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The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended for
muscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated with
aqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solution
without tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones of
the beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin and
ciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels the
differences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappeared
completely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected in
spiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These results
indicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intact
meat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initially
intended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screening
purposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitive
for beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolones
enrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.
Introduction
Microbiological inhibition tests are considered as multi-residue
screening tests for antibiotics in milk, meat or other animal
tissues. Several methods have been described to investigate
disks of frozen tissue, which are laid directly on one or more
agar media, each seeded with a susceptible bacterial strain.15
An inhibition test method is useful for detection of an
antibiotic or a group of antibiotics, if the detection limits of
these antibiotics are below safe levels or maximal residue limits
(MRL). With agar diffusion methods used for animal tissues,
such as the Four Plate Test (FPT), detection limits of antibiotics
have most often been determined using aqueous solutions of
analytical standards.1,4 However, residues are detected directly
in undiluted meat with the FPT and other comparable tests, and
effects of this matrix on detection limits never have been
determined.
Residues of substances other than antibacterials are nowadays most often detected with immunological or chemical
methods. To determine the recovery of the analyte or analytes,
a spiked sample is prepared by adding a known amount of
analytical standard to the sample before it is mixed with
extraction fluid. The extraction is then followed by a clean-up
procedure and a detection step. Inhibition tests such as the FPT
detect residues in intact meat, without any extraction or cleanup procedures; determination of recoveries is not necessary.
During routine testing, spiked samples are not prepared and this
is another reason why matrix effects have never been measured.
The composition and the properties of the medium, used in a
microbiological inhibition test, influence the detection limits of
antibiotics.3,57 For example, the pH of the test medium is an
important factor influencing the detection limits of most
antibiotics,3,5 and inhibitory zones produced by 500 ng sulfadimidin may vary from 0 to more than 10 mm, depending on the
origin of the peptone in the medium.7 It is therefore possible that
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2. Bacterial suspensions
Bacillus subtilis spore suspension: Merck (Darmstadt, Germany) No. 10649 is a ready-to-use suspension.
M. luteus bacterial suspension: the ATCC9341 strain was
prepared as described in the Manual of Reference Materials and
Methods to Detect Veterinary Drug Residues,1 but MD was
used instead of culture broth to maintain the stock inoculum. A
few colonies were suspended in 0.5 ml MD in sterile Eppendorf
tubes; these tubes were kept frozen until needed.
E. coli suspension: A freeze-dried strain of E. coli
ATCC11303 was reconstituted and inoculated onto TSA in a
Petri dish. The plate was incubated for 24 h at 37 C and
inspected for purity. Sterile Eppendorf tubes with 0.5 ml MD
were inoculated with several colonies of the E. coli strain. This
stock inoculum was kept at 220 C for maximally two years.
When needed, the stock inoculum was thawed and inoculated
onto a TSA plate. After overnight incubation at 37 C the plate
was inspected for purity. Ten ml of TSB were inoculated with
several colonies obtained on the TSA plate and incubated
overnight. The TSB culture, which contained at least 5 3 108
colony forming units of the E. coli strain per ml, was diluted 1
+ 9 in sterile TSB and added to the prepared medium cooled to
45-50 C.
Results
All results are summarized in Table 1. Six observations were
obtained with each concentration of antibiotic without meat,
and six with meat. No obvious differences were seen between
the different meat species (data not shown in the table). The
ranges of zones obtained with aqueous solutions of antibiotics
and the ranges of zones obtained with meat spiked with the same
level can be found in Table 1.
1. Beta-lactam antibiotics
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2. Cephalosporins
Ceftiofur was detected on plate I and on plate IV. Addition of
meat affected the detection of ceftiofur substantially on both
plates.
Results on plate I were as follows: at 20 ng no zones occurred
around the disks with meat. At 50 ng, which is five times higher
than the detection limit, inhibition zones larger than 12 mm
were only observed in two of six cases, while the zones
surrounding the six control disks were larger than 23 mm. The
highest level, 100 ng per disk, was only tested on plate I;
inhibition zones were observed around the six meat disks, but
usually they were small and in one case less than 12 mm.
Detection limits of ceftiofur were lower on plate IV. Two of
six meat samples spiked with 20 ng ceftiofur and all six meat
samples spiked with 50 ng ceftiofur were detected on this plate.
Large variations of inhibition zones of samples spiked with the
same level were seen.
3. Tetracyclines
Low quantities, approximately equal to the detection limits,
were not always detected in spiked meat. With higher
quantities, the zones were almost equal around paper disks with
Erythromycin and tylosin were tested with meat at concentrations five-fold higher than the detection limits, 1 ng and 10 ng,
respectively. No inhibition was seen with meat spiked with 5 ng
erythromycin (0.05 mg kg21), while the zones of meat spiked
with 50 ng tylosin (0.5 mg kg21) were small and irregular. Meat
spiked with 40 ng (0.4 mg kg21) erythromycin yielded positive
results; diameters of zones ranged from 17 to > 30 mm.
Erythromycin and tylosin were also detectable on plate III,
but the detection limits were 2 and 20 ng, respectively. The
effects of samples spiked with 5 ng erythromycin (0.05
mg kg21) and 50 ng tylosin (0.5 mg kg21) were analoguous to
those observed on plate IV. Meat spiked with 40 ng (0.4
mg kg21) erythromycin was not always detected.
Table 1 Comparison of inhibition zones observed with paper disks impregnated with aqueous antibiotic solutions and identical disks layered with pork,
beef or chicken meat
Medium
Ceftiofur (9 ng)
Ampicillin (3 ng)
Doxycycline (1 ng)
Chlortetracycline (1 ng)
Tetracycline (5 ng)
Oxytetracycline (8 ng)
II
III
IV
Paper disk
tested/ng
Diameters of zones
without tissue (range of
six observations)/mm
0.4
1.2
1.5
3
20
50
100
4
10
15
30
1
2.5
4
1
2.5
4
5
20
10
20
40
200
400
100
100
10
40
1.2
3
4
10
20
50
50
5
40
80
10
20
50
80
1
2
5
8
1
2
5
8
812
1722
1821
2326
1720
2426
2829
1315
2426
2526
2931
1012
1718
1822
1213
1719
2022
1011
1821
1315
1516
2125
1618
2025
1822
1921
1620
> 30
2325
2226
2529
1720
28 > 30
> 30
2224
1924
> 30
2829
1316
2125
2527
2930
1820
2225
2529
2833
2123
2427
2829
2931
No inhibition
1018
1318
2225
No inhibition
< 815
916
Absent or small and asymmetric
1723
2024
2530
Absent or asymmetric
1518
1722
915
1520
2021
< 812
1821
1215
1317
2025
No inhibition
No inhibition
No inhibition
Absent or small and asymmetric
No inhibition
918
2125
2226
2429
No inhibition
Mostly absent
1325
Absent or small and asymmetric
No inhibition
17 > 30
No inhibition
No inhibition
1322
1822
2529
No inhibition
1825
2529
2729
< 811
2327
2830
2631
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Discussion
Microbiological inhibition tests are intended for screening of
foods for residues of antibiotics. The purpose of such tests is to
Table 2 Maximal residue limits of antibiotics in muscle tissue, and usefulness of the FPT or a fifth medium intended for detection of quinolones for each
antibiotic or antibiotic group
Group
Antibiotic
MRL/mg kg21
Beta-lactam
Penicillin G
Ampicillin
Ceftiofur
0.05
0.05
0.2 (B)
0.5 (P)
0.1
0.1
0.1
0.1
0.1
0.5
0.4
0.1
0.05 (P)
0.05
0.03
0.03
Cephalosporins
Tetracyclines
Sulfonamides
Aminoglycosides
Macrolides
Lincosamides
Quinolones
Oxytetracycline
Tetracycline
Chlortetracycline
Doxycycline
Sulfadimidin
Streptomycin
Erythromycin
Tylosin
Lincomycin
Flumequin
Enrofloxacin
Ciprofloxacin
* B = beef; P = pork.
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Detection of MRL in
aqueous solution?
Yes
Not always on plate I
No
Most often not found
Yes
Yes
Yes
Yes
No
No
Not always on plate III
No
No
No
Yes
Yes
Yes, on plate I
Yes, on plate I
Yes, on plate I
Yes, on plate I
No
Yes, on plate III
Yes, on plates III and IV
Yes, on Plate IV
No
No
Yes, on plate V
Yes, on plate V
Acknowledgements
We thank Martine Boonaert and Ghislaine Vermassen for
technical help, and Chris Puttevils for the photograph. We
appreciated helpful discussion with Rgine Fuselier.
References
1
2
3
4
5
6
7
8
9
10
Paper 8/04903C
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