference, S. aureus is able to ferment mannitol, and this is why it appeared yellow on the MSA plate (FIGURE XX Organism #3).

S. epidermidis was not able to grow on the MacConkey’s Agar plate (FIGURE!!). This is because the crystal violet and bile salts in the MAC media inhibit the growth of Gram-positive organisms. The media selects from Gram-negative organisms to grow, and since S. epidermidis is a Gram-positive organism, it is not able to grow on the MacConkey’s Agar. The cytoplasmic membrane is directly beneath the peptidoglycan layer in the cell wall of a Gram-positive bacterium such as S. epidermidis. This cytoplasmic membrane is bile-sensitive, and when introduced to bile, the cell is not able to grow and survive (Neidhardt et al. 1987). The bile in the medium penetrated to the cytoplasmic membrane of S. epidermis, preventing growth from occurring on the MacConkey’s Agar plate.

E. coli was only able to partially grow on the PEA plate in comparison to the TSA plate (FIGURE AND TABLE!). This is because E. coli is a Gram-negative bacterium. Phenylethyl alcohol breaks down the permeability of cell membrane barriers in Gram-negative bacteria (Silver and Wendt 1966). When exposed to phenylethyl alcohol, large amounts of potassium inside the cell are leaked outward. Inhibition of DNA synthesis also occurs when Gram-negative bacteria are exposed to phenylethyl alcohol. This phenomenon, however, is due to the cell membrane barrier breaking down (Silver and Wendt 1966).

E. coli was able to grow on the Mannitol Salt Agar plate (Figure XX). The media surrounding the organism appeared yellow (FIGURE). This suggests that E. coli is able to survive in a high salt condition and ferment mannitol. Although E. coli is able to ferment mannitol into mannitol-1-phosphate by the enzyme IIMtl, the literature suggests that E. coli is unable to tolerate such high salt concentrations. We suspect that a false positive result occurred on our Mannitol Salt Agar plate. This could have been due to contamination from improper aseptic technique, or due to the concentration of salt in the MSA plate being too low. E. coli cells should dehydrate and should not survive the high concentration of salt in the MSA plate.

E. coli was able to grow on the MacConkey’s Agar plate. The media surrounding the organism appeared hot pink (FIGURE!!!!) Since E. coli is a Gram-negative organism, it is able to grow on MacConkey’s Agar. The crystal violet and bile salts that prevent Gram-positive organisms such as S. epidermidis from growing on the media, do not inhibit the growth of Gram-negative organisms such as E. coli. This is due to the outer membrane of the cell wall in Gram-negative organisms, which is fairly resistant to bile. It provides a protection for the inner cytoplasmic membrane, which is highly sensitive to bile (Neidhardt et al. 1987). The pink color indicates lactose fermentation. E. coli is a known excellent lactose fermenter. This color change is due to acid accumulating from the fermentation, changing the neutral red pH indicator to a red/pink color. This indicates that the pH is below 6.8.

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