Ground Beef Isolates .pdf

Antibiotic Testing in
Ground Beef Isolates
Abstract

Methods and Materials

This experiment explores the microbes that are living in your meat products. The goal is to isolate and identify potentially
harmful bacteria from ground beef and test them for antibiotic resistance. 8 distinct bacterial colonies are chosen and
isolated from a ground beef homogenate sample to run antibiotic testing and identification assays. Gram negative bacteria
are the focus of this experiment because the goal is to find potentially harmful gut bacteria (enteric bacteria) that have
resistance to common antibiotics including penicillin, tetracycline, novobiocin, erythromycin, and ciprofloxacin. Therefore,
selective media (MacConkey agar) is used to select for Gram negatives, and blood agar is used to determine the total
bacterial load. Four distinct colonies are chosen from each of the two plates to isolate and do further testing. Out of the 8
isolates, there were 2 Gram positives and 6 Gram negatives. The antibiotic tests showed various antibiotic
sensitivity/resistant results, but it seemed like tetracycline and ciprofloxacin were the most effective drugs as they inhibited
growth in all of the 8 isolates. The 2 Gram positive isolates were able to be narrowed down to Staphylococcus, however
further testing is needed to determine the 6 Gram negative isolates. Based on the results of this experiment, the most likely
genera for the Gram negatives could include Serratia, Cedecea, Klebsiella, Escherichia, Shigella, and/or Enterobacter,
however further testing is needed to confirm.

Sample Collection and Isolation: 1g of store-bought ground beef was weighed and put into a 10mL sterile
solution. The mixture was then pulverized to make it into a pink, smoothie-like texture. 100uL of the ground beef
homogenate was then plated onto blood agar and MacConkey agar and was spread evenly by using sterile beads.
After 48 hours of incubation at 37° C, 4 distinct colonies were chosen from each plate (non-hemolytic) and reinoculated on the same media for a total of 8 distinct isolates.

Introduction
Antibiotic resistance is an ongoing threat to our health from the overuse of the drugs, especially on the livestock of the
agricultural industry. The meat and poultry industries face economic pressures that encourage wide spread adoption of
antibiotic use. There’s been a huge decrease in farms over the years, causing overcrowdings in the remaining farms.
These concentrated animal feeding facilities are overcrowded, unclean, and the animal feeds are supplemented with
copious amounts of antibiotics to encourage faster growth of the animals and to prevent disease. This overuse of antibiotics
increases the chance of resistant bacteria to grow within the livestock, which then in turn get transferred to humans when we
eat the meat. If we get infected by one of these resistant bacteria, we could get sick and there might not be an antibiotic to
treat it.[1]
Many potentially pathogenic bacteria can be found in the meat products that we buy from the super market, especially
ground meat since it can be a combination of many different animals. These microbes are generally of the
Enterobacteriaceae family, which includes many members of the normal gut flora, like E. coli, along with some notorious
pathogens like Salmonella. The spread of antibiotic resistant Enterobacteriaceae is complicating treatment options for
serious infections like gastroenteritis. There have been strains found to be resistant to third-generation cephalosporins,
quinolones, and even carbapenem, which is supposed to be the last line of defense for multidrug-resistant bacteria.[2], [3]
Many biochemical tests can be performed to help narrow down the enteric species including, but not limited to
oxidative/fermentative assays, phenol red carbohydrate assays, cytochrome c oxidase assay, urease assay, indole
production assay, sulfide production assay, motility assay, and much more. The oxidative/fermentative assay is a simple
way to detect an enteric species because they are all facultative anaerobes, and can also differentiate them from other
Gram negative rods like Pseudomonas. The phenol red carbohydrate assays detect their ability to utilize a specific carbon
source (lactose, sucrose, etc.), and whether or not it can be fermented. The oxidase assay detects the presence of
cytochrome c, an enzyme used for aerobic respiration. SIM agar is a useful media that gives you results for indole/sulfide
production, and motility. Indole producing microbes will turn the agar red when Kovac’s reagent is added, hydrogen sulfide
producing microbes will create a black precipitate within the agar, and motile microbes will swim and spread out farther than
where the inoculation occurred. Other medias, like urea, citrate, etc. will show whether or not they can utilize that carbon
source. BBL Enterotube II tubes are also extremely useful in that it’s a one-time multi test system used to identify among the
Enterobacteriaceae family, and includes some of the tests described above along with some others.

Figure 1. The
bacterial load from
a diluted sample of
ground beef
(700,000 CFU/g)

Figure 2. Isolates #1
and #3 were Gram
positive staphylococci.
This is isolate #1, a
good representation
of both.

Figure 3. Isolates #2,
and #4-8 were Gram
negative rods. This is
isolate #4.

Antibiotic Testing: 8 saline suspensions were made from the 8 different isolates. Then a sterile swab was used
to transfer bacteria from the saline suspensions onto 8 separate blood agar plates, distributing them onto the
whole plate as evenly as possible. Five antibiotic disks were then placed around the plate: penicillin, tetracycline,
novobiocin, erythromycin, and ciprofloxacin. The ciprofloxacin disks were made by transferring 20uL of liquid
ciprofloxacin onto blank disks. The 8 plates were incubated at 37° C for 48 hours and then examined for antibiotic
sensitivity or resistant.
Identification: Differential staining was performed on each of the 8 isolates. The Gram positive isolates were
subjected to a catalase test and were further inoculated onto mannitol salt agar to examine lactose fermenting.
The Gram negatives were subjected to 10 different biochemical tests: oxidative/fermentative glucose assay, phenol
red lactose and sucrose assays, cytochrome c oxidase assay, sulfide production assay, indole production assay,
motility assay, methyl red assay, citrate assay, and urease assay. The Gram negative isolates were also further
inoculated onto EMB agar and Hektoen enteric agar to examine possible heavy lactose fermenters and
Salmonella/Shigella species. Lastly, three of the Gram negative isolates were also tested on BBL Enterotube II
tubes (isolates #2, 5, and 6).

Table 2. Gram Positive Identification Tests
Hemolysis on Blood Agar
Catalase

Results
Plating and Gram Staining: The blood agar was full of bacteria with about 700 CFU (figure 1), while the
MacConkey agar had about 12 CFU. Four colonies were chosen from each plate to be Gram stained and reinoculated. The four isolates chosen from the blood Agar were named numerically 1-4, and the four isolates from
the MacConkey agar were named 5-8. Isolates #1 and #3 were Gram positive cocci in grape-like clusters (figure
2), and the other six (#2,4,5,6,7,8) were Gram negative rods with no arrangement (figure 3).
Calculating Bacterial Load of Sample: 1g of ground beef was added to 10mL of sterile solution, about a 1/10
dilution factor. Then 100uL was taken from this homogenate, which is about another 1/100 dilution factor, and that
was plated onto the blood agar. There were about 700 CFU on the plate after 48hrs of incubation. To calculate
the CFU of the original 1g beef sample, 700 x 10 x 100 = 700,000 CFU for every gram of ground beef (figure 1).
Antibiotic Testing: The Gram positive isolates #1 and #3 were sensitive to every antibiotic, and the Gram
negative isolates varied in antibiotic sensitivity/resistance (table 1, figure 4).
Gram Positive Identification: The Gram positive isolates #1 and #3 were both non-hemolytic, catalase positive,
and sensitive to novobiocin. The mannitol salt agar showed that isolate #3 ferments mannitol while isolate #1 did
not. Based on these results, the two isolates are Staphylococcus, but identification of the specific species is
inconclusive based on the mix results (table 2).
Gram Negative Identification: The six Gram negative isolates (#2, #4-8) were subjected to a variety of
biochemical tests (table 3, figure 5). Further testing is needed to narrow down the possibilities, but based on the
data that was acquired, possible genera include Serratia, Cedecea, Klebsiella, Escherichia, Shigella, and/or
Enterobacter. Three BBL Enterotube II tubes were used for isolates #2, #5, and #6. Based on those results (figure
6), by using the BBL Enterotube II interpretation guide, isolates #2, #4, and #6 are Serratia marcescens, Cedecea
davisae, and Enterobacter cloacae respectively, but these results are not completely reliable because the tubes
have been expired for 5 years. Also, based on all the tests done, isolates #5 and #7 are most likely the same
species, along with isolates #6 and #8.

Table 1. Antibiotic Sensitivity/Resistance of the 8 Beef Isolates.
Penicillin
Tetracycline
Novobiocin
Erythromycin
1 (G+)

Figure 5. The various biochemical assay results for the identification
of the 6 Gram negative isolates. O/F glucose test (top left), PR-L and
PR-S tests (top right), MR and SIM tests (bottom left), urea and
citrate tests (bottom right). See table 3 for positive/negative results.

Figure 6. The BBL Enterotube II assay is a multi test system
used to identify among the Enterobacteriaceae family. The
positive/negative results are color coded and can be looked
up in the BBL Enterotube II interpretation guide. From top to
bottom, these are the results of isolates #2, 5, and 6 after
48hrs of incubation at 37° C. Based on the interpretation
guide, these isolates are Serratia marcescens, Cedecea
davisae, and Enterobacter cloacae respectively, but these
results are not completely reliable because the tubes have
been expired for 5 years

Figure 4. These are the antibiotic
sensitivity assays that were
performed on all 8 beef isolates.
The formation of a ring around
the antibiotic disk shows
sensitivity. If the antibiotic disk
had no effect, then no ring forms
and is therefore resistant. The
top row consists of isolates #1-4
and the bottom row consists of
isolates #5-8. Starting at 12
o’clock and going clockwise, the
antibiotic disks used were
penicillin, tetracycline,
novobiocin, erythromycin, and
ciprofloxacin.

Sensitive

Sensitive

Sensitive

Sensitive

Ciprofloxacin
Sensitive

2 (G-)

Resistant

Sensitive

Mostly Resistant

Resistant

Sensitive

3 (G+)

Sensitive

Sensitive

Sensitive

Sensitive

4 (G-)

Mostly
Sensitive
Resistant

Sensitive

Resistant

Resistant

Sensitive

5 (G-)

Resistant

Sensitive

Mostly Resistant

Resistant

Sensitive

6 (G-)

Resistant

Sensitive

Sensitive

Resistant

Sensitive

7 (G-)

Resistant

Sensitive

Mostly Resistant

Resistant

Sensitive

8 (G-)

Resistant

Sensitive

Resistant

Resistant

Sensitive

The 8 beef isolates were subjected to 5 common antibiotics to test for resistance. The Gram positives were
sensitive to all 5 of the antibiotics, while the Gram negative results varied. Tetracycline and ciprofloxacin were
the most effective drugs, inhibiting growth in all 8 isolates.

Contact

References

CJ Cracchiolo
Merrimack College
cracchioloc@Merrimack.edu

1.
2.
3.

1
3

Non-hemolytic
Non-hemolytic

+
+

Mannitol
Fermentation
+

Novobiocin
Sensitivity/Resistance
Sensitive
Sensitive

This table shows the identification test results for the 2 Gram positive isolates. It narrows them down to the
Staphylococcus genus, but the mixed results leaves the identification inconclusive at the species level.

Table 3. Gram Negative Identification Tests
O/F Glucose Oxidase PR-L PR-S Indole
2
Facultative
+ (gas)
Anaerobe
4
Facultative
+
+
+
Anaerobe
5
Facultative
+ (gas)
Anaerobe
6
Facultative
Anaerobe
7
Facultative
+ (gas)
Anaerobe
8
Facultative
Anaerobe

H2S
-

Motile
-

MR
-

Urea
-

Citrate
+

-

-

+

+

-

-

-

-

-

+

-

-

+

Weak +

+

-

-

-

-

+

-

-

+

Weak +

+

This table shows the biochemical assay results for the 6 Gram negative isolates. Further testing is needed for
identification, but they are most likely in the genera Serratia, Cedecea, Klebsiella, Escherichia, Shigella, and/or
Enterobacter based on these tests, and the BBL Enterotube II tests.

Discussion/Conclusions
Antibiotic resistance is an ongoing health risk, especially with the meat and poultry industries overusing antibiotics
in their livestock. I decided to sample some ground beef from the super market and run antibiotic testing with
some common drugs. The goal of this experiment was to choose 8 distinct colonies from a 1g sample of ground
beef, test them against penicillin, tetracycline, novobiocin, erythromycin, and ciprofloxacin, and to run identification
tests in an attempt to put a name to these isolates. My focus was on the Gram negative bacteria because many
coliforms and other enteric species can cause very serious infections in humans, and if the strains are resistant to
our drugs, then the infection cannot be treated. Tetracycline and ciprofloxacin seemed to inhibit all of my 8
isolates, however, many of the Gram negative strains were resistant to either penicillin, novobiocin, and/or
erythromycin.
For identification, I used biochemical assays to start narrowing down each of the isolates. I was able to narrow
down the 2 Gram positive isolates to the Staphylococcus genus, but was only able to narrow down the Gram
negative isolates to the most likely genera of Serratia, Cedecea, Klebsiella, Escherichia, Shigella, and/or
Enterobacter. There was simply not enough time to continue further testing on my isolates, but if I were to
continue this experiment, some other identifications assays would include nitrate assay, spore staining, and
especially 16s rRNA sequencing.

Resistance documentary by Michael Graziano
Paterson, David L. “Resistance in Gram-Negative Bacteria: Enterobacteriaceae.” The American Journal of Medicine , Volume 119 , Issue 6 , S20 - S28 (2006)
Falagas ME, Lourida P, Poulikakos P, Rafailidis PI, Tansarli GS. “Antibiotic treatment of infections due to carbapenem-resistant Enterobacteriaceae: systematic evaluation of the available evidence.” Antimicrobial Agents and Chemotherapy 2014; 58(2): 654-663.