final senior research paper

Matching Bite Mark Bacterial DNA to the Mouth of the Biter and Matching Dental

Impressions to Bite Marks Left on the Body

Brittany Cottingham

Abstract

The goal of this study was to investigate the possibility of matching oral bacterial DNA

from a fresh human bite mark to the oral bacterial DNA of the biter. When little to no evidence is

left at a crime scene, this protocol could provide a new insight into crime scene investigation. In

this experiment, bacterial samples were collected from 5 volunteers. They were instructed to bite

their biceps and the bite marks were then swabbed after the bite, after an hour had passed, and

again 12 hours later. All swabs were plated, then bacterial DNA was extracted from each sample

and PCR was run using primers designed to identify the bacterial species. The bacteria were not

conclusively identified, so the mouth band bite mark bacterial DNA could not be conclusively

matched. Each participant also had dental impressions made, which were used to compare and

match each volunteer’s impression to the picture of their bite mark. Out of the five dental

impressions, only one was able to be matched correctly. This study provides evidence that with

more time, microbial analysis of bite marks may be a promising tool used in forensics.

Introduction

Assault cases are being dismissed if there is little to no human DNA or if the victim

cannot identify their attacker (FBI, 2010). Cases in which bite marks are present can yield

desirable results in catching the perpetrator. One of the most famous cases in history that used

bite mark evidence was the Ted Bundy case. A main component in this conviction was the

examination and analysis of a bite mark he left on one of his victims. The odontologist for the

case was able to match his dental records to a photo of the bite marks found on the victim

(Bundy vs. State 1984).

Although bite mark analysis can be a useful tool, problems can occur when a bite mark is

left on a body. One of the major problems that can occur is that the bite mark can be distorted by

the movement of the dentition, but also because the skin is a poor material used for impressions

(Rothwell 1995). There are a set of characteristics that forensic odontologists consider when

examining bite marks. These characteristics include the presence or absence of each tooth, shape

of the teeth, and any abnormalities, such as fractured teeth (Glass et al 1980). The problem is that

any distortions in the bite mark, such as sliding the teeth down the skin, can mask any of the

characteristics, allowing for a false ID. Bite mark distortion is common, so to prevent a wrong

conviction, oral bacterial DNA may be used as evidence in cases which are otherwise thought to

have no evidence. The enzymes in saliva take longer to deteriorate the bacteria left behind

because it takes longer to digest the cell wall (Rahimi, et al 2005). Human DNA lacks a cell wall,

so the enzymes found in saliva deteriorate the DNA much quicker. By collecting the oral bacteria

left behind on a bite mark and amplifying their bacterial DNA by means of Polymerase Chain

Reaction, it can be compared to oral bacteria from the mouth, and used as evidence (Rahimi, et al

2005).

Though oral bacteria’s greatest use will be in aiding in the identification of a suspect, it

can also be helpful in determining the age of the bite mark. There isn’t a specific amount of time

known for how long oral bacteria can survive on the skin before it is either outcompeted or the

saliva degrades the cell wall, but the amount of bacteria collected from the skin can give

scientists a better understanding of the bacterial survival rate. This is important to determine

because most women who have been assaulted won’t report it for up to 12 hours after the attack,

on average (Peipert and Domagalski 1994). In this experiment, very little bacteria were found

after 12 hours, however, one study (Rahimi et al 2005) had a group of participants bite

themselves, and then waited 24 hours to gather the bacteria. Their results showed that even after

24 hours had past, oral bacteria from the biter were still able to be gathered from the marks. As

stated in similar studies, Streptococcus is the most prevalent genus found in the human mouth

(Rahimi 2005). Using this information, both Rahimi et al. (2005) and Kennedy (2011) swabbed

the bacteria that was collected from bite marks on a plate and allowed it to grow. Without

identifying the genus of the bacteria, they proceeded to perform PCR to amplify the bacterial

DNA. Next they ran gel electrophoresis, which showed the DNA profile needed to match the

samples. By comparing the bacteria from the mouths of the participants to the unknown bite

mark, they were able to correctly match the bacterial DNA from the unknown bite mark to the

bacterial DNA from the mouth of the biter (Rahimi et al. 2005)).

In order for PCR to be successful, primers are required. Primers act as starting points in

the amplification of bacterial DNA, which is then visualized through the bands that appear

during gel electophoresis. In an experiment performed by Chen et al. (2007), the primers

Sm479F and Sm479R were used. This primer pair is typically species specific for Streptococcus

mutans. S. mutans is the major bacteria species found in oral cavities. The purpose of Chen’s

(2007) experiment was to research S. mutans effect on dental caries and used the primer pair to

identify the bacteria. Using the primer pair Sm479F/R, Chen (2007) was able to show that the

primer pair was species specicific to S. mutans by identifying the species from a mixed sample.

Using this as a reference, bacteria was collected from the mouths of five volunteers and

from their bite marks after they were directed to bite themselves, one hour after, and 12 hours

afterward. DNA was extracted using a protocol specific for Streptococcal species. PCR was used

to amplify specific regions of Streptococcus mutans DNA and the DNA was then ran using gel

electrophoresis. No PCR products appeared, though DNA was clearly present in the gel. If more

time were allowed, the bacteria would have been identified and S. mutans would have been

specifically isolated. The primer pair Sm479F/R would have been used to identify S. mutans and

another primer would have been used to amplify the bacterial DNA and the bands would have

made a DNA profile that could have be used to match the DNA from the bite mark to the mouth.

When comparing the dental impressions to the bite marks, looking for the aforementioned

characteristics, distortions in the bite marks caused problems in matching the impressions to the

bite mark in all but one case. Overall, a new and an old method were attempted to match a bite

mark to the biter.

Materials and Methods

Before beginning the experiment, an IRB form was submitted, explaining the use of the

human subjects and the task they had to accomplish. It took about two months to get approved

and the experiment was allowed to begin. A blind study was performed using five volunteers.

Each volunteer signed a consent form stating that they understood any dangers involved in

participating in this study. For the purpose of the experiment, each volunteer bit themselves on

the bicep hard enough to leave an impression that lasted roughly 10 minutes. The section of the

bicep that was bitten was degermed with an alcohol wipe prior to biting so the bacteria collected

were strictly from the mouth. Photos of the bite mark were taken for comparison purposes. The

incisors and canines, the teeth that most prominently leave marks on the skin, were swabbed with

a sterile cotton swab, along with the saliva, for bacteria. The participants’ skin was swabbed after

the bite, one hour after the bite, and again 12 hours after the bite occurred with a sterile cotton

swab.

After all of the samples had been collected, the assistant randomly assigned each piece of

evidence a number, 1-5, each bite mark photo a number, 6-10, and each swab from mouth and

teeth, 21-40. The code was unknown to the experimenter.

Each of the collected samples was swabbed onto Nutrient Agar (Carolina, Burlington, NC)

and the plates were placed in an incubator for 48 hours at 37°C. After the 48 hours, the different

types of bacterial colonies grown were counted and gram stained to determine whether the

colony was gram positive or negative. Every colony was then grown in its own tube of nutrient

broth, which was also incubated for 48 hours at 37°C. After the 48 hours, a sample total of 5mL

of bacteria was taken from the tubes originating from the same plate and were mixed together,

combining all bacterial species (for example, plate 7 had five bacterial samples growing on it.

1mL was taken from each of the five tubes to make a total of 5mL of bacteria from plate 7). All

plates and tubes were placed at 4°C for storage.

Bacterial DNA Extraction

Sigma GenElute Bacterial Genomic DNA Kit (St. Louis, MO) was used to extract

bacterial DNA with the addition of Lysozyme from chicken egg white. A lysozyme solution was

made by diluting 2,115,000U/mL of lysozyme in 10mL of Gram-Positive Lysis Solution, making

a lysozyme solution stock of 4200µL. In order to extract the bacterial DNA, 1.5mL of bacterial

broth was pelleted by centrifuging for 2 minutes. The pellet was then resuspended by adding

200µL of Lysozyme Solution and then incubated at 37°C for 30 minutes. After 30 minutes, 20µL

of Proteinase K solution was added to the suspension, followed by 200µL of Lysis Solution C.

The sample was vortexed for 15 seconds and then incubated at 55°C for 10 minutes. This created

a lysate and was done for all 20 tubes. After 10 minutes of incubation, 500µL of Column

Preparation Solution was added to each binding column assembled within each tube. Each tube

containing this solution was centrifuged for 1 minute. 200µL were then added to the lysate and

vortexed for 10 seconds. All the contents in the tube were transferred into a clean binding

column tube, centrifuged for 1 minute at 12,000 X g, and the binding column was placed in a

new tube. 500µL of Wash Solution 1 was added to the column, centrifuged for 1 minute at 6500

X g, and the binding column was placed in a new tube. 500µL of Wash Solution was then added

to the tube, centrifuged for 3 minutes at maximum speed, and then placed in a new tube. 200µL

of Elution Buffer was added to the center of the column and centrifuged for 1 minute at

maximum speed in order to elute the bacterial DNA and the extraction was complete. This

protocol was specific for Streptococcus, which is why the primers used were for S. mutans. To

determine the amount of bacterial DNA extracted from each sample, 5µL of bacterial DNA and

45µL of water was measured using the spectrophotometer (BioRad, Hercules, CA).

Polymerase Chain Reaction

The primer pair Sm479F: 5` TCGCGAAAAAGATAAACAAACA-3`and Sm479R: 5`

GCCCCTTCACAGTTGGTTAG-3` were used. Sm479F/R is species specific for Streptococcus

mutans, meaning bands will only be produced if S. mutans is present in the bite mark.. S. mutans

is the major pathogen found in the oral cavity and the primers amplify specific regions in the 16S

rRNA genes, which are unique in S. mutans compared to other species of Streptococcus (Chen et

al 2007). In a 2mL tube, 10µL of DNA, 1µL Mg (25mM), 1µL Sm479F (10pmol/µL), 1µL

Sm479R (10pmol/µL), 0.6µL dNTP (10mM each), and 2.5µL 1X buffer were all added together.

This was done for all 20 tubes of extracted bacterial DNA. In an icycler (Bio-Rad, Hercules,CA),

the PCR was performed as follows: 95°C for 2 minutes, then 0.2µL Taq was added to each tube;

it was then followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds,72°C for 1

minute, and then 5 minutes at 72°C. The PCR amplified bacterial DNA was then placed in a 1%

agarose gel in TBE buffer and stained with 4µL of ethidium bromide (0.5µg/mL). All the

samples taken from the mouth and saliva were placed on one gel, swabs taken directly after the

bite were placed on a separate gel, swabs taken 1 hour after the bite were placed on a separate

gel, and swabs taken 12 hours after the bite were placed on a separate gel, creating a total of 4

gels. The gels were ran at 200 volts for 5 minutes, then reduced to 90 volts for 45 minutes. PCR

was ran 3 times previous to the final end products. The gel photos were documented by digital

camera.

Dental Impressions

All 5 volunteers were taken to Dr. Todd at C Clark Todd Dental Office in Canton, MO.

The volunteers were fitted for a metal maxillary impression plate. Putty, provided by the dentist,

was mixed and poured into the plate. The plate was placed in the mouth of the volunteer and

pushed against their top teeth until the putty hardened. This was repeated with the bottom set of

teeth and for each volunteer. Once all molds were made, they were left to dry for 30 minutes.

After 30 minutes, a plaster was poured into the molds to create a stronger mold of the teeth sets.

The new molds were left to dry for 30 minutes. The molds were used as a comparison to the bite

mark photos.

Results

The volunteers were asked to bite their bicep in order to collect bacterial samples. The

bite marks were swabbed directly after the bite, 1 hour after the bite, and 12 hours after the bite.

Photographs were taken (figure 1).

A. B. C.

D. E.

The bacteria from the bite marks were plated and incubated. Figure 2 shows the average

bacterial samples. On average, 3 colonies were found on each plate. They ranged from white to

yellow in color and the morphology was mostly coccus with few spirillum. As clearly depicted in

figure 2, the amount of bacteria found on the skin decreases as the time increases, with the plate

almost completely covered in the initial swab of the bitemark to very colonies found after 12

hours. The different colonies found were gram stained to determine whether the bacteria were

gram-positive or gram-negative. The results from the gram stain are shown in Table 1. The

results are 7 out of 40 colonies were gram-negative and 2 out of 40 were spirillum in shape.

Streptococcus is typically gram-positive and cocci in shape, which gives the possibility that the

majority of the bacteria found were species of Streptococcus, which is what this experiment was

specific for.

Figure 1. A-E are photographs of the bite marks on the five volunteers. Each was swabbed for bacteria at 3 time intervals.

A. B. C.

D.

Plate the colony was found on

and letter representing it

Gram-positive or Gram-

negative

Shape

Initial Bite Mark:

1A Gram positive Coccus

1B Gram positive Coccus




4B Gram negative Coccus

4C Gram positive Coccus

11 Gram positive Coccus

16A Gram negative Coccus


Figure 2. 1A represents the average plate of bacteria swabbed from the mouth. 1B is the average of bacteria swabbed from the initial bitemark. 1C is the average of bacteria swabbed from the bitemark after 1 hour. 1D is the average of bacteria swabbed from the bitemark after 12 hours.

1 hour Swab:

6 Gram negative Coccus



7C Gram negative Coccus

7D Gram positive Spirillum

7E Gram positive Coccus






12 hour Swab:



8A Gram positive Spirillum


8C Gram positive Coccus





Mouth:





19A Gram negative Coccus






Bacterial DNA Extraction

The bacterial DNA was extracted following the Streptococcus specific protocol from the

Sigma GenElute Bacterial Genomic DNA kit (St. Louis, MO). Once the bacterial DNA from

each plate was extracted, it was analyzed with the spectrophotometer to quantify the isolated

DNA. There was no correlation between a large quantity of DNA read by the machine and the

presence of bands on the gel. The most DNA in a sample was 1981.9/µL and the least amount in

a sample was 4.2775ng/µL

Tube number Machine reading (ng/mL) Actual amount (ng/µL)

1 2.4717 61.7925

2 1.0325 25.8125

3 72.5055 1812.6375

4 0.7220 18.05

5 0.2715 6.7875

Table 1. Gram stain results for the bacteria colonies found on the plates and bacterium morphology.

6 1.0623 26.5575

7 1.8288 45.72

8 2.9382 74.58

9 1.2495 31.2375

10 79.2760 1981.9

11 1.8985 47.4625

12 2.4956 62.39

14 0.3941 9.8525

15 0.1926 4.8150

16 5.5345 138.3625

17 0.9063 22.6575

18 0.1711 4.2775

19 0.4811 12.0275

20 0.4158 10.3950

22 5.0632 126.5800

Polymerase Chain Reaction

A. B.

Table 2. Results from the spectrophotometer, determining the amount of bacterial DNA sample from each plate.

C. D.

The Polymerase Chain Reaction was performed 3 times previous to the last. In the first 3

attempts, no bacterial DNA was amplified. In the last trial, all bacterial reserves were used,

which was a larger amount of DNA used previously. Figure 3A was from the initial bite mark

swab. The size of the band in the figure was greater than 10kb, which is too large to be specific

fragments of bacterial DNA. The bands were genomic DNA, which means that DNA was

isolated and that there was a large quantity, but the PCR did not work. No PCR products were

collected in this experiment.

Dental Impressions

A. B.

Figure 3. Gels resulting from gel electrophoresis. A is from the initial swab of the bitemark. B is the swab after an hour. C is the swab after 12 hours. D is the swab from the mouth.

C. D.

E.

The dental impressions were matched to the bite marks based on characteristics in the dentition.

These characteristics include the shape of the teeth, the presence or absence of teeth, and any

abnormalities. Based on these characteristics, only one set was able to be matched with the

bitemark. The bottom half of the bitemark shown in Figure 5D was made by the upper teeth (Fig.

5C). The curvature of the two front teeth on the impression matches the curvature in the

bitemark. It can also be matched by the unique structure on the bottom row of teeth. One tooth

on the bottom set is set further back than the other teeth, which is clearly shown in the figure.

The rest of the bite marks were too distorted to correctly match.

Figure 4. A-E are the dental impressions from each volunteer had made.

A. B.

C. D.

Conclusion

Overall, this project attempted to compare two methods to match a biter to their bite

mark. Multiple methods can be useful for future forensic studies and to have more accurate

results in crime cases.

In previous research, Rahimi (2005) yielded positive results, with 100% matching, when

matching the bacterial DNA from a bite mark to the mouth of the biter during the experiment.

The primer pairs used in that experiment was the OPA 02 and OPA13 pair. In terms of this

experiment, the results weren’t as adequate. The primer used was Sm479F/R. This primer is said

to be species specific for Streptococcus mutans. When tested in a study, 10 S. mutans strains and

10 non-S. mutans strains were tested and only S. mutans strains were amplified (Chen et al

Figure 5. Matching the bitemark to the dental impressions.

2007). However, it was unknown during this experiment that Sm479F/R was a primer pair used

to identify a specific bacterial species (S. mutans) and another primer pair was used to actually

amplify the bacterial DNA. When gel electrophoresis was performed, the gel didn’t produce

bands because no PCR product was produced. The band shown was genomic DNA, meaning that

bacterial DNA was extracted; however, it wasn’t amplified in a specific region. Without detailed

results from PCR and gel electrophoresis, the bacterial DNA from each source was unable to be

linked back to the person it originated from.

The second aim of this experiment was to match dental impressions of each volunteer to

the bite mark photos. With today’s technology and training, forensic odontologists are able to

correctly match teeth to bite marks, though no actual percentage has been calculated (Pretty and

Sweet 2001). In order to successfully match them, odontologists look for unique teeth markers,

use dental records, x- rays, and any other tool they deem beneficial. The bite marks obtained

during this experiment were too distorted to match, except for one bite mark, which was able to

be match to the impression because of a notable tooth variation.

For future research, it would be beneficial to determine the various species of bacteria

grown on the agar plates. This would give the researcher a specific bacterium to run PCR and gel

electrophoresis on, leading to the possible correct identification of the biter. It would also narrow

down the best primer to use in order to amplify the bacterial DNA. In order to match the

impressions, better photos would be needed, along with more training in the field of odontology.

With a more in depth protocol, the research on this topic may prove beneficial in its purposes.

This protocol would open a new door in forensics in terms of solving cases with no DNA other

than bacterial DNA found in saliva. It would also be beneficial because there are few ethical

issues that could be brought up, mainly because it is only bacterial DNA being used, not human

DNA.

Acknowledgments

I would like to thank all my volunteers for donating their time, Dr. LaFerriere for going

over every little thing throughout the whole process and for making sure everything was on

track, all the other science faculty at Culver-Stockton College, Laken Workman for assisting this

experiment, Michelle Hall for helping when needed, and Dr. Todd for donating all the dental

equipment needed.

References

Bundy v. State 455 So 2d 330 (Fla. 1984): 349.

Chen, Z., Saxena, D., Caufield, P.W., Ge, Y., Wang, M., et al (2007). Development of species- specific primers for detection of Streptococcus mutans in mixed bacterial samples. FEMS Microbial Lett., 272, 154-162.

Glass, R. T., Andrews, E. E., and Jones, K. (1980). Bite mark evidence: a case report using accepted and new techniques. Journal of Forensic Sciences, JFSCA, 25, 638-645.

Kennedy, D. (2011) Forensic dentistry and microbial analysis of bite marks. APJ, 6-15.

Peipert, J.F. and Domagalski, L.R. (1994) Epidemiology of adolescent sexual assault. Obstet Gynecol 84, 867–871.

Pretty, I. A., and D. Sweet. "A Look at Forensic Dentistry — Part 1: The Role of Teeth in the Determination of Human Identity." British Dental Journal 190.7 (2001): 359-66. Web. 10 Dec. 2012.

Rahimi, M., Heng, N.C.K., Kieser, J.A. and Tompkins, G.R. (2005) Genotypic comparison of bacteria recovered from human bite marks and teeth using arbitrarily primed PCR. Journal of Applied Microbiology, 99, 1265-1270.

Rothwell, B.R. (1995) Bite marks in forensic dentistry: a review of legal, scientific issues. J Am Dent Assoc 126, 223–232.

U.S. Department of Justice, FBI (2010). Preliminary semiannual uniform crime report, january-june, 2011.

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