Virtual Bacterial Identification Introduction

Welcome to the Virtual Bacterial Identification Lab. The purpose of the lab is to familiarize you
with the science and techniques used to identify different types of bacteria based on their DNA
sequence. Not long ago, DNA sequencing was a time-consuming, tedious process. With readily
available commercial equipment and kits, it is now routine. The techniques used in this lab are
applicable in a wide variety of settings, including scientific research and forensic labs.

Basic Steps

• Prepare a sample from a patient and isolate whole bacterial DNA.

• Make many copies of the desired piece of DNA.
• Sequence the DNA.
• Analyze the sequence and identify the bacteria.

The piece of DNA used for identifying bacteria is the region that codes for a small subunit of the
ribosomal RNA (16S rRNA). We will refer to this piece as 16S rDNA. Different bacterial species
have unique 16S rDNA sequences. The identification relies on matching the sequence from
your sample against a database of all known 16S rDNA sequences. (Learn more about
ribosomal RNA.)

Learning Objectives

• What kind of patient samples are used for the purpose of identifying possible
pathogens?
• What does PCR do, how does it work, and why is it useful?
• How do you separate the desired DNA from all others?
• How does an automatic DNA sequencer work?
• Why is it possible to use a DNA sequence to identify bacteria?

Throughout each exercise, this window will display information explaining what you are doing.
All the interactions, however, will be done inside the graphic window to the left. The small white
box below the graphic will give you specific instructions on what objects to click on.

Note: Ribosomal RNA

Ribosomes are the sites of protein synthesis in a cell. They are among the most fundamental
organelles and are found both in procaryotes and eucaryotes, albeit in slightly different form.
The cytoplasm of a procaryote contains tens of thousands of free-floating ribosomes.

Ribosomes are made up of both proteins and pieces of RNA. The ribosomes of procaryotes
measure 70S (S = Svedberg unit; it describes the sedimentation rate during centrifugation,
which depends on the size, weight, and shape of a particle). The 70S ribosome consists of two
subunits--50S and 30S--each containing a variety of proteins and ribosomal RNA (rRNA)
molecules. The smaller 30S subunit contains a 16S rRNA molecule and the larger 50S subunit
contains a 5S and a 23S rRNA molecule.

This lab describes a technique to identify bacteria by using the sequence of the gene that codes
for the 16S rRNA.

Part 1 - Sample Preparation

The key to a successful identification is to start with a "good" sample. Many pathogenic bacteria
do not grow well on solid culture medium, making identification by traditional means difficult.
(Why?) Even poorly growing bacterial cultures, however, can be identified with the methods
used in this lab.

In this lab, you will act as a pathologist or perhaps a pathology lab technician at a well-equipped
research hospital. Your task is to identify a bacterial sample received from a clinician.

Assuming that you have managed to grow bacterial colonies on a solid medium culture dish, the
first step is to pick up a single colony and drop it into a microcentrifuge tube.

The process of extracting bacterial DNA consists of dissolving the cell wall with a digestive
buffer (in the white-capped bottle) available as a commercial kit. The buffer contains proteolytic
enzymes that "eat" the cell wall. This step may take several hours.

Since we will be using other enzymes in the next step, we need to get rid of the proteolytic
enzymes before we can proceed. The enzymes are denatured by heating the sample in a water
bath at 100°C. Next, the cellular debris is spun down in the centrifuge and appears as a solid
deposit (pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid),
which is then transferred to the PCR tube.

Note: Limitations of the traditional methods of identification

Over the years, a battery of tests has been developed to categorize and identify bacteria. Tests
include staining and growing bacteria under a variety of conditions. Such procedures typically
require vigorously and reliably growing bacterial cultures.

Many pathogens grow poorly on solid medium while others grow only in liquid culture, making
identification through traditional techniques difficult or impossible. With the aid of molecular
methods, however, these limitations can be overcome.

In addition, some species of bacteria cannot be differentiated from closely related species
through traditional methods. For these species, molecular methods offer the only reliable and
convenient means of identification.

Part 2 - PCR Amplification

Click here to learn What is PCR?

Step 1: Add Master Mix

To prepare the polymerase chain reaction (PCR), we will add the PCR Master Mix solution to
our sample DNA. The PCR Master Mix solution (in the red-capped bottle) contains the following:
water; a buffer to keep the mixture at the correct pH for the PCR reaction; large quantities of the
four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide
DNA primers that bind the 16S rDNA region to initiate the replication process (What are
primers?); and a heat-stable DNA polymerase that extends the copy DNA strand.

At the same time as the test reaction, we will prepare negative and positive control reactions.
Instead of the sample DNA, the positive control reaction contains positive control DNA (the
solution of 16S rDNA in the green-capped bottle) while the negative control reaction contains
sterile deionized water. Both reactions contain the PCR Master Mix solution.

Step 2: Run PCR

Once the reaction tubes are loaded onto the thermocycler (the "PCR machine"), the automatic
process of DNA replication starts (refer to the animation). The machine used in this lab has
readouts that describe what is happening:

(from left to right: temperature, time remaining, cycle number, melt, anneal, and extend)

The temperature control is set up as follows:

Initial incubation step - 95°C for 10 minutes
30 cycles of the following sequence of steps:
 o Melt - 95°C 30 seconds
o Anneal - 60°C 30 seconds
o Extend - 72°C 45 seconds

Final extension Step - 72°C 10 minutes

Final step - 4°C store at this temperature
During each cycle, the first step (melt) is to separate the two DNA chains in the double helix by
heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. The primers
cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this
temperature, the primers bind (anneal) to the single-stranded DNA. (The reason the two
separated strands of DNA do not reanneal is that there is a large excess of primers in the
solution; therefore, it's more likely for the DNA strands to bind to the primers instead of to each
other.) The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand
by raising the temperature to 70°C for 45 seconds.

The three steps—the separation of the strands, annealing the primer to the template, and the
synthesis of new strands—take less than two minutes. Each is carried out in the same vial. At
the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be
repeated 30 or more times, and each newly synthesized DNA piece acts as a new template.
After 30 cycles, 1million copies of the initial DNA piece can be produced.

Note: What is PCR?

PCR (Polymerase Chain Reaction) is a technique that allows many copies of DNA to be made.
It is one of the cornerstones of the current revolution in molecular genetics. Before the advent of
PCR, making sufficient copies of DNA for sequencing was a tedious process. Now, PCR makes
obtaining copies of DNA from a small original sample routine, with far-reaching consequences.
For example, it made DNA finger-printing widely available, for use not only in forensic science
for identifying potential suspects but also for use by behavioral biologists to trace paternities in a
population of wild birds to monitor what type of behavior leads to success in mating.

In normal cells, the double-stranded DNA is unzipped with an enzyme to start the replication
process. In PCR, single-stranded DNA is made by heating a chromosome fragment to 95°C. It
is then cooled so that the primers anneal to the original DNA strands and DNA polymerase can
bind and copy each strand. With repeated heating and cooling, millions and even billions of
copies of DNA can be made in a few hours.

An interesting twist was the discovery of chemoautotrophic bacteria that live in the hydrothermal
vents in the deep ocean floor. These creatures live in the vents where temperatures exceed
100°C; consequently, their DNA polymerases remain functional at such temperatures that would
have denatured an ordinary creature's DNA polymerases. DNA polymerases isolated from
these bacteria have made it possible to develop automatic thermocycler machines without the
need to add new polymerases each cycle.

To obtain only the desired portion of the DNA, this lab uses oligonucleotide primers that
specifically bind to regions flanking the 16S rRNA gene. (For further explanation, watch the PCR
animation in the main lab.)

Note: What are primers?

Primers are small pieces of DNA that bind to specific sequences—in this case, sequences
within the 16S rRNA gene. In this lab, the two primers used, 27F and 1525R, bind to opposite
ends of the gene, one on each DNA strand. Once the primers bind, DNA polymerase extends
the DNA from the 5' end to the 3' end. The primers are selected so that as the two new copies
are made, they overlap in the region of interest (see animation for illustration).

These primers are "universal," meaning that they bind to and thus copy the 16S rRNA gene
from any bacterial species (except perhaps for the most unusual ones). This is because the
sequences to which the primers bind are extremely similar among all bacterial species.
You might ask, If they are similar, how can you use them to identify different bacterial species?
Doesn't that require uniquely different signatures? The answer is that some parts of a gene are
extremely similar among different species (i.e., highly conserved) while others are highly
variable. Universal primers bind to the highly conserved regions of genes so that they can be
used to copy DNA from a variety of species of bacteria. The variable regions, which differ
between species, are used for identification.

Part 3 - Purify PCR Product

The tube should now contain many copies of 16s rDNA, each about 1,500 base pairs (bp) long.
At this time, it is prudent to run a gel to confirm that the PCR reaction worked. The gel should
contain three lanes: one for the negative control (i.e., water), which should not have a product
unless the water was contaminated; another for positive control (PCR product of a known DNA
sequence) to make sure that the PCR itself worked; and the last lane for your sample.

If you are confident that the PCR worked, you can proceed to purifying the PCR product.
Running a gel is actually one method of purification. Once the PCR product is in the gel, you
can cut out the band corresponding to the PCR product and isolate the DNA from the gel.
Nowadays, you can buy compact microfilters to filter the DNA from the PCR tube without
running a gel. We will use such microconcentrator columns in our procedure:

1. Insert the microconcentrator column of appropriate size into a collection tube.

2. Add 400 µl of buffer to the column.
3. Add the entire PCR content (~100 µL) to the column.
4. Spin the column at 3,000 rpm in a fixed-angle centrifuge for 15 minutes.
5. The PCR product should be trapped in the column while the collection tube should
contain all the primers, nucleotides, and other small compounds that we no longer
need. Remove the collection tube and discard it.
6. Invert the column and attach it to a new collection tube.
7. Add 50 µl of buffer to the inverted column. This step should loosen the DNA from the
column into the collection tube.
8. Spin the inverted column at 3,000 rpm for 2 minutes to collect the sample in the
collection tube. Discard the column.

The final collection tube should now have many pieces of 1,500bp-long 16S rDNA, with a very
small amount of longer DNA strands (which are contaminants).

Part 4 - Prepare for Sequencing

The DNA sample has been purified; your PCR tube should now contain almost nothing but
copies of the 16S rDNA. Now, we can prepare the sample for automatic sequencing. DNA
sequencing technology is another area of molecular biology that has seen an impressive
amount of refinement. The predominant method, illustrated here, is called PCR cycle
sequencing. (Learn about cycle sequencing before proceeding.)

In this lab, we use a set of 12 primers; six for each strand of the double-stranded DNA. It is
theoretically possible to use a single primer in PCR cycle sequencing, but it is not feasible for
long sequences. With multiple primers, short, overlapping stretches of DNA are sequenced to
obtain the complete sequence. In addition, it is not absolutely necessary to sequence both
strands, although sequencing both strands generates redundant data, thereby reducing error.
The exact number and location of primers used in a reaction depend on the availability of
suitable primers. The primers used here are available from a commercial source and bind to
conserved regions of the 16S rDNA gene. They should thus be able to bind to the sequence
regardless of the bacterial source.

Each green and blue tube contains a "sequencing brew" consisting of buffers, primers (a
different one in each tube), DNA polymerases, nucleotides, and fluorescence-tagged
terminators in suitable proportions. The PCR product from the previous step is added to each
tube and another PCR is run. This time the aim is not to produce identical copies of DNA but
many copies of variable length. The animation illustrates what is happening in one tube
containing the primer 651R. Each DNA strand binds the primer at one end and will have a
fluorescence-tagged terminator at the other end.

Note: What is cycle sequencing?

In cycle sequencing, the first step is to use a thermocycler to create many copies of the target
DNA but with one twist: you terminate the replication process at random places so the copies
are all partial sequences with different lengths. The reaction mixture contains normal
deoxynucleotides (A, C, G, and T) as well as some special dideoxynucleotides (A*, C*, G*, and
T*) that are tagged with fluorescent markers. The fluorescent markers differ for each base, and
are designed to fluoresce with different colors (G* is yellow, T* is green, for example).
Dideoxynucleotides can substitute for normal deoxynucleotides during replication, but if such a
substitution occurs by chance, that chain can no longer be extended, terminating that DNA
strand. Dideoxynucleotides are thus called terminators. (See the pictorial representation in the
lab animation.)

Let's use a specific example. To sequence a six-base-long single-stranded DNA (consisting of

the sequence 3' A-C-G-T-T-G 5') with this method, you might start by generating pieces that are
one to six bases long. Because DNA polymerase extends the copy DNA from the 5' end to the
3' end (i.e., from the 3' end to the 5' end of the template DNA), the pieces are going to be
(remember that A pairs with T and C pairs with G)
T*
T-G*
T-G-C*
T-G-C-A*
T-G-C-A-A*
T-G-C-A-A-C*
In all of these pieces, the last (3' end) base is a dideoxynucleotide. Once you have these
pieces, you can separate them based on their size by means of a very sensitive version of gel
electrophoresis. Because each differently sized strand terminates in a specific base (for
example, T-G-C* ends in a C*), it will fluoresce with a specific color. By looking at the
fluorescent colors of DNA pieces of increasing size, you can compile the sequence for the
complementary DNA (T-G-C-A-A-C), from which you can infer the original sequence.

Of course, for this to work, replication must start at the same place on the target DNA. In the
above example, you do not want pieces such as G-C-A* (position 2-3-4) floating around. In
addition, the normal sample contains double-stranded DNA, which means that you not only
have the template we want (3' A-C-G-T-T-G 5') but also the complementary strand (5' T-G-C-A-
A-C 3'). The complementary strand can create pieces such as G-T-T*, which is also
undesirable. These problems are avoided by using primers that bind to specific known
sequences of only one strand.In our example, instead of the six bases, consider a ten base
strand with four more bases in the 3' end direction so the sequence is actually (3' T-G-T-A-A-C-
G-T-T-G 5'). Using a primer sequence of 3' A-C-A-T, the replication will always start at the same
place, and the resulting DNA pieces are going to be
A-C-A-T-T*
A-C-A-T-T-G*
A-C-A-T-T-G-C*
A-C-A-T-T-G-C-A*
A-C-A-T-T-G-C-A-A*
A-C-A-T-T-G-C-A-A-G*

Given that the incorporation of the terminator occurs by chance, it is difficult to make a very long
DNA copy if you also want to make a short copy in the same reaction. Further, separating long
chains by electrophoresis where the difference is only one base is difficult. Thus, to sequence a
long section of DNA, many different primers are used, each in its own reaction tube. This way,
many overlapping sections are sequenced in parallel and the result compiled to generate a
complete sequence.
Part 5 - DNA Sequencing

From the last step, you have 12 tubes that contain the final PCR product, a mix of DNA pieces
of variable length. All DNA pieces in each tube start with the same primer but end with a
different nucleotide tagged with a fluorescent marker (different color for each nucleotide A, T, G,
C). What remains to be done is to separate the individual DNA pieces and identify the end
nucleotide.

This is done by using an automatic sequencer that performs gel electrophoresis on the DNA in
each tube. Gel electrophoresis is a method to separate molecules based on differences in size.
The sequencer used in this lab has a thin capillary tube attached at one end to a syringe
mechanism that contains a buffer solution. The tube is filled with the buffer solution and the
other end inserted into one of the tubes containing the DNA pieces. Then, an electric current is
applied so that the end of the tube in contact with the DNA has a negative charge and the
syringe end a positive charge. Since DNA molecules are negatively charged, they start to move
through the tube toward the positively charged syringe end, with the smaller pieces moving
faster than the larger ones. Near the syringe end, the capillary tube passes through a laser
beam that excites the fluorescent markers, and optical detectors detect the color of the
fluorescence.

We can assume that a complete set of DNA pieces, all differing in size by exactly one
nucleotide, were generated in the previous step. The smallest piece of DNA that has a
fluorescent tag attached to it is the primer. This DNA fragment will travel faster than the other
ones. By reading the color (in our example, red), we determine that the first nucleotide beyond
the primer sequence is thymidine (T). The next smallest piece of DNA will fluoresce with the
color (green) representing the next nucleotide in the sequence (A) and so on (see the
animation). By reading the sequence of nucleotides based on their fluorescence as the DNA
pieces pass through the laser beam, the sequence of the DNA can be reconstructed.

The sequencer automatically flushes out the buffer from the tube, moves the tray, and runs the
electrophoresis again, repeating the program until all 12 tubes have been examined. The
resulting sets of sequences are collated by a computer program to build the complete sequence
of the 16S rRNA gene.

Part 6 - DNA Sequence Analysis

After the sequence information has been gathered from all the reaction tubes, the computer
builds the actual sequence by matching together different pieces. You now have the 16S rDNA
sequence for this bacterial species, which can be compared with all other known 16S rDNA
sequences for identification.

Learn about the science behind sequence matching.

There are many sequence databases in existence. Some databases are sold commercially as
part of an identification kit. In this example, we will use the GenBank public database available
through the National Library of Medicine. The matching algorithm is known as BLAST (Basic
Local Alignment Search Tool). A direct match of the DNA sequence determines the exact
bacterial species you have found. When the DNA sequence is not an exact match but a close
match to another found in the sequence database, you need to assess whether it is a new
species or a variation of an existing one.

Learn more about BLAST search results.

Now follow these steps to identify your sample:

Step 1: Click here to see the data output from the sequencer. Select the all data in the window
and copy (ctrl-c on the PC or cmd-c on the Mac).

Step 2: Click here to go to the NCBI site to perform your search. (This will pop open a new
window)
Step 3: On the NCBI site's BLAST page, paste your data (ctrl-v on the PC or cmd-v on the Mac)
in the box labeled "Enter accession number, gi, or FASTA sequence," in the "Enter Query
Sequence" near the top of the page. In the "Choose Search Set" section, Database: "Others (nr
etc.)" and "Nucleotide collection (nr/nt)" should already be selected. When you are ready, click
on the blue "BLAST" button. (updated October 21, 2007)

Step 4: Follow the instructions provided by the NCBI site to obtain your results.

Step 5: Come back to this page (which should remain open) and identify the bacteria in the lab
window to the left.

Note: The science behind sequence matching

Analysis of a newly isolated DNA molecule by comparing its sequence with all other known
sequences in search of a match involves database searching and sequence alignment. The
ultimate goal of this type of analysis is to determine whether the new sequence bears a
significant degree of similarity (or homology) to another known sequence.

Perhaps the most important tool necessary for sequence matching is access to a
comprehensive and up-to-date sequence database. GenBank, the EMBL nucleotide sequence
database, and the DNA Database of Japan (DDBJ) are three partners in a longstanding
collaboration to collect all publicly available sequence data. Sites in Bethesda, Maryland (USA),
Hinxton (UK), and Mishima (Japan) exchange new sequence data and updates over the Internet
everyday and make the information immediately available to everyone by e-mail, anonymous
ftp, and the World Wide Web.

Next in importance is the computer program used to search the database. Several different
mathematical programs allow two sequences to be compared with each other and determine
the degree of similarity between them. The BLAST programs (BLAST is an acronym for Basic
Local Alignment Search Tool) are among the most popular programs. They offer a good
combination of speed, sensitivity, flexibility, and statistical rigorousness. (See interpreting
BLAST search results.)

In what situations would a scientist search sequence databases? As an example, sequence

matching can be used to determine whether a newly identified DNA sequence is part of a known
gene. In the simplest scenario, if a new sequence is identical or almost identical (except for a
few nucleotide changes) to that of a gene in the sequence database, it is reasonable to
conclude that the new sequence is either part of the same gene or of a closely related gene. But
what if two sequences which appear to be different share sections that are identical? How do
you know whether the identical sections are due to chance or indicate some meaningful
relationship between the two sequences? Sequence analysis using BLAST or another program
provides a "similarity score" to help answer this question.

If the function of a particular DNA sequence is already known–for example, the 16S rRNA gene
we have been working with in this lab–comparing its sequence with that of the same gene from
another species of bacteria provides information about the evolutionary relationship between the
two bacterial species. The assumption here is that the number of positions that differ in the
nucleotide sequence is proportional to the time elapsed since the two species formed their own
lines of descent from a common predecessor.

However, not all DNA sequences change at a constant rate over time. For example, it is not at
all clear whether all organisms experience similar mutation rates from purely environmental
factors (from increased UV exposure, for example). If the DNA sequence has or has had at
some point in evolution a functional role, the rate of evolution and selection—which may be
related to population size among other things—can affect its rate of change. And, in some
cases, mutations are caused by deletions, insertions, and substitutions of long sequences of
DNA rather than by single nucleotide changes. Finally, some sequences of DNA encode
proteins with very specific structural requirements, and any change may prove unfavorable to
the organism. Such sequences therefore do not tolerate change well and tend to remain the
same for long periods of time. These are referred to as "conserved" regions. In contrast,
sequences that can accommodate change more easily are referred to as "variable" regions.

Note: Interpreting BLAST search results

Let's consider how one might go about assigning a numerical value to the degree of similarity
between two DNA sequences. Suppose we have two sequences as follows:
CGGCAT
CGCGAT
Let's assign 1 point for each base pair that matches exactly and 0 point for each base pair that
does not. We have C-C (match), G-G (match), G-C (no match), C-G (no match), A-A (match),
and T-T (match) for a total of 4 points. Under this hypothetical system, the more nucleotides that
match up, the higher is the score.

When comparing two DNA sequences, it's important to remember that because of evolutionary
history, the sequences may have diverged not only by substitution of bases but also possibly by
deletions or insertions of bases. This means that the sequences that are being matched may
not be exactly the same length but might have gaps. In practical terms, for these two
sequences, the best match is
CGGC-AT
CG-CGAT
for a total of 5 points.
Another possible alignment is
CG-GCAT
CGCG-AT
for a total of 5 points.

From the simple example above, you can imagine how rapidly sequence comparisons can
become complicated as DNA length increases. The statistics for comparing two sequences of
DNA are thus highly complicated. Here we cover just the bare essence of the topic so that you
can interpret the response from your sequence query.

Let's suppose you do a BLAST search of the following sequence:

TATCGCGTATTGCC
BLAST will come back with a result, starting with the reference of the search program, the
number of letters in your sequence, the number of letters in the database, a graphic
representation of the sequence matches, and a list of matches. The list of matches is sorted
with the best matching sequences shown first. For the sequence we used, the list starts with the
following:

Score E
Sequences producing significant alignments:(bits) Value

gb|AC012156.14|AC012 Homo sapiens chr 12.. 28 5.8

ref|NC_001142.1 Saccharomyces cerevisiae... 28 5.8

What does this mean? "Score" is a numerical score assigned by BLAST. In the simple example,
we used earlier, we simply assigned 1 point for matches, 0 point for non-matches. In BLAST,
the scoring system uses "bits" as the measure of information. For DNA, each position can be
occupied by either T, A, C, or G. Each match therefore contains 2 bits of information (only 1 is
correct out of 4 possibles). For a 14-nucleotide-long sequence like ours, the maximum match
score then is 28 bits. The higher the score, the better is the match.

"E-value" is the number of hits one can expect to see just by chance when searching a
database of a particular size. The value is defined as
E = N/n * m * n * 2-S
where m and n are the length of the two nucleotide sequences (measured in base pairs), S is
the bit score, and N refers to the total length of all sequences in the database. The formula
should make intuitive sense. For example, if S is higher (i.e., better matches), you would expect
to see fewer "hits." On the other hand, if m or n are larger (i.e., one or the other sequence is
longer), then you would expect to see more hits purely by chance. Finally, if the database
contains more sequences (i.e., N is larger), then you would expect to see more hits. In any
case, if BLAST returns an E-value that is very small or close to zero, then you probably have a
meaningful match that is not due to random chance.

To interpret the matches, you therefore need to pay attention to whether the E-value is
reasonably small. E-value is related to the P-value by the following formula:
P = 1 - e-E
So for a P-value of 0.95 (the statistically significant level), the E-value is around 3. Thus, in your
search, an E-value of 3 or less would be an acceptable match.

You should also keep in mind that there are a lot of sequences in the database and that some of
them are from the same species and therefore might be very similar. In some cases, the name
of the organism may have changed after it was originally reported; accordingly, two or more
sequences may match extremely well but appear to belong to completely different species.

Current Sample:
Sample A - Fluid from Lymph Node

This sample was obtained from tissue and fluid aspirated from a lymph node of a patient
complaining of a fever, fatigue, and swollen lymph glands. The sample was chemically treated
and plated on agar that supports the growth of bacteria. Petri dishes containing clearly visible
colonies of bacteria were sent to the laboratory for identification of the specific type of bacteria
by PCR amplification and sequence analysis.

Other samples from different patients. Click the sample links below to identify the bacteria.

• Sample A - Fluid from Lymph Node

Identification Correct - Bartonella henselae
This sample was obtained from tissue and fluid aspirated from a lymph node of a
patient complaining of a fever, fatigue, and swollen lymph glands.

• Sample B - Stool Sample

This sample was obtained from the stool of a patient with a normal temperature
complaining of severe abdominal cramps and watery diarrhea.

• Sample C - Urine Sample

This sample was obtained from the urine of a patient complaining of pain during
urination and a discharge from the urethra.

• Sample D - Blood Sample

This sample was obtained from the blood of a patient complaining of fever, headache,
joint pain, constipation, loss of appetite, and abdominal pain.

• Sample E - Sputum Sample

This sample was obtained from the sputum of a patient complaining of chills, high fever,
a cough with labored breathing and sputum with flecks of blood.

• Sample F - Stool Sample

This sample was obtained from the stool of a child who has fever, abdominal pain, and
bloody diarrhea.

Congratulations! You have correctly identified the bacteria.

*** The BLAST search displays the matching sequences in the database in descending order
of the degree of the match. Most of the top scorers are either Bartonella henselae or
Rochalimaea henselae. The latter is an older name for this species. Bartonella species are
demanding in their nutritional requirements for culture; as a result, normal culturing techniques
do not yield vigorously growing bacterial colonies.

Here is the description of this species from our reference section:

Bartonella henselae Various species of Bartonella that are pathogenic to humans are
transmitted via a vector, or directly from an animal reservoir. For example, B. bacilliformis via
sandflies causes Oroya fever; B. quintana via body lice causes trench fever; and B. henselae
via cats causes cat scratch disease (CSD). CSD typically manifests as swellings of the lymph
glands, possibly with skin lesions at the site of inoculation and possibly accompanied by fever,
fatigue, and other symptoms. Immunocompromised patients may be particularly susceptible and
can develop a different disease, bacillary angiomatosis, as a result of infection by B. henselae
or B. quintana.

*** The BLAST search displays the matching sequences in the database in descending order of
the degree of the match. The top scorer is Escherichia coli.

Here is the description of this species from our reference section:

Escherichia coli Probably the most famous bacterium, E. coli is one of the most common
inhabitants of the intestinal tract. Generally, they are not considered pathogenic, but they can
cause disease under certain conditions. There are pathogenic strains that can cause diarrhea or
other serious conditions. (Visit www.biointeractive.org, where there is an animation clip of an
enteropathogenic strain in action.)

***The BLAST search displays the matching sequences in the database in descending order of
the degree of the match. The top scorer is Pseudomonas aeruginosa.

Here is the description of this species from our reference section:

Pseudomonas aeruginosa Pseudomonads are commonly found in soil, water, and other such
natural environments. To healthy individuals, these bacteria do not generally constitute a health
threat. They have been found to be remarkably adept at growth by using most unusual sources
of nutrients such as soap residues or glue. In hospitals, P. aeruginosa has been known to cause
problems in debilitated patients and is a common cause of disease in children with cystic
fibrosis. These persistent creatures have been found in unlikely places such as air vents, water
hoses, and even in detergent holding tanks in the very machines that are used to clean medical
instruments.

*** The BLAST search displays the matching sequences in the database in descending order of
the degree of the match. The top scorer is Salmonella typhimurium.

Here is the description of this species from our reference section:

Salmonella typhimurium Many strains of Salmonella are potentially pathogenic. They are
commonly found in the intestinal tract of mammals, birds, and reptiles. Under some conditions,
they can lead to contamination of food. The most severe illness caused by any Salmonella is
typhoid fever (pathogen: Salmonella typhi). Other strains, of which S. typhimurium is one, cause
less serious gastrointestinal diseases, collectively known as Salmonellosis. The bacteria invade
the cells of the intestinal tract of the host (see www.biointeractive.org for an animation of how
this occurs), multiply, and sometimes escape to the bloodstream and the lymphatic system.
Symptoms include fever, nausea, abdominal pain, and diarrhea. Nowadays, uncooked eggs are
a particularly common source of salmonellosis. Soft-boiled eggs, incompletely cooked yolk in
fried eggs, or use of raw or incompletely cooked eggs in sauces and desserts can all present a
risk.

***The BLAST search displays the matching sequences in the database in descending order of
the degree of the match. The top scorer is Yersinia pestis.

Here is the description of this species from our reference section:

Yersinia pestis This is the bacteria that causes the plague, which has had a huge impact on
human history. In the 14th century, possibly 25% of the total population of Europe was
destroyed. The plague is still with us, even in the United States. The plague is ordinarily a
disease of the rat, carried both by urban and rural rodents. The bacteria is transmitted from one
rat to another by fleas. However, if the host rat dies, then the fleas seek another host, and can
attach to a human and transmit the disease. When the bacteria enter the human bloodstream,
they multiply in the lymph and blood, causing the lymph nodes in the groin and armpit to
become swollen, accompanied by fever. If untreated, death can result within a week. The
plague can also become pneumonic by entering the lungs which is particularly dangerous with a
nearly 100% mortality rate. This form of plague can also be contagious by air.

*** The BLAST search displays the matching sequences in the database in descending order
of the degree of the match. The top scorer is Yersinia enterocolitica.

Here is the description of this species from our reference section:

Yersinia enterocolitica is a species of bacteria that are often found in the intestines of many
domestic animals including sheep, rabbits, pigs, cattle, horses dogs and cats. Bacteria can be
transmitted in meat and milk or other contaminated food or water. They are unusual in that they
can grow at 4°C, which is the temperature of refrigerators. When transmitted, the bacteria cause
Yersinia gastroenteritis, or yersiniosis. The symptoms of this disease are fever, diarrhea,
headache and abdominal pain. Pain is often severe and can be mistaken for appendicitis. There
may be a post-infection reaction of inflamed joints. Incidences of yersiniosis is relatively rare in
the US, but more common in Northern Europe.