Antimicrobial in Milk PDF

PREVALENCE OF ANTIMICROBIAL RESIDUES IN MILK AND
DAIRY PRODUCTS IN THE STATE OF KUWAIT
HUSAM ALOMIRAH1,4, HANI AL-MAZEEDI1, SAMEER AL-ZENKI1,

TAREQ AL-AATI1, JAMLA Al-OTAIBI1, MAHA AL-BATEL2
and JIWAN SIDHU3
1
Biotechnology and Food Department
Food Resources and Marine Sciences Division
Kuwait Institute for Scientific Research
PO Box 24885, 13109-Safat, Kuwait
2
Dairy and Fodder Analysis Laboratory
Public Authority for Agriculture and Fisheries Resources
PO Box 21422, Safat, Kuwait
3
Department of Family Sciences
College for Women, Kuwait University
PO Box 5969, Safat, Kuwait
ABSTRACT
Approximately, 1,000 locally produced and imported milk and dairy

product samples were collected at different seasonal periods from differ-
ent farms and retail outlets in Kuwait, and screened for the presence of
four antimicrobial residues (beta-lactams, tetracyclines sulfonamides and
chloramphenicol) using Charm II system. Results indicated that 29.1% of the
analyzed local fresh milk samples were above the maximum residue level
(MRL) for tested residues with tetracycline as predominant residue. The preva-
lence of antimicrobial residues in imported pasteurized milk samples (5.4%)
was higher than that of local pasteurized milk samples (3.4%). No residues
were detected in powdered and condensed milk samples imported from the
European Union (EU) countries with the exception of tetracyclines. Results
also showed that 9.4% of the analyzed imported cheese samples were above
the MRL for tested residues, while tested ice cream samples were negative for
antimicrobial residues. The significant presence of antimicrobial residues,
particularly chloramphenicol, in raw and pasteurized milk, clearly implies the
misuse of antimicrobial drugs in animal farms in Kuwait and neighboring
4
Corresponding author. TEL: (965) 488-9177; FAX: (965) 488-9069; EMAIL: omirah@kisr.edu.kw
Journal of Food Quality 30 (2007) 745–763. All Rights Reserved.

© 2007, The Author(s) 745
Journal compilation © 2007, Blackwell Publishing
746 H. ALOMIRAH ET AL.
countries, and implies the need for stricter regulations on antimicrobial drug
usage in the dairy industry.
PRACTICAL APPLICATIONS
This article addresses the generation of a baseline data on the current

presence of four antimicrobial residues in locally produced and imported milk
and dairy products in the state of Kuwait. The outcome of this study will
provide valuable baseline information for local governmental authorities for
effective monitoring for the use and misuse of agricultural antimicrobial drugs.
Such data will also be important for other Gulf Cooperation Council countries,
because such countries share similar farming practices and import comparable
foodstuffs.
INTRODUCTION
Antimicrobial drugs are a chemically heterogeneous group of small

organic molecules of microbial origin that, at low concentrations, are delete-
rious to the growth or metabolic activities of other microorganisms
(Thomashow and Weller 1996). Although the use of antimicrobial drugs has
significantly improved the health and production efficiency of food-producing
animals, antimicrobial resistance is of concern. The continuous presence of the
antimicrobial drugs in animals has led to the evolution of antimicrobial-
resistant microbes that are pathogenic to animals and humans. Moreover,
antimicrobial residues in food products of animal origin may potentially cause
allergic reactions and alter the dynamics of the microflora in the intestinal
tracts of humans (Witte 1998).
Presently, the reference test method used for the detection of antimicro-
bial residues in milk and dairy products is the microbiological inhibition test.
These microbiological tests depend on the rapid growth and acid production of
Bacillus stearothermophilus. However, the microbiological test is quite
complex and lengthy to carry out as it involves the continuous growth of B.
stearothermophilus spores. Moreover, microbiological inhibitor tests are not
specific for antimicrobial pharmacological group, and there are occasional
reports of false-positive reactions associated with other inhibitors such as
lactoferrin, lysozyme and/or sanitizers. Because of the disadvantages of the
microbiological inhibitor tests, several companies have offered more rapid
commercial antimicrobial residue tests such as Beta Star, Charm II, Delvo-X-
Press bL-II, Penzyme 50 and SNAP Beta-lactam. Kroll et al. (2000) have
evaluated these commercial rapid tests and showed that three rapid tests,
ANTIMICROBIAL RESIDUES IN MILK 747
namely, Beta Star, Charm II and SNAP Beta-lactam, were found to be accept-
able, with the Charm II and SNAP Beta-lactam rapid tests showing more than
98% agreement with the microbiological reference methods.
The Charm II assay for b-lactams, tetracyclines, macrolides, aminogly-
cosides, sulfonamides and chloramphenicol in milk became AOAC-A1
method in 1989 (Charm and Chi 1988). The principle of the Charm II
system for class drug residue analysis is based on the use of two reagents:
a radio-labeled [14C] or [3H] antimicrobial drug (tracer) and a binding
reagent (microbial or immunochemical receptor). When the receptor is
added to milk containing antimicrobial residue, the contaminating residue
binds to the receptor. When the tracer is added, any antimicrobial residue in
the sample bound to the receptor will prevent the tracer from binding to
these sites. The bound receptor/tracer complex is then separated from the
unbound tracer by centrifugation and measured with a liquid scintillation
counter (counts per minute). Count reading of sample is then compared
either with counts of a negative or a positive standard sample.
The most effective way to reduce the rate of evolution of drug resistance
and to prevent toxic residues from appearing in consumed foods of animal
origin is to control the use of antimicrobial drugs. Control of the use of
antimicrobial drugs and prevention of their misuse rely primarily on an effec-
tive, well-planned and conducted, national drug residue surveillance pro-
gram(s). In the state of Kuwait, at the present time, information on the levels
of antimicrobial residues in imported and locally produced milk and dairy
products remains unknown. No surveys have been undertaken to assess residue
concentrations or seasonal variations, and the decision on the safe use of
antimicrobial drugs in animal production is left to the farming community.
Because of the lack of data on the presence of antimicrobial residues in
local and imported milk and dairy products, the outcome of this study will
provide valuable baseline information for local governmental authorities for
effective monitoring for the use and misuse of agricultural antimicrobial drugs.
Such data will also be important for other Gulf Cooperation Council countries,
because such countries share similar farming practices and import comparable
foodstuffs.
The objective and quantitative data of antimicrobial residues presented
in this article are essential for risk analyses and management, and is helpful
in evaluating the effectiveness of the national food control system. Further-
more, these data can also be utilized by the concerned expert committees in
international organizations such as the World Organization for Animal
Health and Food and Agriculture Organization/World Health Organization of
the United Nations in their efforts to help local and regional countries in
developing their monitoring and surveillance programs to meet international
standards. This will facilitate international harmonization and trade.
MATERIALS AND METHODS
Sampling
The snapshot sampling protocol plan used in this study is presented in
Table 1. The objective of the snapshot survey was to collect a total of 1,038
milk and dairy product samples from as many outlets as practically possible in
a short time period as possible. The snapshot sample collection was repeated
during the period from April 2004 to February 2005 to ensure that time of
production and seasonal variation have been taken into consideration, and
therefore, comparison will be valid, and any trends in the use of antimicrobials
on a state scale can be determined.
TABLE 1.
COLLECTION SAMPLING PLAN FOR LOCALLY PRODUCED AND IMPORTED MILK AND
DAIRY PRODUCTS IN THE STATE OF KUWAIT DURING THE YEAR 2004/2005
Food item Testing Sampling plan Total

frequency samples
Summer Winter
Local raw milk 11 6 (30 farms) 5 (30 farms) 330

Local pasteurized milk
Full cream milk 36 20 (3 brands) 16 (3 brands) 108
Low-fat milk 11 6 (3 brands) 5 (3 brands) 33
Skimmed milk 23 13 (3 brands) 10 (3 brands) 69
Subtotal 210
Imported pasteurized milk
Full cream milk 20 10 (10 brands) 10 (10 brands) 200
Low-fat milk 18 9 (6 brands) 9 (6 brands) 108
Skimmed milk 5 3 (4 brands) 2 (4 brands) 20
Subtotal 328
Imported condensed milk
Full cream milk 5 5 (5 brands) 25
Light cream milk 1 1 (4 brands) 4
Subtotal 29
Imported powdered milk
Full cream milk 2 2 (8 brands) 16
Infant formula 2 2 (8 brands) 16
Subtotal 32
Cheese
Imported 5 3 (15 brands) 2 (15 brands) 75
Local 2 1 (2 brands) 1 (2 brands) 4
Subtotal 79
Ice cream
Imported 3 2 (6 brands) 1 (6 brands) 18
Local 3 2 (3 brands) 2 (3 brands) 12
Subtotal 30
Grand total 1,038
A minimum of three raw milk samples were collected from the pooled
milk from dairy farms. The raw milk samples were stored in polycarbonate
containers, kept in ice and then transported to the laboratory. Similarly, three
samples of pasteurized milk or dairy products of selected brands were col-
lected from outlets according to the sampling plan (Table 1). Of the three milk
samples, one sample was used for the screening test, while the remaining
two samples were used for further confirmation tests, if necessary. Although
(1,038) milk and dairy product samples were collected for the analysis, it is
envisaged that the results of the analysis of 997 samples tested in duplicate
should give an indication of the current use and/or misuse of antimicrobial
residues in Kuwait.
Sample and Standard Assay Procedure

The Charm II 7600 system (Charm Sciences Inc., Lawrence, MA) was
used to screen for the presence of antimicrobial residues (beta-lactams, tetra-
cyclines, sulfonamides and chloramphenicol) in milk and dairy products from
Kuwaiti dairy farms and outlets. The used Charm II kits were sensitive to
detect antimicrobial residues at the EU maximum residue limits (Council
Regulation (EEC) No. 2377/90.1990 and amendments). Sample and standard
preparation, as well as Charm II test sequential assay procedure, for beta-
lactams, tetracyclines, sulfonamides and chloramphenicol class drugs in milk
and dairy product samples was carried out according to the protocols provided
by the manufacturer. Control points for each class of antimicrobial residues
analyzed, as well as for each new lot of reagents and kits, were determined.
The control point is a measurement (counts per minute) of the radio-labeled
drug (tracer) binding to the specific binding sites (receptor) using a liquid
scintillation counter and determined from a negative reference or positive
spiked sample.
Confirmation of Charm II System Results with Liquid

Chromatography-Mass Spectrometry (LC/MS/MS) and Gas
Chromatography-Mass Spectrometry in Negative Chemical Ionization
(GC/MS/NCI) Mode
Selected samples analyzed by the Charm II system for tetracycline (eight
samples), chloramphenicol (six samples) and sulfonamide (five samples) resi-
dues were confirmed using GC/MS/NCI and LC/MS/MS in a private laboratory
(Maxxam Analytics Inc., Mississauga, Ontario, Canada). Samples intended to
be analyzed for tetracycline residues were carried out by LC/MS/MS with a
method detection limit (MDL) of 10 ppb for chloratetracycline, and 5 ppb for
tetracycline and oxytetracycline. Chloramphenicol residues were analyzed by
LC/MS/MS and GC/MS/NCI with a MDL of 1 ppb and 0.15 ppb, respectively.
While sulfonamide residues were analyzed by GC/MS/NCI with a MDL of

0.02 ppb for sulfadiazine, sulfadimethoxine, sulfamethazine, sulfaquinoxaline
and sulfathiazole. All confirmation tests were carried out in duplicate.
RESULTS AND DISCUSSION
Local Raw Milk

Antimicrobial residue families detected in local raw milk samples are
shown in Table 2. Samples were collected from 30 local farms of different
sizes during the period from April 2004 to February 2005. Less number of raw
milk samples was collected during the period of August to November 2004
because cows were mainly dry. Antimicrobial residues detected in raw milk
samples were beta-lactams, tetracyclines and chloramphenicol. Sulfonamide
residues in these samples were not analyzed for the complete sampling period
because of stock shortage of sulfonamide analysis kit, and therefore, sulfona-
mide results are not presented. Out of the 308 samples analyzed for beta-
lactams, 62 samples (20.1%) were above the maximum residue level (MRL)
(4 ppb). One hundred twenty-one samples (37.3%) were above the tetracycline
MRL (100 ppb) from a total of 324 analyzed samples. Sixty-one samples
(29.4%) were positive for chloramphenicol (no MRL, prohibited antimicrobial
drug) from a total of 207 analyzed samples.
The percentage of contaminated local raw milk samples with beta-lactam,
tetracycline and chloramphenicol residues in relevance to the collection period
is shown in Fig. 1. It is clear that a higher percentage of contaminated milk
samples occurs at the interval between the months of August to November. As
we mentioned previously, local cows are dry during this season. These results
are similar to those reported by Radostitis et al. (1994) who indicated that
milking dry cows represents a major source of antimicrobial residues in raw
milk samples. The significant presence of antimicrobial residues, particularly
chloramphenicol, in tested raw milk could be of public health significant to
consumer.
Local Pasteurized Milk

Antimicrobial residue families detected in local pasteurized milk samples
are shown in Table 3. These samples represent the products of the only three
local milk-producing companies in Kuwait. Emphasis was made to collect an
equal number of full cream, low fat and skimmed milk samples from each of
these local companies. Approximately 50% of the collected milk samples were
full cream milk, 35% were skimmed milk and only 15% were low-fat milk.
Sample size was based on personal communication with managers of various
TABLE 2.
ANTIMICROBIAL RESIDUE FAMILIES DETECTED IN LOCAL RAW MILK SAMPLES COLLECTED DURING THE YEAR 2004/2005
Month Beta-lactams Tetracycline Chloramphenicol
Samples Samples Samples Samples Samples Samples Samples Negative Positive

analyzed below MRL above MRL analyzed below MRL above MRL analyzed samples samples
April–May 47 41 6 46 26 20 7 5 2
June–July 114 107 7 122 49 73 49 28 21
August–September 4 0 4 14 3 11 4 0 4
October–November 14 9 5 9 3 6 4 0 4
December–January 84 56 28 88 78 10 99 80 19
February 45 33 12 45 44 1 44 33 11
Total 308 246 62 324 203 121 207 146 61
ANTIMICROBIAL RESIDUES IN MILK
MRL, maximum residue level.

751
Beta-Lactams
100%
% of Contaminated Samples
80%
60%
40%
20%
0%
Apr.- Jun.- Aug.- Oct.- Dec.- Feb.
May Jul. Sep. Nov. Jan.
Months
Tetracyclines
100%
80%
60%
40%
20%
0%
Months
Chloramphenicol
100%
80%
60%
40%
20%
0%
Months
FIG. 1. PERCENTAGE OF CONTAMINATED LOCAL RAW MILK SAMPLES WITH

b-LACTAMS, TETRACYCLINE AND CHLORAMPHENICOL RESIDUES AS A FACTOR OF
COLLECTION PERIOD
TABLE 3.
ANTIMICROBIAL RESIDUE FAMILIES DETECTED IN LOCAL PASTEURIZED MILK SAMPLES COLLECTED DURING THE YEAR 2004/2005
Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol
Samples Samples Samples Samples Samples Samples Samples Samples Samples Samples Negative Positive
analyzed below above analyzed below above analyzed below above analyzed samples samples
MRL MRL MRL MRL MRL MRL
Full cream milk 108 106 2 108 101 7 97 97 0 93 92 1

Low-fat milk 33 32 1 33 29 4 27 27 0 26 26 0
Skimmed milk 68 63 5 67 62 5 57 57 0 56 55 1
Total 209 201 8 208 192 16 181 181 0 175 173 2

753
cooperative societies in Kuwait, who indicated such consumption pattern for

local pasteurized milk products. Samples were analyzed for beta-lactams,
tetracyclines, sulfonamides and chloramphenicol. Out of the 209 samples
analyzed for beta-lactams, eight samples (3.8%) were above the MRL (4 ppb).
Sixteen samples (7.7%) were above the tetracycline MRL (100 ppb) from a
total of 208 analyzed samples, and two samples (1.1%) were positive for
chloramphenicol from a total of 175 analyzed samples. All of the 181 local
pasteurized milk samples collected were tested negative for sulfonamide resi-
dues. Although it is difficult to correlate between the results of local raw and
pasteurized milk samples, it is evident that local milk was frequently contami-
nated with tetracycline residues. These results coincide with the outcome of
our field survey in local dairy farms, which revealed that tetracycline was the
prevalent drug used in most farms. Furthermore, the notable reduction in the
incidence of antimicrobial residues in pasteurized milk sample to that of raw
milk samples may be associated with the effect of pasteurization. Various
studies have reported that severe heating can inactivate antimicrobial residues
in foods (Rose et al. 1996; Moats 1999; Fang 2004). Moreover, Kang et al.
(2005) have demonstrated that the heat treatment of milk samples over with-
drawal times at 82C for 5 min can denature natural inhibitors resulting in
reducing false-positive outcomes by 89% when using the microbiological
inhibition test (Delvotest SP assay). Kang and Kondo (2001) have also related
the increase in the somatic cell count with increased probability of a false-
positive result using the Delvotest SP assay. Although Charm II test utilizes
specific receptors or an antibody in immunobinding assay formats, the reduc-
tion of natural inhibitors by heat treatment cannot be totally ignored.
Imported Pasteurized Milk

Antimicrobial residue families detected in imported pasteurized milk
samples are shown in Table 4. Because of the short shelf life of pasteurized
milk samples, all of the collected imported brands were from neighboring
countries. In this snapshot survey, 11 brands of full cream milk samples
(9 Saudi Arabia, 1 Bahrain and 1 Iran), six brands of low-fat milk samples
(5 Saudi Arabia and 1 Bahrain) and four brands of skimmed milk samples
(2 Saudi Arabia, 1 Bahrain and 1 Iran) were collected. Emphasis was made to
collect comparable number of milk samples from each brand in the full cream,
low fat and skimmed milk products. Approximately, 60% of the collected
samples were full cream milk, 30% were low-fat milk and the remaining 10%
were skimmed milk samples. The collected imported milk samples were
screened for beta-lactams, tetracyclines, sulfonamides and chloramphenicol.
Out of the 313 samples analyzed for beta-lactams, 28 samples (8.9%) were
above the MRL (4 ppb), 22 samples were above the tetracycline MRL
TABLE 4.
ANTIMICROBIAL RESIDUE FAMILIES DETECTED IN IMPORTED PASTEURIZED MILK SAMPLES COLLECTED DURING THE YEAR
2004/2005
Full cream milk 198 186 12 192 178 14 182 179 3 178 171 7
Low-fat milk 96 86 10 95 90 5 84 83 1 82 82 0
Skimmed milk 19 13 6 19 16 3 19 19 0 19 16 3
Total 313 285 28 306 284 22 285 281 4 279 269 10

755
(100 ppb) from a total of 306 analyzed samples (7.1%), and 10 samples (3.6%)
were positive for chloramphenicol from a total of 279 analyzed samples.
Results also revealed that out of the 285 imported milk samples tested for
sulfonamides, four samples (1.4%) were above the MRL (100 ppb). As in the
case for local pasteurized milk samples, the percentage of tetracycline residue
contamination in imported pasteurized milk samples was approximately 7%.
However, the percentage of residue contamination for beta-lactams, chloram-
phenicol and sulfonamide in imported pasteurized milk samples was higher to
that in local pasteurized milk samples.
In general, the prevalence of antimicrobial residues in imported pasteur-
ized milk samples (5.4%) was higher than that of local pasteurized milk
samples (3.4%). High levels of antimicrobial residues in imported and local
pasteurized milk samples clearly imply the misuse of antimicrobial drugs in
animal farms in Kuwait as well as in the neighboring countries. These prac-
tices may result in the spread of drug-resistant microbes, which are considered
to be a serious threat to the public health. Therefore, effective and well-
planned national antimicrobial residues surveillance programs must be con-
ducted at regular intervals to ensure that farmers are adhering to the guidelines
for antimicrobial use and withdrawal.
Condensed and Powdered Milk
Antimicrobial residue families detected in imported condensed and pow-
dered milk samples are shown in Tables 5 and 6, respectively. Four brands of
imported condensed milk and eight brands of imported powdered milk that are
highly consumed in Kuwait were selected in this snapshot survey. All brands
were imported from the EU countries. For the condensed milk samples, only
full cream and light cream samples were collected, and for the powdered milk,
only full cream and infant formula samples were collected, as low fat and
skimmed condensed and powdered milk samples were not available. Samples
were screened for beta-lactams, tetracyclines, sulfonamides and chloram-
phenicol. All of the 96 condensed milk samples tested were negative for
beta-lactams, sulfonamides and chloramphenicol. For tetracycline, two
samples (8.7%) were above the MRL (100 ppb). Furthermore, all of the 106
powdered milk samples were negative for all the tested antimicrobial residues.
The low incidence of antimicrobial residues in imported condensed and pow-
dered milk samples indicates good control and proper use of antimicrobial
drugs in EU countries. Such appropriate use of antimicrobial drugs is highly
recommended in animal farms in Kuwait and in the neighboring countries.
Cheese
Antimicrobial residue families detected in imported and local cheese
samples are shown in Tables 7 and 8, respectively. In this snapshot survey, 72
TABLE 5.
ANTIBIOTIC RESIDUE FAMILIES DETECTED IN IMPORTED CONDENSED MILK SAMPLES COLLECTED DURING THE YEAR 2004/2005
Full cream milk 21 21 0 20 18 2 21 21 0 20 20 0

Light cream milk 4 4 0 3 3 0 4 4 0 3 3 0
Total 25 25 0 23 21 2 25 25 0 23 23 0
TABLE 6.
ANTIBIOTIC RESIDUE FAMILIES DETECTED IN IMPORTED POWDERED MILK SAMPLES COLLECTED DURING THE YEAR 2004/2005

analyzed below above analyzed below above analyzed below Above analyzed samples samples
Full cream milk 11 11 0 11 11 0 11 11 0 11 11 0

Infant formula 15 15 0 16 16 0 16 16 0 15 15 0
Total 26 26 0 27 27 0 27 27 0 26 26 0

757
758
TABLE 7.
ANTIBIOTIC RESIDUE FAMILIES DETECTED IN IMPORTED CHEESE SAMPLES COLLECTED DURING THE YEAR 2004/2005
Soft cheese
Domiati (Egyptian 6 6 0 6 6 0 6 5 1 6 4 2
white soft cheese)
Feta 35 34 1 35 35 0 35 27 8 35 29 6
Danish white cheese 8 8 0 8 8 0 8 8 0 8 7 1
Baladi (soft white 13 12 1 13 13 0 13 11 2 13 12 1
cream cheese)
H. ALOMIRAH ET AL.
Arish (Egyptian 6 6 0 6 6 0 6 4 2 6 6 0
white soft cheese)
Hard cheese
Mozzarela 1 1 0 1 1 0 1 1 0 1 1 0
Halumi 2 2 0 2 2 0 2 2 0 2 2 0
Cheddar 1 1 0 1 1 0 1 0 1 1 0 1
Total 72 70 2 72 72 0 72 58 14 72 61 11

TABLE 8.
ANTIBIOTIC RESIDUE FAMILIES DETECTED IN LOCAL CHEESE SAMPLES COLLECTED DURING THE YEAR 2004/2005
Soft cheese
Baladi (soft white 4 4 0 4 4 0 4 4 0 4 4 0
cream cheese)
Total 4 4 0 4 4 0 4 4 0 4 4 0
TABLE 9.
ANTIBIOTIC RESIDUE FAMILIES DETECTED IN IMPORTED AND LOCAL ICE CREAM SAMPLES COLLECTED DURING THE YEAR
2004/2005
Imported ice cream 13 13 0 13 13 0 13 13 0 13 13 0

Local ice cream 10 10 0 10 10 0 10 10 0 10 10 0
Total 23 23 0 23 23 0 23 23 0 23 23 0
759

imported and 4 local cheese samples were collected from retail outlets in
Kuwait. Because of the limited production of local cheese in Kuwait, only four
local cheese samples were collected. The collected imported brands were from
Egypt, Iran, Saudi Arabia and the EU countries. Samples were grouped into
soft or hard cheese. The soft cheese group included Domiati (Egyptian white
soft cheese), Feta, Danish white cheese, Baladi (soft white cream cheese) and
Arish (Egyptian white soft cheese). While the hard cheese group included
Mozzarella, Halumi and Cheddar cheese. Emphasis was made to collect com-
parable number of cheese samples from each brand in the full cream, low-fat
and low-salt cheese products. The collected cheese samples were screened
for beta-lactams, tetracyclines, sulfonamides and chloramphenicol. Out of the
72 imported cheese samples analyzed for antimicrobial residues, 2 samples
(2.7%) were above the beta-lactams MRL (4 ppb), 14 samples (19.4%) were
above the sulfonamides MRL (100 ppb), 11 samples (15.2%) were positive for
chloramphenicol and no sample tested were positive for tetracycline residues.
This trend of residue contamination is different from that of imported pasteur-
ized milk where beta-lactam residues were the most frequent (8.9%), followed
by tetracyclines (7.1%), chloramphenicol (3.6%) and sulfonamides (1.4%).
This can be related to the fact that all brands of imported pasteurized milk
samples tested were from Saudi Arabia, Bahrain and Iran, while imported
cheese brands were from Egypt, Iran, Saudi Arabia and the EU countries. This
confirms our previous observation that variation in sample origin results in
different trends of antimicrobial residues. Furthermore, more than 90% of the
tested cheese samples with residues exceeding the MRL were from the soft
cheese group. The low contamination found in hard cheese group could be
attributed to the reduced solubility of hard cheese during sample preparation,
which may have affected residue detection. Table 8 illustrates that all four
local soft cheese samples tested were negative for antimicrobial residues.
However, because of the limited number of tested samples, one cannot con-
clude that local cheese samples are free from antimicrobial residues, especially
with the high incident of antimicrobial residues in local raw and pasteurized
milk samples. Therefore, more routine investigation of local cheese samples is
needed to ensure that the milk used in local cheese manufacturing is properly
controlled.
Ice Cream
Antimicrobial residue families detected in imported and local ice cream
samples are shown in Table 9. In this snapshot survey, 13 imported and 10
local ice cream samples were collected from retail outlets in Kuwait. Imported
brands were from U.S.A., Iran, Saudi Arabia and EU. All collected ice cream
samples were full cream products, because low-fat ice cream was not available
in most brands. Antimicrobial families analyzed in the collected ice cream

samples were beta-lactams, tetracyclines, sulfonamides and chloramphenicol.
Table 9 illustrates that all of the 13 imported and 10 local ice cream samples
were negative for the analyzed antimicrobial residues. Although the total
number of tested ice cream samples was relatively limited, the absence of
antimicrobial residues in the tested samples could be related to interference
made by residual fat, preservatives, emulsifiers and thickeners used in ice
cream manufacturing rather than the controlled use of antimicrobial drugs.
This explanation is supported by our previous observation of misuse of anti-
microbial drugs in milk samples.
Confirmation of Charm II System Results with LC/MS/MS and

GC/MS/NCI
Four presumptive negative and four presumptive positive milk samples
for tetracycline residues as determined by the Charm II system were confirmed
by LC/MS/MS. Results indicated that all presumptive negative samples were
also found negative by LC/MS/MS. However, only two presumptive positive
milk samples were found to have oxytetracycline at a concentration less than
the MDL of 5 ppb, while the remaining two presumptive positive milk samples
tested were negative by LC/MS/MS.
For chloramphenicol, two presumptive negative and six presumptive
positive milk samples as determined by the Charm II system were confirmed
by LC/MS/MS and GC/MS/NCI. Results indicated that the two presumptive
negative samples were also negative by LC/MS/MS and GC/MS/NCI.
However, only two presumptive positive milk samples were found to have
chloramphenicol at a concentration less than the MDL of 0.15 ppb, while the
remaining two presumptive positive milk samples tested were negative by
LC/MS/MS and chloramphenicol.
For sulfonamides, one presumptive negative and four presumptive posi-
tive milk samples as determined by the Charm II system were confirmed by
GC/MS/NCI NCI for sulfadiazine, sulfadimethoxine, sulfamethazine, sul-
faquinoxaline and sulfathiazole with an MDL of 0.02 ppb. Results indicated
that all presumptive negative and presumptive positive samples were found
negative by GC/MS/NCI. This might be due to the presence of other groups
of sulfonamide residues in the milk samples than the five tested groups by
GC/MS/NCI.
The confirmation results indicate that the Charm II system was reliable
for rapid screening of antimicrobial residues, and the few cases of false-
positive results obtained by Charm II system necessitate the need to conduct a
confirmatory analysis with conventional methods. Overall, the rapid screening
ability of the Charm II system is essential to rule out negative results and
reduce the cost of analyses using highly sophisticated and time-consuming

conventional methods.
ACKNOWLEDGMENT
The authors are grateful to the Kuwait Foundation for Advancement of

Science (KFAS) for the partial funding and continued support for the project.
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PREVALENCE OF ANTIMICROBIAL RESIDUES IN MILK AND

DAIRY PRODUCTS IN THE STATE OF KUWAIT

HUSAM ALOMIRAH1,4, HANI AL-MAZEEDI1, SAMEER AL-ZENKI1,

Approximately, 1,000 locally produced and imported milk and dairy

Journal of Food Quality 30 (2007) 745–763. All Rights Reserved.

This article addresses the generation of a baseline data on the current

Antimicrobial drugs are a chemically heterogeneous group of small

MATERIALS AND METHODS

Food item Testing Sampling plan Total

Local raw milk 11 6 (30 farms) 5 (30 farms) 330

Sample and Standard Assay Procedure

Confirmation of Charm II System Results with Liquid

While sulfonamide residues were analyzed by GC/MS/NCI with a MDL of

RESULTS AND DISCUSSION

Local Raw Milk

Local Pasteurized Milk

Month Beta-lactams Tetracycline Chloramphenicol

Samples Samples Samples Samples Samples Samples Samples Negative Positive

MRL, maximum residue level.

FIG. 1. PERCENTAGE OF CONTAMINATED LOCAL RAW MILK SAMPLES WITH

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

Full cream milk 108 106 2 108 101 7 97 97 0 93 92 1

MRL, maximum residue level.

cooperative societies in Kuwait, who indicated such consumption pattern for

Imported Pasteurized Milk

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

MRL, maximum residue level.

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

Full cream milk 21 21 0 20 18 2 21 21 0 20 20 0

MRL, maximum residue level.

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

Full cream milk 11 11 0 11 11 0 11 11 0 11 11 0

MRL, maximum residue level.

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

MRL, maximum residue level.

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

MRL, maximum residue level.

Commodities Beta-lactams Tetracycline Sulfonamides Chloramphenicol

Imported ice cream 13 13 0 13 13 0 13 13 0 13 13 0

MRL, maximum residue level.

in most brands. Antimicrobial families analyzed in the collected ice cream

Confirmation of Charm II System Results with LC/MS/MS and

reduce the cost of analyses using highly sophisticated and time-consuming

The authors are grateful to the Kuwait Foundation for Advancement of

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