THE CULTURING OF STAPHYLOCOCCUS EPIDERMIDIS AND STREPTOCOCCUS

SALIVARIUS WITH ESCHERICHIA COLI DOES NOT INCREASE ANTIBIOTIC

RESISTANCE IN ESCHERICHIA COLI

Sarah Weber

Bio 4211

3 March 2013

ABSTRACT

Antibiotic resistance in bacteria is developed over many generations with the

bacteria building up a resistance until completely desensitized to the antibiotic. This

can occur slowly over time or by the combination of two different bacteria with

different resistances. This process could create a new strain that could be resistant

to both antibiotics to which the parents were separately resistant. Whether the

combined treatment strains of the two bacteria or the parent stain of Escherichia

coli acquired resistance to one of the three antibiotics used faster was clear. This

study investigated the effect of culturing E. coli with Streptococcus salivarius or

Staphylococcus epidermidis (treatments) versus E. coli alone on the rate of

desensitization to one of three antibiotics: penicillin, streptomycin, and

erythromycin. Over the subsequent generations, de-sensitivity to erythromycin

increased significantly from having no resistance in the first generation to being

fully resistant to erythromycin by the end of the experiment. A significant increase

was seen in the resistance to penicillin and streptomycin for both the treatments as

well as the control. This data does not support the hypothesis that the antibiotic

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resistance in the E. coli with S. epidermidis and E. coli with S. salivarius would

increase faster than in the E. coli.

INTRODUCTION

The broad use of antibiotics exposes many different bacteria strains to various

antibiotics. As the number of exposures increases, the resistance of the bacteria to the

antibiotic would increase because the bacteria would begin to mutate, making it less

affected by the antibiotic.

According to the CDC, “These [antibiotic] drugs have been used so widely and for

so long that the infectious organisms the antibiotics are designed to kill have adapted to

them, making the drugs less effective. People infected with antimicrobial-resistant

organisms are more likely to have longer, more expensive hospital stays, and may be more

likely to die as a result of the infection.”

As this occurred many of the antibiotics that were used as broad spectrum

antibiotics would become ineffective and would no longer treat the various bacterial

infections that are currently treated. Ochman et al. (2000) found that incorporating

antibiotic resistance genes allowed bacteria to expand their ecological niche and grow in

the presence of particular toxic compounds. This implies that as the bacteria become

resistant to more antibiotics, they are able to reproduce in new environments that they

could not previously, allowing the bacteria to influence more organisms.

Neu (1992) found that bacteria are now more resistant to antimicrobial agents due

to chromosomal changes and recombination processes. Resistance can be measured by the

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zones of inhibition that occur when bacteria is plated with a disk of the antibiotic. After

growth the zone of inhibition would either form an area around the disk that is clear from

bacteria or the bacteria would grow around the disk indicating that it would not work as a

treatment.

DeBoy II et al. (1980) found that resistances to particular antibiotics happened

more often, secondary resistance patterns that were often combined or coexisted in

Escherichia coli to make tertiary drug resistance patterns, allowing for multiple drug

resistances to form. This research investigated the effectiveness of antibiotics on the

bacteria used. Okeke et al. (2000) found that as the level of bacteria not resistant to the

antibiotic decreases, the level of bacteria resistant to the antibiotic increases.

Bacteria interact in communities in their natural environment. This allows hundreds

of different strains of bacteria to interact with one another and exchange genetic

information. The exchange of genetic information could be due to the presence of a

compound toxic to some of the bacteria but not others. Some toxic compounds induce

mutations, but many do not. Usually it is random mutations caused by the toxic compound

inducing selection for the randomly mutated bacterial cells. This toxic compound will

induce mutations causing selection of those mutant cells to further propagate while those

not mutated would die off. As genetic information is exchanged between different strains,

greater diversity will arise allowing the bacteria to expand their ecological niche.

Many of the bacteria used for this study are commonly found on or in the human

body, as a natural requirement to maintain homeostasis. E. coli is a member of a large and

diverse group of gram negative rod-shaped bacteria. Most of the strains are harmless, but

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some are pathogenic and can cause diseases. It is normally found in the intestines of

humans and animals. In humans E. coli functions to prevent the establishment of hostile

bacteria and aids in digestion. The bacterium, Staphylococcus epidermidis, is a gram

positive spherical shaped bacterium that forms in grape-like clusters. Similar to E. coli

some of the strains are harmless, while others can cause diseases. S. epidermidis is a found

on the skin of humans as a part of the normal flora. This bacterium has been found to be

the leading cause of nosocomial infections due to the biofilm it produces when colonizing

on biomaterials implanted in the human body or instruments. In comparison to S.

epidermidis, Streptococcus salivarius is a gram positive spherical shaped bacterium. S.

salivarius is the principal commensal bacterium of the human oral cavity. It is the pioneer

in colonizing dental plaque, allowing additional bacteria to associate with it to form

cavities. If these bacteria were to enter the blood stream it would cause disease. All the

bacteria used in this study are commonly found on the human body, thus there is a chance

of them intermingling.

The antibiotics used in this study were penicillin, streptomycin, and erythromycin.

Penicillin was one of the earliest discovered and now most widely used antibiotics. It was

synthesized by mold that grew on cheese. It is used to treat many different types of

infections caused by bacteria. Penicillin kills bacteria by interfering with the ability of the

cell to synthesize the cell wall. This causes a weakening in the structure of the cell that

eventually will kill it off or prevent it from growing. Erythromycin is a macrolide

antibiotic that is used to treat many different types of bacterial infections. It functions by

slowing the growth, sometimes killing, sensitive bacteria. This occurs by the antibiotic

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reducing the production of important proteins needed by the bacteria to grow and survive.

Streptomycin is an aminoglycoside antibiotic that is used to treat many different kinds of

bacterial infections. It prevents growth of bacteria by inhibition of protein synthesis. This

antibiotic cannot be taken orally as the previous two mentioned can, it must be injected

regularly intramuscularly until treatment with it is finished. All of the antibiotics used in

the study are considered broad-spectrum, meaning they are used to treat a variety of

different infections. For this reason they were selected and used.

While antibiotic testing has previously been done, this study looked at the changes

in antibiotic resistance in response to different combinations of bacteria. The aim of the

research was to study the rate at which bacterial resistance occurred in a pure culture

versus a mixed culture.

The potential value of this research was to determine if the use of antibiotics

should be more limited rather than used for broad spectrum treatment for a multitude of

bacterial diseases. The results would show whether the bacterial mutations were occurring

at a fast pace implying that new antibiotics need to be found as soon as possible or if there

is time to create and/or discover new antibiotics. I hypothesize that the antibiotic

resistance in the E. coli with S. epidermidis and E. coli with S. salivarius would increase

faster than in the E. coli.

METHODS

Antibiotic effectiveness was determined by the disk diffusion assay. The zone of

inhibition was the area of clearance that was found around the individual disks of

antibiotics. The larger the zone size the more effective the antibiotic on the bacteria. The

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zone of inhibition was measured in radius and diameter. The radius was measured from the

edge of the antibiotic disk to the edge of clearance. The diameter was measured from the

edge of clearance to the opposite edge of clearance. The size of the zone of inhibition

showed whether the strains of S. epidermidis and S. salivarius mixed into the E. coli by

decreasing in size in comparison to the parent zone size. The three antibiotics used were:

penicillin (10 IE), erythromycin (15 µg), and streptomycin ((10 µg). One disk each of

penicillin, erythromycin, and streptomycin antibiotics were placed on each blood agar plate

using an automated dispenser. Thus the placement of the antibiotics on the plates was

random.

In the first week bacterial colonies of S. epidermidis and S. salivarius were mixed

with E. coli in tryptic soy nutrient broth. Tube one contained only the E. coli and acted as

the control. Tube two contained the E. coli plus S. epidermidis only. Tube three contained

the E. coli plus S. salivarius only. The mixtures from each tube were tested on tryptic soy

with 5 percent blood agar plates with disks of antibiotics to test for a clear zone of

inhibition. In week two the mixtures from each tube, were plated on blood agar plates. On

each of the three blood agar plates one antibiotic disk each of penicillin, streptomycin, and

erythromycin were placed. The plates were incubated for two days at 37 °C. Colonies from

around each of the antibiotic disks were collected from the three different plates and

cultured in tryptic soy broth to grow a new generation. The cultures were grown for one

day at 37 °C. The cultures were plated and tested with disks of antibiotic. This procedure

was repeated for every subsequent generation’s cultures.

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The research was carried out from late August 2012 through November 2012 over a

period of ten weeks. An ANOVA (General Linear Model) analysis was performed using

Minitab-16 software. An analysis of the data collected throughout the research was

performed using an analysis of variance that analyzed the diameter of zones of inhibition

versus antibiotics and generations.

RESULTS

The data for the diameter of the zone of inhibition of the parent strains to the three

different antibiotics used was shown in Figure 1. This figure showed that E. coli alone was

least sensitive to erythromycin of all the strains used; was most susceptible to streptomycin

and penicillin. The individual strain of S. salivarius was most susceptible to erythromycin

and penicillin. S. salivarius was the least susceptible to streptomycin of the three strains

used. In contrast to the other two strains, S. epidermidis showed no susceptibility to

erythromycin. In addition, S. epidermidis was the most susceptible to streptomycin and the

least susceptible to penicillin. The data for the diameter of the zone of inhibition for the

control of E. coli alone over the course of the entire study was shown in Figure 2. In the

control by the first generation, the bacteria had already mutated and lost the slight

susceptibility to erythromycin. The data for the diameter of the zone of inhibition for the

treatment of E. coli with S. salivarius was shown in Figure 3. This treatment lost

sensitivity to erythromycin first, which showed recombination did occur as S. salivarius on

its own, was the most sensitive to erythromycin. For the treatment of E. coli with S.

epidermidis the data for the diameter of the zone of inhibition over the entire study was

shown in Figure 4. Erythromycin sensitivity lasted for four generations, until the fifth

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generation. Both treatments and the control showed a significant decrease in the zone of

inhibition over time. As the number of generations increased the zone of inhibition

decreased.

The analysis showed a significant difference in the three antibiotics and generations

during the time the research was performed with a p-value of 0.000 (Appendix I). No

significant difference was found in the treatment, culturing E. coli with one of the other

bacteria, with a p-value of 0.140 (Appendix I). There was a significant difference found

between penicillin and streptomycin. There was not a significant difference between

streptomycin and erythromycin, and erythromycin and penicillin (Table 1). There was a

significant difference between generations 1, 3-17 (Table 2). There was not a significant

difference between generations 1-2, 2-5, 3-12 and 14-17, and 4-17 (Table 2). The

decreasing trend of the zone of inhibition over time is represented in Figures 2-4.

Table 1: Duncan’s Means Separation for the diameter of the zone of inhibition for all three

treatments combined at the end of research. Means with the same superscript are not

significantly different from each other.

Antibiotic MeanStreptomycin 0.4371a

Erythromycin 0.3400a,b

Penicillin 0.2961b

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Table 2: Duncan’s Means Separation for the diameter of the zone of inhibition for all three

treatments combined generations of the research. Means with the same superscript are not

significantly different from each other.

Generation Mean

1 0.5875a

2 0.4714a,b

3 0.4286b,c

4 0.3857b,c,d

5 0.3667b,c,d

6 0.3333c,d

7 0.30002c,d

8 0.3167c,d

9 0.3833c,d

10 0.3167c,d

11 0.3167c,d

12 0.3000c,d

13 0.2833d

14 0.3000c,d

15 0.3500c,d

16 0.3500c,d

17 0.3167c,d

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E. coli S. salivarius S. epidermidis0

0.5

1

1.5

2

2.5

3

3.5

4

Diameter of the Zone of Inhibition for the Parent Generation

PenicillinErythromycinStreptomycin

Parent Generation

Zone

of I

nhib

ition

(mm

)

Figure 1: The diameter of the zone of inhibition in the parent generation of E. coli, S.

epidermidis, and S. salivarius plated with the three antibiotics: streptomycin,

erythromycin, and penicillin, to establish a base line of sensitivity for each of the

individual species to the antibiotics used.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

0.2

0.4

0.6

0.8

1

1.2

1.4

Diameters of the Zone of Inhibition for E. coli Control

PenicillinStreptomycin

Generations

Zone

of I

nhib

ition

(mm

)

Figure 2: The diameter of the zone of inhibition in the control for E. coli plated with the

two antibiotics: streptomycin and penicillin, over the different generations. By the first

generation E. coli showed no sensitivity to erythromycin.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Diameters of the Zone of Inhibition for S. salivarius and E. coli

PenicillinErythromycinStreptomycin

Generations

Zone

of I

nhib

ition

(mm

)

Figure 3: The diameter of the zone of inhibition in the treatment for E. coli plus S.

salivarius plated with the three antibiotics: streptomycin, erythromycin, and penicillin,

over the different generations.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

0.5

1

1.5

2

2.5

Diameters of the Zone of Inhibition for S. epidermidis and E.coli

PenicillinErythromycinStreptomycin

Generations

Zone

of I

nhib

ition

(mm

)

Figure 4: The diameter of the zone of inhibition in the treatment for E. coli plus S.

epidermidis plated with the three antibiotics: streptomycin, erythromycin, and penicillin,

over the different generations.

DISCUSSION

The hypothesis stated that the E. coli antibiotic resistance when treated with other

bacteria would increase faster than the control of E. coli alone. The results of the research

did not support the tested hypothesis. My experimental results showed that mixtures of S.

salivarius with E. coli and of S. epidermidis with E. coli did not gain antibiotic resistance

faster than E.coli alone. Out of the two treatments, the S. salivarius plus E. coli developed

resistance to erythromycin the quickest. E. coli alone had already mutated by the first

generation and was resistant to erythromycin. This is supported by the findings of Okeke et

al. (2000) who showed that E. coli can rapidly develop resistance to an antibiotic on its

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own. In contrast, S. epidermidis and E.coli took four generations to become completely

desensitized to erythromycin which was surprising as S. epidermidis on its own was

resistant to erythromycin. This could have been due to non-mixed colonies growing around

the erythromycin disk. Another possible explanation could be that a pure colony of non-

mutated E.coli grew around the erythromycin.

For antibiotics used, erythromycin was the only one to which the treatments and the

control all became completely desensitized (see Figures 2-4). The sensitivity of the control

and the treatments to the penicillin and streptomycin slowly decreased over the generations

but did not become completely desensitized to either antibiotic. At the eleventh generation

an increase of a tenth of a millimeter occurred, but at the next growth decreased by a tenth

of a millimeter. By the fourth generation no significant difference was found between all of

the following generations up to the final generation. The data was supported by Ochman et

al. (2000) as lateral gene transfer of antibiotic resistance between the bacteria was a

possible explanation of the increase in resistance to the antibiotic.

The slow rate at which the antibiotic resistance occurred implies that antibiotics are

not as effective at treating bacterial infections as when initially introduced. This slow trend

of decreasing effectiveness of an antibiotic is reflected in Figures 2-4. The decrease further

supports the idea that while antibiotics may not be as effective as when first introduced,

they still are effective against bacteria.

Possible limitations that exist with this research are that the DNA from S.

epidermidis and S. salivarius and the E. coli DNA would not combine when grown

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together causing separate colonies of both to form on the same plate, with some

combinations.

The broad-spectrum antibiotics used in this study were still effective on the bacteria

used. The effectiveness of these antibiotics decreased as the time of the study progressed.

While there is not a pressing need for new antibiotics to be made, new antibiotics should

be developed to replace those used in this study. The fact that the bacteria all showed less

sensitivity to the antibiotics means that people with poor immune systems would take

longer to heal from infections with these antibiotics and need stronger antibiotics or more

specific ones than those used in this research in order to compensate for an inadequate

immune system. People with compromised immune systems would need the additional

boost to help fight off infections as their bodies are more susceptible to infection. Reasons

for the immune system to be compromised could range from the system not being

developed yet in the case of infants; elderly people have chronic disease and declining

nutritional health; and other people who are immune compromised can be from birth

defects or diseases that attack the immune system. The need for new antibiotics, while not

immediate, is for elderly, infants, and those immune compromised people who do not have

a healthy immune system to assist the less effective antibiotics. This is supported by the

works of Otters et al. (2004), Hohenadel et al. (2001), and Bonomo (2000).

In conclusion, the results of my research show that antibiotics after multiple

generations are not as effective on bacteria as when first introduced. The results did not

support my hypothesis that two different bacteria grown together will become resistant to

antibiotics faster than one bacteria on its own. Significant differences were found for the

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development of resistance to antibiotics used in the research and in the generations over

time but not in the treatment of growing two different bacteria together (Appendix I). The

final results of the research showed that the antibiotics used today are adequate for a young

healthy person’s immune system; but, would possibly be insufficient in an immune

compromised individual. Further research that could be done based on this study could be

to change the bacteria used, change the antibiotics used, or to allow for more generations to

determine how many generations are needed for the bacteria to become completely

desensitized to the antibiotic.

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REFERENCES

Bonomo RA. 2000. Multiple Antibiotic- resistant Bacteria in Long-term-care Facilities: An

Emerging Problem in the Practice of Infectious Diseases. J Clin Infect Dis. 31(6): 1414-

1422.

[CDC] Centers for Disease Control and Prevention. 2011. Antibiotic/Antimicrobial Resistance

[Internet]. 2011. Atlanta (GA): Centers for Disease Control and Prevention (US); [updated

2011 Oct 4; cited 2012 Mar 2]. Available from:

ht tp://www.cdc.gov/drugresistance/index.html

E. coli [Internet]. 2013. Encyclopedia Britannica.; [cited 2013 Feb 10]. Available from:

http://www.britannica.com/EBchecked/topic/192351/E-coli

DeBoy II JM, Wachsmuth KI, Davis BR. 1980. Antibiotic Resistance in Enterotoxigenic and Non-

enterotoxigenic Escherichia coli. J Clin Microbiol. 12 (2): 264-270.

Hohenadel IA, Kiworr M, Genitsariotis R, Zeidler D, Lorenz J. 2001. Role of bronchoalveolar

lavage in immunocompromised patients with pneumonia treated with a broad spectrum

antibiotic and antifungal regimen. Thorax. 56(2): 115-120.

Neu HC. 1992. The Crisis in Antibiotic Resistance. Science. 257 (5073): 1064-1073.

Ochman H, Lawrence LG, Groisman EA. 2000. Lateral Gene Transfer and the Nature of Bacterial

Innovation. Nature. 405: 299-304.

Okeke IN, Fayinka ST, Lamikanra A. 2000. Antibiotic Resistance in Escherichia coli from

Nigerian Students, 1986-1998. Emerg Infect Dis. 2000-Aug 6 (4): 1-5.

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Otters HBM, van der Wouden JC, Schellevis FG, van Suijlekom-Smit LWA, Koes BW. 2004.

Trends in prescribing antibiotics for children in Dutch general practice. J Antimicrob

Chemother. 53(2): 361-366.

Ross-Flangian N. 2006. “Erythromycins” [Internet]. Gale Encyclopedia of Medicine, 3rd ed.; [cited

2012 Feb 10]. Available from: http://www.encyclopedia.com/doc/1G2-3451600598.html

Ross-Flangian N. 2006. “Penicillins” [Internet]. Gale Encyclopedia of Medicine, 3rd ed.; [cited

2012 Feb 10]. Available from: http://www.encyclopedia.com

Staphylococcus [Internet]. 2013. Encyclopedia Britannica.; [cited 2013 Feb 10]. Available from:

http://www.britannica.com/EBchecked/topic/563360/Staphylococcus

Streptococcus [Internet]. 2013. Encyclopedia Britannica.; [cited 2013 Feb 10]. Available from:

http://www.britannica.com/EBchecked/topic/568809/Streptococcus

Streptomycin [Internet]. 1997. Medical Discoveries; [cited 2013 Feb 10]. Available from:

http://www.encyclopedia.com

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APPENDIX I: ANOVA TABLE

Analysis of variance on the diameters of Zones of Inhibition versus Antibiotic, Treatment,

and Generation.

Source DF F P-valueAntibiotic 2 51.72 0.000Treatment 2 2.01 0.140Generation 16 8.07 0.000Error 86