Food and Nutrition Sciences, 2013, 4, 1-9
http://dx.doi.org/10.4236/fns.2013.47A001 Published Online July 2013 (http://www.scirp.org/journal/fns)
Quantification of Antibiotic Residues and Determination
of Antimicrobial Resistance Profiles of Microorganisms
Isolated from Bovine Milk in Lebanon
Kassaify Zeina1*, Abi Khalil Pamela1, Sleiman Fawwak2
1Department of Nutrition and Food Sciences, American University of Beirut, Beirut, Lebanon; 2Department of Animal and Veteri-
nary Sciences, American University of Beirut, Beirut, Lebanon.
Received March 18th, 2013; revised April 18th, 2013; accepted April 28th, 2013
Copyright © 2013 Kassaify Zeina et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The rapid growth of dairy sectors in the Middle East, particularly in Lebanon, led to extensive use of antibiotics to en-
hance the health and productivity of animals. Prolonged usage may lead to antibiotic residues in foods of animal origin;
hence, the emergence of antimicrobial resistant microorganisms. Accurate data on the antibiotic usage in livestock
treatment, antibiotic residues and antimicrobial resistances in raw milk in Lebanon are lacking. This study aimed to in-
vestigate the types and usages of antibiotics in cattle, their residual levels and the potential microbial resistances in raw
milk samples. A questionnaire-based survey identified Gentamicin and Streptomycin as the most frequently used anti-
biotics. Selected raw milk samples from main dairy farms were then analyzed in duplicate by quantitative ELISA for
the antibiotics residual levels. The mean residual levels of Gentamicin and Streptomycin were 90 and 80 μg/L, respec-
tively; which are below the allowable maximum residue limit of 200 μg/L as set by the FAO/WHO. Staphylococcus
aureus, Listeria monocytogenes, E. coli and total aerobic microorganisms isolated from the milk samples were then
tested for resistance against Gentamicin and Streptomycin by the disc agar diffusion method. All the S. aureus, E. coli,
and L. monocytogenes isolates showed high resistance to Gentamicin. However, 95% of S. aureus, 60% of E. coli and
58% of L. monocytogenes isolates were resistant to Streptomycin. The obtained results provide evidence that antimicro-
bial resistant strains of the above pathogens have become remarkably widespread in raw milk. This requires better
management for antibiotic usages among livestock farmers to control sources of food contamination and reduce the
health risks associated with the development of resistant microbial strains.
Keywords: Milk; Antibiotics; Residues; Resistant Pathogens
1. Introduction
Antibiotics, the microbiologically produced compounds,
are used in humans to treat or prevent certain diseases
caused by infectious agents. However, the major antibi-
otics used for humans either belong to the same general
classes or have the same mode of action as those used for
animals [1]. A questionnaire-based survey conducted
preliminarily by the authors in this study showed that the
most commonly used antibiotics in livestock among ma-
jor Lebanese dairy farmers are Gentamicin and Strepto-
mycin which belong to the Aminoglycoside group of an-
tibiotics. Furthermore, a limited survey conducted by
Choueiri [2] on 17 main dairy farms in Lebanon as part
of her thesis work also found that Streptomycin and
Gentamicin are used in all the surveyed farms. It is also
worth mentioning that the exact amounts of antibiotics
used by farmers in livestock production in Lebanon are
not known since they are not regulated. Streptomycin is
mainly used to treat plague and infrequently Brucellosis.
It is also combined with penicillin in treating enterococ-
cal and Listeria monocytogenes infections. Gentamicin
sulfate is quite effective against several types of bacterial
infections mainly those caused by gram-negative bacteria.
It is mainly used against Pseudomonas, E. coli, Entero-
bacteria, Proteus and Serratia [3].
These antibiotics, despite their effectiveness, can leave
residues in the treated animal and contaminate its edible
parts; the muscle meat and milk [4]. Environmental and
*Corresponding author.

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
2
human health risks are associated with these residues.
They range from direct toxicity on consumers exhibiting
allergic reactions to indirect problems through the gen-
eration of resistant strains of pathogenic bacteria and the
residual contamination of manures used in crop produc-
tions [5]. To ensure consumer safety, worldwide regula-
tory authorities have set MRL’s (Maximum Residual Li-
mit) for several veterinary drugs [6,7]. These MRL’s, are
expected to regulate the maximum permitted levels of the
drug residue for each antibiotic which is considered
safely acceptable in food of animal origin [8].
The major worldwide public concern and health hazard
associated with antibiotic residues is the development of
the antimicrobial resistant bacterial strains of animal origin
and its consequent effect on human health [9-13] regarding
the efficacy of antimicrobial therapy [14]. As the pathogens
have become more resistant to the used doses of antibiotics,
incidences of morbidity have increased, therapeutic effi-
ciency has failed, healthcare costs are on the increase, and
the antimicrobial doses have increased with scientists oc-
cupied searching for alternatives to relieve the burden [15].
According to Prescott and Baggot [16], microbial resis-
tance to aminoglycosides, particularly Streptomycin, Neo-
mycin, and Kanamycin is very common and pathogens
present in the milk mainly S. aureus, E. coli O157:H7 and
L. monocytogenes may easily develop antimicrobial resis-
tance [17-19].
This study aimed to determine the residual levels of the
two most commonly used antibiotics, Gentamicin and
Streptomycin, in milk samples collected from 24 random
dairy farms in Lebanon. The enzyme linked immunosor-
bant assay (ELISA) being user friendly, sensitive and can
be economical when many samples need to be analyzed [20]
was the method of choice. Another main objective of this
work was to determine and link the resistance prevalence of
pathogenic Staphylococcus aureus, Escherichia coli and L.
monocytogenes isolated from the same milk samples to
Streptomycin and Gentamicin residues.
2. Materials and Methods
2.1. Questionnaire-Based Survey on Major
Farms
A Questionnaire-based survey was conducted on twenty
six Lebanese dairy cattle farms to identify the most com-
monly used antibiotics, their dosage, timing of use and
the practiced withholding times prior to dispatch. In this
survey, farms located only in the Bekaa Valley and
Mount Lebanon were visited because the southern region
was not accessible. Between July and August, several
farms were contacted, only 26 agreed to answer the sur-
vey divided as 54.1% in the Bekaa Valley and 45.8% in
Mount Lebanon. Two of the farms were for meat produc-
tion; therefore, the collected data were eliminated. The
remaining 24 were divided as nine farms for both milk
and meat production and 15 for only milk production.
Cattle farms varied in capacity; two were large-scale
farms with >1000 cows, six were medium-scale with
cattle capacity 100 - 400 and 16 were small-scale with
cattle capacity <100. Interviews were conducted with the
owners and/or the farmers and the questions were close-
ended. For instance, farmers were asked about the milk-
ing techniques used and their corresponding schedule,
the sanitary conditions of the flock, percentage mortality
and the feed (composition was important for the medi-
cated feed). In addition, farmers were asked about any
treatment administered including the brand name or ac-
tive compound of the medication, treatment period (date
of beginning and end of treatment), withdrawal time and
identification number of the veterinary prescription.
2.2. The Controlled Study
A 10 days controlled study was performed at the Agri-
cultural Research and Education Center (AREC), Faculty
of Agricultural and Food Sciences of the American Uni-
versity of Beirut in the Bekaa of Lebanon. In the study,
four Holstein cows aged between 34 and 39 months, that
yielded approximately the same daily amount of milk (16
- 18 kg) were selected. The cows weighed around 450 -
500 kg and had no signs of disease when inspected. They
tested negative for mastitis and were not exposed to any
antibiotic treatment for a minimum of eight weeks. Each
cow was placed in a labeled separate pen and were all
given the same amount of water and feed. The two anti-
biotics Gentamicin and Streptomycin were selected for
this study based on the data acquired from the survey that
was performed during this study and described above.
Two cows were treated with the antibiotic Gentamicin
(Gentaprim, Invesa, Veterinary Industry, Barcelona, Spain)
at a concentration of 4 ml/100kg three times a day for 3
days, and the other two were treated with the antibiotic
Streptomycin (Pen and Strep, Norbrook Laboratories Li-
mited, Newry, BT35 6JP) at a concentration of 1 ml/25kg
once a day for five days. Treatments were administered
by deep intramuscular injection using sterile disposable
syringes (20 ml syringe, Luer-Lok and BD Microlance
18 G needles) to prevent possible contamination. Milking
was performed twice per day; at 2:00 am and 2:00 pm.
The milk samples used for analysis consisted of a mix-
ture of both milking times and were collected in 100 ml
sterile plastic containers in duplicate. Aseptic measures
were taken during sample collection by cleaning and
drying the udders before milking and sanitizing the milk
buckets before and between every milking session. All
samples were analyzed for antibiotic residues and antim-
icrobial susceptibility.

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
3
2.3. The Experimental Study
In order to validate the information provided by the farm-
ers in the Questionnaire-based survey with respect to the
withholding periods and the proper dosage of antibiotics
used, analyses were conducted on random milk samples
collected from the different farms. The 26 farms that
were visited for the survey were contacted again and only
22 farms had agreed to provide samples to be analyzed.
These farms varied from large-scale (9%) to medium-
scale (27%) and small-scale (64%). Microbiological, an-
tibiotic residues and antimicrobial susceptibility analyses
were performed on all the samples in duplicate.
2.4. Analysis of Antibiotic Residues
Enzyme linked immunosorbant assay (ELISA) test kits
were used to quantitatively analyze both the control and
the experimental milk samples for the presence of the
two most commonly used antibiotics by the farmers, the
aminoglycoside antibiotics Streptomycin and Gentamicin
(BIO SCIENTIFIC Austin, TX 78744 USA. ELISA kits
1014 and 1027, respectively). Reagents and samples were
prepared according to the ELISA kits instructional man-
ual. Six standard antibiotic concentrations of the Gen-
tamicin (negative control, 0.25 ng/ml, 0.75 ng/ml, 1.5
ng/ml, 3.0 ng/ml and 15 ng/ml) and 42 prepared milk test
samples (20 samples from the control study and 22 ex-
perimental samples collected from various farms) were
pipetted in duplicate into different wells. ELISA proce-
dure was followed according to the provided manufac-
turers’ instructional material. Absorbance was read on a
micro titer plate reader with 450 nm wavelength. The
same procedure was followed for Streptomycin analyses
but the standards provided were; negative control, 0.5
ng/ml, 1 ng/ml, 2.5 ng/ml, 5 ng/ml and 10 ng/ml.
2.5. Microbiological Analysis of Milk Samples
A total of 30 raw milk samples collected from the vari-
ous farms and from the control study were microbiologi-
cally analyzed following the procedure of the bacterio-
logical analytical manual [21]. The samples were tested
for the presence of Staphylococcus aureus, E. coli, Lis-
teria monocytogenes, Listeria innocua, total aerobic count
and total coliforms. In the procedure, for the enumeration
of E. coli and the total aerobic microorg anisms, a 10 ml
portion of each milk sample was aseptically diluted with
90 ml of sterilized peptone-water (PW)1. Serial dilutions
from 10−1 to 10−3 were prepared and an aliquot of 0.1 ml
of the homogenate was spread on Plate Count Agar1
plates for the total aerobic count while Rapid E. coli
(REC 2/agar)1 agar was used by the pour plate technique
for the enumeration of E. coli. Plates were then incubated
for 48 hours at 37˚C. All colonies on PCA and REC were
enumerated; purple-pink colonies were identified as E.
coli, whereas blue colonies as coliforms. For S. aureus, a
25 ml portion of each sample was treated in an equiva-
lent manner, but diluted with 225 ml PW and plated on
Rapid Staphylococcus (R. Staph)1 agar plates. Selected
black colonies with a white ring were enumerated and
tested for coagulase and catalase activity using the Pas-
torex (Staph plus/Latex)1 test as a biochemical confirma-
tion test [21]. Finally, the presence of L. monocytogenes
was assessed by taking an additional 25 ml portion of
each sample and diluted with sterile Listeria Enrichment
Broth (Fraser 1 broth)1 supplemented with Listeria En-
richment Broth Supplement (Fraser 12 broth supple-
ment)1 and spread on plates of Palcam agar (Palcam)1
supplemented with Palcam supplement1. Black colonies
were identified as presumptive L. monocytogenes, whereas
white colonies were presumptive L. innocua. Then, Fra-
zer One supplement (Frazer 1 broth supplement), (0.1
ml/1ml) was added to the homogenate to further enrich
the remaining Listeria species for complete detection and
were incubated for another 24 hours at 30˚C. 0.1 ml of
serial dilutions from 10−1 to 10−3 were then plated on
Palcam agar plates in duplicate. The suspected colonies
of L. monocytogenes, L. innocua, S. aureus, E. coli and
coliforms were then isolated and frozen stocks of each
isolate were prepared in sterile microtubes with 1 ml of
30% glycerol and stored at −20˚C to be used in the sub-
sequent susceptible tests.
2.6. Antimicrobial Susceptibility Tests
Bacterial isolates from the milk samples were tested for
antimicrobial susceptibility using the disc agar diffusion
method according to the procedures recommended by the
Clinical and Laboratory Standards Institute (CLSI) [22].
Gentamicin and Streptomycin, the most commonly used
antibiotics, were chosen to assess the resistance of the
isolated pathogens. Bacterial isolates from the above mi-
crobiological analyses were grown on PCA plates. Strep-
tomycin and Gentamicin (10 μg, BIORAD) antibiotic
discs and empty discs as control were tested on duplicate
plates. The plates were incubated for 24 hours at 37˚C
and the zones of inhibition were measured with a metric
ruler and interpreted as resistant or sensitive according to
the CLSI guidelines. Predisposed strains did not grow in
the area around the disc; whereas, resistant strains en-
dured the antibiotic.
2.7. Statistical Analyses
Data subjected to statistical analysis were analyzed using
the software SPSS 15.0 for Windows Evaluation Version.
Gentamicin and Streptomycin residue levels were com-
1BIO-RAD; boulevard Raymond-Poincare 92430, Marnes-La-Coquette-
France.

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
4
pared by means of 1-way ANOVA at P < 0.05 among the
milk samples from the selected farms to determine if
antibiotic usage among farmers were similar. Concerning
the antimicrobial resistance patterns in milk, statistical
analyses (Chi-square analysis at P < 0.05) were con-
ducted to compare resistance patterns to Gentamicin with
resistance patterns to Streptomycin. To verify if a rela-
tion between Streptomycin or Gentamicin residue levels
and microorganism levels in the milk samples exists,
regression analysis was conducted at P < 0.05.
3. Results
The main recurrent antibiotics used among the inter-
viewed farmers were Gentamicin, Canamycin, Penicillin,
Oxytetracyclin, Tetracycline and Streptomycin with Gen-
tamicin and Streptomycin being the most frequently used
(88% and 92% respectively). All farmers reported treat-
ing their cattle only during illness in doses specified ei-
ther by the veterinarian or according to the manufac-
turer’s instructions on the label. They also claimed to
give the antibiotic treatment for as long as specified and
withhold as directed.
3.1. Analysis of Antibiotic Residues
3.1.1. Controlled Study
This aimed to determine from a controlled study of a
known antibiotic dosage, the residual levels of antibiotics
during withholding days to compare with the experimen-
tal samples and validate farmers’ claims. Both cows that
were treated with Gentamicin had similar antibiotic resi-
due levels in their milk samples throughout the 10-day
interval of the study. On average, an initial residue level
of 107 ng/ml of Gentamicin was noticed in the milk sam-
ples on day one followed by a peak residue level of 132
ng/ml on day 4 and a decrease to 57 ng/ml on the 10th
day (Figure 1). However, on the 7th day, the final day of
withholding period according to the directions, Gen-
tamicin residue levels reached a mean concentration of
71.6 ng/ml (Figure 1).
Similarly, the initial Streptomycin residue level was
108 ng/ml on day one followed by a peak of 120 ng/ml
on day 2 and a decrease to 74 ng/ml of Streptomycin re-
sidue levels on the 10th day. At the 6th day, the final day
of withholding period as directed, Streptomycin residue
levels reached a mean of 107.91 ng/ml (Figure 2).
3.1.2. Experimental Samples
The random experimental milk samples were also ana-
lyzed for Gentamicin and Streptomycin.
The levels of Gentamicin and Streptomycin residues in
all the tested samples were less than the recommended
maximum residue limit as set by the FAO/WHO; MRL =
200 μg/L (Table 1). This result in milk indicates that the
a = Gentamicin residue level at day 7, the international specified day for
withholding after treatment of cattle with Gentamicin; Sample detection
limit = 2.5 ng/ml; Number of samples = 20 (10 from each cow).
Figure 1. Mean Gentamicin residue levels in milk samples
collected for 10 days after treating 2 control cows with Gen-
tamicin.
Streptomycin residue levels in milk after cow treatment with
st re pt om ycin
a
y = -0. 3689x
2
- 0.9245x + 116.46
R
2
= 0. 7926
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
0246810
Days
Streptomycin residue
levels ng/ml
12
a = Streptomycin residue level at day 7, the international specified day for
withholding after treatment of cattle with Streptomycin; Sample detection
limit = 2.5 ng/ml; Number of samples = 20; 10 from each cow.
Figure 2. Mean Streptomycin residue levels in milk samples
collected for 10 days after treatment of 2 cows with Strep-
tomycin.
application doses by Lebanese farmers may not be ex-
ceeding the recommendations. In addition, the milk sam-
ples collected from the selected farms had their Gen-
tamicin and Streptomycin residue levels equivalent to
those obtained from the milk samples in the controlled
study post the required withholding period (~71 and 107
ng/ml, respectively).
3.2. Microbiological Analysis of Milk Samples
3.2.1. Controlled study
All tested samples were contaminated with S. aureus (103
to 104 CFU/g) and L. monocytogenes (106 to 107 CFU/g);
while 62.5% of the milk samples had their total aerobic
counts ranging between 103 and 104 CFU/g. None of the
samples had E. coli; whereas, 12.5% of samples had total
coliform levels between 104 and 105 CFU/g. Finally, L.
innocua was detected in 62.5% of the tested samples.
3.2.2. Experimental Samples
Microbiological results of the raw milk samples collected
from the various selected farms showed a mean value of

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
5
Table 1. Gentamicin and Streptomycin residue levels in raw
milk samples collected from the selected farms.
Selected
farms Streptomycin (ng/ml) Standard error Gentamicin (ng/ml) Standard error
F 1 48.92 0.18 63.91 0.06
F 2 31.27 0.09 48.66 0.06
F 3 63.05 0.06 49.71 0.33
F 4 40.80 0.08 46.32 0.02
F 5 53.76 0.07 50.13 0.03
F 6 59.53 0.04 41.81 0.17
F 7 55.61 0.11 53.52 0.14
F 8 52.48 0.06 72.77 0.01
F 9 48.08 0.05 69.86 0.04
F 10 29.43 0.01 45.27 0.06
F 11 48.08 0.06 75.89 0.00
F 12 60.89 0.04 58.42 0.02
F 13 9.22 0.16 29.44 0.02
F 14 67.61 0.06 61.26 0.01
F 15 65.41 0.08 60.72 0.12
F 16 13.43 0.01 21.39 0.02
F 17 45.16 0.38 52.97 0.07
F 18 65.21 0.10 71.22 0.00
F 19 36.52 0.18 85.07 0.08
F 20 5.10 0.02 39.20 0.06
F 21 51.64 0.18 67.99 0.05
F 22 65.25 0.14 72.07 0.02
Sample detection limit = 2.5 ng/ml; N = 22 farms; F = farm.
total aerobic counts >106 CFU/g in 58% of the tested
samples. Most of the analyzed samples (95%) contained
Staphylococcus aureus in the range of 105 - 106 CFU/ml.
As for L. monocytogenes, it was detected in 19 (86%) of
the 22 milk samples while L. innocua was detected only
in 46% of the samples. Finally, 55% and 28% of the raw
milk samples had a mean value of 105 CFU/g of coli-
forms and 104 CFU/g of E. coli respectively. The distri-
butions of bacteriological counts obtained are summa-
rized in Table 2.
3.3. Antimicrobial Susceptibility Tests
3.3.1. Controlled Study
The possibility of the development of resistant microor-
ganisms to the two selected antibiotics was investigated
in the control milk samples. A total of 15 (78%) micro-
bial isolates showed some resistance to Gentamicin and
six out of the 19 isolates (32%) showed resistance pat-
terns to Streptomycin. Only six out of the 19 microbial
isolates (32%) showed antimicrobial multi-resistant pat-
terns to both Streptomycin and Gentamicin.
3.3.2. Experimental Samples
Antimicrobial resistance of microorganisms isolated
from the experimental milk samples was also tested and
the resistance patterns of the isolates are summarized in
Table 3. A significantly (P < 0.05) high number of coli-
forms (9 isolates) and Listeria monocytogene (16 isolates)
that were isolated from different milk samples showed
resistance to Gentamicin; whereas, only 5 isolates of co-
liform and 10 isolates of L. monocytogenes were resis-
tant to Streptomycin. The remaining isolates of L. inno-
cua, S. aureus and E. coli isolated were resistant to both
Gentamicin and Streptomycin (Table 3).
4. Discussion
The rapid growth of the dairy sector in the Middle East
and the lack of accurate data on the usage of antibiotics
particularly in Lebanon necessitated the investigation of
the potential presence of antibiotic residues in milk and
the adverse health risks associated with the development
of antimicrobial resistant pathogens. The questionnaire-
based survey identified the most commonly used antibi-
otics among the Lebanese dairy farmers and generated
data that were used in the consequent analytical parts of
this study. This approach helped to envision the antim-
icrobial usage on a detailed basis by interviewing the
farmers. The information collected from the various
owners and/or supervisors of the farms about the applica-
tion dosage, timing of intervention, adherence to with-
holding times and other international standards were va-
lidated by the experimental part of this study. The data
that were analyzed for the most commonly used two an-
tibiotics Gentamicin and Streptomycin showed insignifi-
cant discrepancy between the provided information and
the experimental results. This finding is important to-
wards alleviating concerns about the improper usage of
antibiotics in cattle and their residues in dairy products.
4.1. Analysis of Antibiotic Residues
Various studies have indicated that the exposure of ani-
mals to antimicrobial agents result in microbial resistance
to antibiotics which is possibly transferred to human pa-
thogens [23-26]. In addition, human exposure to signifi-
cant levels of antibiotic residues from animal products
may aggravate immunological responses in susceptible
individuals and negatively affect the intestinal microbiota
[26,27]. Therefore; in this preliminarily study the possi-
ble presence and level of antibiotic residues in milk sam-
ples from selected farms were investigated. The farmers’
compliance to withholding periods and proper usage of
such drugs were also established by comparing antibiotic
residue levels in the experimental samples with those ob-
tained from the controlled study. Although the results

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
6
Table 2. Percentage of samples tested positive for L. monocytogenes, L. innocua, S. aureus, E. c oli, coliforms and total aerobic
counts found in raw milk samples collecte d from 22 r a ndomly selec ted c a ttle far ms in Lebanon.
Type of bacteria 0 ND* (%) <103 CFU/g
(%) 103 to 104 CFU/g (%) 104 to 105 CFU/g (%) 105 to 106 CFU/g (%) 106 to 107 CFU/g (%) Total (%)****
TAC** 0 9*** 14 14 5 58 22 (100)
S. aureus 14 27 9 41 4 21 (95)
Coliform 45 18 18 19 0 0 12 (55)
E. coli 72 23 5 0 0 0 6 (28)
L. mono 14 23 9 27 18 9 19 (86)
L. innocua 54 5 14 27 0 0 10 (46)
*ND not detected in 25 g of sample tested; **Total aerobic count; ***Percentage of samples having a mean count in the specified range; ****Total number of
samples tested positive—number of samples tested negative for the microorganism; N: number of milk samples = 22.
Table 3. Resistance, multiresistance and intermediate resis-
tance patterns of microorganisms in the milk samples to
Gentamicin and Streptomycin.
**N (%) isolates resistant to
Microorganism
*Number of
isolates GM SM
S. aureus 21 21(100)a 20 (95)a
E. coli 10 10 (100)a 6 (60)a
Coliforms 9 9 (100)a 5 (55)b
L. monocytogenes 17 16 (94)a 10 (58)b
L. inocua 7 7 (100)a 6 (85)a
Percentages in same row with different superscripts are significantly differ-
ent at P < 0.05; GM = Gentamicin; SM = Streptomycin. *Number of isolates
isolated from the milk samples for each microorganism. **Number and
percentage of isolates of each microorganism resistant to Gentamicin and
Streptomycin.
obtained in this study show a widespread usage of Strep-
tomycin and Gentamicin in Lebanon, a general compli-
ance with international regulations for their uses and
proper withdrawal times were evident. However, since
all the samples collected from the visited farms contained
a certain level of antibiotic residues, this may imply a
repeated and prolonged treatment of dairy cattle with
these antibiotics. Hence, despite the fact that the usage in
general is in compliance with regulations with respect to
dosages and withholding periods, the excessive and ex-
tended applications among farmers may result in the de-
velopment of antimicrobial resistant microorganisms in
raw milk. This hypothesis was further investigated in this
study for the main pathogenic microorganisms isolated
from the various milk samples. In other studies, for in-
stance, S. aureus has been reported to frequently show
multiple antimicrobial resistance patterns [28-31].
4.2. Microbiological Analysis of Milk Samples
The unnecessary use of therapeutic doses of antibiotics
or as growth promoters in producing animals may be a
main cause for the selection of multiple resistant strains
of bacterial pathogens which can result in serious human
and animal infections [32,33]. The microbiological anal-
yses of both the experimental and control raw milk sam-
ples in this study allowed for the selection of the com-
monly present microorganisms in milk to be tested for
antibiotic resistance against Gentamicin and Streptomy-
cin. Interestingly, when comparing the microbiological
levels in the samples to the microbiological criteria as set
by the Commission Regulation [34], total aerobic counts,
L. monocytogenes and S. aureus exceeded the limit al-
lowed by the legislation for raw milk samples. As for E.
coli and coliforms, although present in the milk samples,
their levels were acceptable. These findings although not
the main scope of this study, indicate a possible health
risk because S. aureus may produce a heat stable toxin in
raw milk [35,36]. Furthermore, S. aureus has been known
to be the most prevalent pathogen to cause intramam-
mary infections in dairy ruminants leading to major eco-
nomic losses [37,38]. However, the S. aureus isolated
from the above samples were resistant to both Gentami-
cin and Streptomycin (Table 3) and this result is quite
alarming because if the drugs were or are to be used to
treat and control the condition, regular doses may no
longer be effective; thus, promoting a high health and
residual levels risk on the animals and humans. Another
serious pathogen, L. monocytogenes, was also isolated
from all collected milk samples. L. monocytogenes has
been linked with numerous outbreaks associated with
milk and milk products [39-41]. This pathogen may have
contaminated the milk samples through inadequate sani-
tation during milking, storage and transport or from in-
fected cows on the farms [42]. Thus, proper animal mo-
nitoring and handling techniques for milk should be ap-
plied on Lebanese farms. Furthermore, poor environ-
mental sanitation noticed during the farm visits may be
the cause for the elevated levels of total coliforms and E.
coli in the analyzed milk samples. Studies have shown
that E. coli, a normal habitat of human and animal intes-
tines, when constantly gets exposed to antibiotics; it de-

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
7
velops resistance in order to survive. When these resis-
tant isolates are excreted to the environment by feces,
they tend to spread resistance genes by vertical gene
transfer to pathogens [43-45]. Thus, this will result in re-
sistance to antimicrobial drugs used in treating infec-
tious diseases leading to serious health implications in
both humans and animals [46-48].
The above risks are reflected in the results that showed
all isolated microorganisms from the experimental and
the controlled study samples resistant to Gentamicin (Ta-
ble 3). With respect to Streptomycin, the findings were
as alarming because all microorganisms, except for E.
coli and coliforms isolated from the controlled milk sam-
ples, showed various resistance patterns. This may indi-
cate that a high percentage of the milk supply in the Le-
banese market may contain resistant strains of major pa-
thogens against the two drugs, Gentamicin and Strepto-
mycin, commonly used in therapeutic treatments; thus,
incurring a major public health concern. The excessive
use of Gentamicin and Streptomycin among the farms
may be a main cause for the emerging of antimicrobial-
resistant bacterial pathogens [49]. Furthermore, accord-
ing to Prescott and Baggot [16] resistance of animal pa-
thogens to aminoglycosides, particularly Streptomycin is
very common.
4.3. Conclusion
The findings of this study preliminarily validated the
claims of dairy Lebanese farmers about the proper usages
of antibiotics with respect to doses and withholding pe-
riods. However, the results highlighted a potential public
health problem reflected in the development of multi-
resistant pathogenic bacteria to both Gentamicin and Strep-
tomycin. A nationwide study is necessary in the near
future with more in depth analyses of the isolated resis-
tant pathogens perhaps at the genotypic levels.
5. Acknowledgements
The researchers would like to thank the University Re-
search Board at the American University of Beirut for
funding this study, the Agricultural Research and Educa-
tion Center (AREC), Faculty of Agricultural and Food
Sciences of the American University of Beirut for mak-
ing the controlled study possible, farmers and our labo-
ratory technician for helping in the analysis.
REFERENCES
[1] S. Joshi, “HPLC Separation of Antibiotics Present in
Formulated and Unformulated Samples,” Journal of Phar-
maceutical and Biomedical Analysis, Vol. 28, No. 5, 2002,
pp. 795-809. doi:10.1016/S0731-7085(01)00706-3
[2] M. Choueiri, “Emerging Contaminants in Lebanon: Anti-
biotic Residues in Cow Manure and Soil,” MS Thesis,
American University of Beirut, Beirut, 2008.
[3] W. C. John and E. H. Fred, “Antibiotics: III; Mechanism
of Action of Antimicrobial and Antitumor Agents,” Sprin-
ger-Verlag, New York, 1975.
[4] A. A. Bergwerff and J. Schloesser, “Antibiotics and Drugs
Residue Determination,” Encyclopaedia of Food Science,
Food Technology and Nutrition, 2003, p. 254.
[5] M. Dubois, D. Fluchard, E. Sior and P. H. Delahaut,
“Identification and Quantification of Five Macrolide An-
tibiotics in Several Tissues, Eggs and Milk by Liquid
Chromatography-Electrospray Tandem Mass Spectrome-
try,” Journal of Chromatography B: Biomedical Sciences
and Applications, Vol. 753, No. 2, 2001, pp. 189-202.
doi:10.1016/S0378-4347(00)00542-9
[6] The Council of the European Communities, “Council Re-
gulation (EEC) No.2377/90,” Official Journal of the Eu-
ropean Communities, No. L 224/P, 1990, pp. 1-8.
[7] Food and Drug Administration, “Tolerances for Residues
of New Animal Drugs in Food,” 2008.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/C
FRSearch.cfm?fr=530.41
[8] K. N. Woodward, “Antibiotics and Drugs Uses in Food
Production,” Encyclopedia of Food Science, Food Tech-
nology and Nutrition, 1993, p. 249.
[9] F. M. Aarestrup, “Antimicrobial Resistance in Bacteria of
Animal Origin,” ASM Press, Washington DC, 2006.
[10] L. Guardabassi, S. Schwarz and D. H. Lloyd, “Pet Ani-
mals as Reservoirs of Antimicrobial-Resistant Bacteria,”
Journal of Antimicrobial Chemotherapy, Vol. 54, No. 2,
2004, pp. 321-332. doi:10.1093/jac/dkh332
[11] I. Phillips, M. Casewell, T. Cox, B. De Groot, C. Friis, R.
Jones, C. Nightingale, R. Preston and J. Waddell, “Does
the Use of Antibiotics in Food Animals Pose a Risk to
Human Health? A Critical Review of Published Data,”
Journal of Antimicrobial Chemotherapy, Vol. 53, No. 1,
2004, pp. 28-52. doi:10.1093/jac/dkg483
[12] D. L. Smith, J. Dushoff and J. G. Morris, “Agricultural
Antibiotics and Human Health,” PLoS Medicine, Vol. 2,
No. 8, 2005, p. 232. doi:10.1371/journal.pmed.0020232
[13] E. J. Threlfall, “Antimicrobial Drug Resistance in Salmo-
nella: Problems and Perspectives in Food- and Water-
Borne Infections,” FEMS Microbiology Reviews, Vol. 26,
No. 2, 2002, pp. 141-148.
[14] A. Casadevall, “Crisis in Infectious Diseases: Time for a
New Paradigm?” Clinical Infectious Diseases, Vol. 23,
No. 4, 1996, pp. 790-794. doi:10.1093/clinids/23.4.790
[15] T. C. Eickhoff, “Antibiotics and Nosocomial Infections,”
In: J. V. Bennett and P. S. Brachman, Eds., Hospital In-
fections, 3rd Edition, Little, Brown and Company, Boston,
1992, pp. 245-264.
[16] J. F. Prescott and J. D. Baggot, “Antimicrobial Therapy in
Veterinary Medicine,” Iowa State University Press, Ames,
1993.
[17] F. Peles, M. Wagner, L. Varga, I. Hein, P. Rieck, K. Gut-
ser, P. Keresztúri, G. Kardos, I. Turcsányi, B. Béri and A.
Szabó, “Characterization of Staphylococcus aureus Strains

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
8
Isolated from Bovine Milk in Hungary,” International
Journal of Food Microbiology, Vol. 118, No. 2, 2007, pp.
186-193. doi:10.1016/j.ijfoodmicro.2007.07.010
[18] P. M. Griffin and A. V. Tauxe, “The Epidemiology of
Infections Caused by Escherichia coli O157:H7, Other
Enterohemorrhagic E. coli and the Associated Hemolytic
Uremic Syndrome,” Epidemiologic Reviews, Vol. 13, 1991,
pp. 60-98.
[19] H. P. R. Seeliger and D. Jones, “Genus Listeria,” In: J. G.
Holt, Ed., Bergey’s Manual of Systematic Bacteriology,
Vol. 2, 8th Edition, Williams and Wilkins, Baltimore,
1986, pp. 1235-1245.
[20] B.-C. Ye, S. Y. Li, P. Zuo and X.-H. Li, “Simultaneous
Detection of Sulfamethazine, Streptomycin, and Tylosin
in Milk by Microplate-Array Based SMM-FIA,” Food
Chemistry, Vol. 106, No. 2, 2007, pp. 797-803.
doi:10.1016/j.foodchem.2007.06.006
[21] H. A. Wallace, G. A. June, P. S. Sherrod, T. S. Hammack
and R. M. Amaguana, “Salmonella,” In FDA Bacteriolo-
gical Analytical Manual, 8th Edition, Washington DC,
1995.
[22] Clinical and Laboratory Standards Institute, “Performance
Standards for Antimicrobial Susceptibility Tests,” Na-
tional Committee for Clinical Laboratory Standards, Wayne,
2009.
[23] C. Chauvin, S. Le Bouquin-Leneveu, A. Hardy, D. Hag-
uet, J. P. Orand and P. Sanders, “An Original System for
the Continuous Monitoring of Antimicrobial Use in Poul-
try Production in France,” Journal of Veterinary Phar-
macology and Therapeutics, Vol. 28, No. 6, 2005, pp.
515-523. doi:10.1111/j.1365-2885.2005.00697.x
[24] A. L. Aronson, “Potential Impact of the Use of Antim-
icrobial Drugs in Farm Animals on Public Health,” Pre-
sented at the Meeting on Pharmacology in the Animal
Health Sector, Colorado State University, 1975.
[25] H. D. Mercer, “Antimicrobial Drugs in Food Producing
Animals,” Veterinary Clinics of North America, Vol. 5,
1977, pp. 3-5.
[26] S. D. Holmberg, M. T. Osterholm, K. A. Senger and M. L.
Cohen, “Drug-Resistant Salmonella from Animals Fed
Antimicrobials,” New England Journal of Medicine, Vol.
311, No. 10, 1984, pp. 617-622.
doi:10.1056/NEJM198409063111001
[27] P. H. Archimbault, “Persistence in Milk of Active An-
timicrobial Intramammary Substances. Veterinary Phar-
macology and Toxicology,” MTP Press Ltd., Lancaster,
2005.
[28] M. C. Enright, “The Evolution of Resistant Pathogen—
The Case of MRSA,” Current Opinion in Pharmacology,
Vol. 3, No. 5, 2003, pp. 474-479.
doi:10.1016/S1471-4892(03)00109-7
[29] N. H. Kwon, K. T. Park, J. S. Moon, W. K. Jung, S. H.
Kim and J. M. Kim, “Staphylococcal Cassette Chromo-
some Mec (SCCmec) Characterization and Molecular Ana-
lysis for Methicillin-Resistant Staphylococcus aureus and
Novel SCCmec Subtype IVg Isolated from Bovine Milk
in Korea,” Journal of Antimicrobial Chemotherapy, Vol.
56, No. 4, 2005, pp. 624-632. doi:10.1093/jac/dki306
[30] R. O’Mahony, Y. Abbott, F. C. Leonard, B. K. Markey, P.
J. Quinn and P. J. Pollock, “Methicillin-Resistant Staphyl-
ococcus aureus (MRSA) Isolated from Animals and Vet-
erinary Personnel in Ireland,” Veterinary Microbiology,
Vol. 109, No. 3-4, 2005, pp. 285-296.
doi:10.1016/j.vetmic.2005.06.003
[31] J. L. Wylie and D. L. Nowicki, “Molecular Epidemiology
of Community- and Health Care-Associated Methicillin-
Resistant Staphylococcus aureus in Manitoba, Canada,”
Journal of Clinical Microbiology, Vol. 43, No. 6, 2005,
pp. 2830-2836. doi:10.1128/JCM.43.6.2830-2836.2005
[32] WHO, “Surveillance for the Prevention and Control of
Health Hazards Due to Antibiotic Resistant Enterobacte-
ria,” Technical Rep ort Series 624, 1978.
[33] D. A. Barber, G. Y. Miller and P. E. McNamara, “Models
of Antimicrobial Resistance and Food-Borne Illness: Ex-
amining Assumptions and Practical Application,” Journal
of Food Protection, Vol. 66, No. 4, 2003, pp. 700-709.
[34] The Council of the European Communities, “Council Re-
gulation (EEC) No. 2073/2005,” Official Journal of the
European Communities, No. L 338, 2005, pp. 1-26.
[35] G. Normanno, G. La Salandra, A. Dambrosio, N. C.
Quaglia, M. Corrente, A. Parisi, G. Santagada, A. Firinu,
E. Crisetti and G. V. Celano, “Occurrence, Characteriza-
tion and Antimicrobial Resistance of Enterotoxigenic Sta-
phylococcus aureus Isolated from Meat and Dairy Prod-
ucts,” International Journal of Food Microbiology, Vol.
115, No. 3, 2007, pp. 290-296.
doi:10.1016/j.ijfoodmicro.2006.10.049
[36] M. L. Marco and M. H. J. Wells-Bennik, “Impact of Bac-
terial Genomics on Determining Quality and Safety in the
Dairy Production Chain,” International Dairy Journal,
Vol. 18, No. 5, 2008, pp. 486-495.
[37] O. Akineden, C. Annemüller, A. Hassan, C. Lämmler, W.
Wolter and M. Schöck, “Toxin Genes and Other Charac-
teristics of Staphylococcus aureus Isolates from Milk of
Cows with Mastitis,” Clinical and Diagnostic Laboratory
Immunology, Vol. 8, No. 5, 2001, pp. 959-964.
[38] A. Pengov, C. V. Flajs, T. Zadnik, J. Marinsek and M.
Pogacnik, “Distribution of Chloramphenicol Residues in
Lactating Cows Following an External Application,”
Analytica Chimica Acta, Vol. 529, No. 1-2, 2005, pp.
347-351. doi:10.1016/j.aca.2004.07.035
[39] D. W. Flemming, S. L. Cochi, K. L. Mackonald, J. Bron-
dum, P. S. Hayes and B. D. Plikaytis, “Pasteurised Milk
as a Vehicle of Infection in an Outbreak of Listeriosis,”
New England Journal of Medicine, Vol. 312, 1985, pp.
404-407. doi:10.1056/NEJM198502143120704
[40] M. J. Linnan, L. Mascola, X. D. Lou, V. Goulet, S. May,
C. Salminen, D. W. Hird, M. L. Yonekura, P. Hayes, R.
Weaver, A. Audurier, B. D. Plikaytis, S. L. Fannin, A.
Kleks and C. V. Broome, “Epidemic Listeriosis Associ-
ated with Mexican Style Cheese,” New England Journal
of Medicine, Vol. 319, 1988, pp. 823-828.
doi:10.1056/NEJM198809293191303
[41] O. Lyytikainen, T. Autio, R. Maijala, P. Ruutu, T. Hon-
kanen-Buzalski and M. Miettinen, “An Outbreak of Lis-
teria Monocytogenes Serotype 3a Infections from Butter

Quantification of Antibiotic Residues and Determination of Antimicrobial Resistance
Profiles of Microorganisms Isolated from Bovine Milk in Lebanon
9
in Finland,” Journal of Infectious Diseases, Vol. 181, No.
5, 2000, pp. 1838-1841. doi:10.1086/315453
[42] N. Bemrah, M. Sanaa, M. H. Cassin, M. W. Griffiths and
O. Cerf, “Quantitative Risk Assessment of Human Liste-
riosis from Consumption of Soft Cheese Made from Raw
Milk,” Preventive Veterinary Medicine, Vol. 37, No. 1-4,
1998, pp. 129-145. doi:10.1016/S0167-5877(98)00112-3
[43] V. Babak, J. Schlegelova and H. Vlkova, “Interpretation
of the Results of Antimicrobial Susceptibility Analysis of
Escherichia coli Isolates from Bovine Milk, Meat and
Associated Foodstuffs,” Food Microbiology, Vol. 22, No.
4, 2004, pp. 353-358. doi:10.1016/j.fm.2004.08.010
[44] H. Sorum and M. Sunde, “Resistance to Antibiotics in the
Normal Flora of Animals,” Veterinary Research, Vol. 32,
No. 3-4, 2001, pp. 227-241. doi:10.1051/vetres:2001121
[45] B. Catry, H. Laevens, L. A. Devriese, G. Opsomer and A.
de Kruif, “Antimicrobial Resistance in Livestock,” Jour-
nal of Veterinary Pharmacology and Therapeutics, Vol.
26, No. 2, 2003, pp. 81-93.
doi:10.1046/j.1365-2885.2003.00463.x
[46] H. Oppegaard, T. M. Steinum and Y. Wasteson, “Hori-
zontal Transfer of a Multi-Drug Resistance Plasmid be-
tween Coliform Bacteria of Human and Bovine Origin in
a Farm Environment,” Applied and Environmental Mi-
crobiology, Vol. 67, No. 8, 2001, pp. 3732-3734.
doi:10.1128/AEM.67.8.3732-3734.2001
[47] B. R. Berends, A. E. Van den Bogaard, F. Van Knapen
and J. M. Snijders, “Human Health Hazards Associated
with the Administration of Antimicrobials to Slaughter
Animals. Part II. An Assessment of the Risks of Resistant
Bacteria in Pigs and Pork,” Veterinary Quarterly, Vol. 23,
No. 1, 2001, pp. 10-21.
doi:10.1080/01652176.2001.9695069
[48] J. W. Chow, V. Kak, I. You, S. J. Kao, J. Petrin, D. B.
Clewell, S. A. Lerner, G. H. Miller and K. J. Shaw, “Ami-
noglycoside Resistance Genes aph(200)-Ib and aac(60)-
Im Detected Together in Strains of Both Escherichia coli
and Enterococcus faecium,” Antimicrobial Agents and
Chemotherapy, Vol. 45, No. 10, 2001, pp. 2691-2694.
doi:10.1128/AAC.45.10.2691-2694.2001
[49] S. Magnet and J. S. Blanchard, “Molecular Insights into
Aminoglycoside Action and Resistance,” Chemical Re-
views, Vol. 105, No. 2, 2005, pp. 477-498.
doi:10.1021/cr0301088