43 Post-Screening Classification of Tetracycline …randd.defra.gov.uk/Document.aspx?Document=vm02142_7196_FRA.pdf1 Post-Screening Classification of Tetracycline Positives Detected

FD04\43

Post-Screening Classification of

Tetracycline Positives Detected by the

Microbial Inhibition Test

REPORT

1

Post-Screening Classification of Tetracycline Positives Detected by the


Signatories:

………………………………………. …………………………………

Mrs. S.L.Stead Mr.M.Sharman

Senior Analytical Chemist Team Leader

Central Science Laboratory

Sand Hutton York

Y041 1LZ

2

Post-Screening Classification of Tetracycline Positives Detected by the


FINAL REPORT

Report Number: FD04\43

Author: Mrs.S.L.Stead

Date: 29 March 2005

Project Title: Post-Screening Classification of

Tetracycline Positives Detected by the


Sponsor: Veterinary Medicines Directorate

Sponsor’s Project Number: VM002142

CSL Contract Number: L5BV1000

CSL file Reference: PPC 1979

Principal Workers: Mrs. Anisha Sharma, Miss Helen Ashwin,

Mrs. Sara Stead

Distribution: 1. Dr.J.Kay (VMD)

2. Dr.N.Renn (CSG)

3. Mr.M.Sharman (CSL)

Central Science Laboratory

Sand Hutton

York

Y041 1LZ

Telephone: 01904 462551

Fax: 01904 462111

Email: [email protected]

3

SUMMARY

The overall aim of this research project was to develop and validate a tetracycline

selective assay to be used in combination with the previously reported, integrated

Premi®Test methodology [1].

AIMS

The key stages of this project were: -

��To conduct a literature search in order to identify all suitable candidate

proteins and other mechanisms for inhibiting the action of tetracyclines.

��To develop / optimise a post-antimicrobial screening methodology to

confirm the identity of suspect tetracycline positive samples. Initial work

will be conducted using the optimum candidate from the prototype work.

��To devise an integrated post-antimicrobial screening strategy for class

specific identification of the penicillins, sulphonamides and tetracyclines

to be used in combination with the optimised PremiTest methodology.

��To conduct a validation study in accordance with 2002/657/EC [2].

RESULTS

1. Three mechanisms for the selective reversal of the tetracycline response on

the Premi®Test have been identified through a literature study, including the

matrix metalloproteinases, Tet efflux proteins and metal ion chelation.

2. The metal ion chelation approach has proved to be the most cost-effective and

technically simple format for the tetracycline selective post-antimicrobial

screening (AMS) assay. The TCN assay has been validated in accordance

with 2002/657/EC guidelines for qualitative screening assays.

3. The TCN assay is complementary to the sulphonamide and penicillin selective

class confirmatory assays previously reported in FD 02/12 and the

methodologies have been fully integrated.

4

LIST OF CONTENTS

PAGE

SUMMARY 3

LIST OF TABLES 5

LIST OF FIGURES 6

1. INTRODUCTION 7

2. EXPERIMENTAL 10

2.1. Chemicals and Reagents 10

2.2 Equipment 10

2.3. Preparation of Solutions and Buffers 10

2.4. Standards and Spiking Solutions 11

2.5. Fortified Samples 12

2.6. Development and Optimization of Post-Antimicrobial Screening,

Tetracycline Selective Assay 12

2.7. Validation Study 13

3. RESULTS AND DISCUSSION 15

3.1. Evaluation of approaches for the tetracycline selective reversal assay

for use with the Premi®Test 15

3.2. Optimization of the chelation tetracycline selective reversal assay 18

3.3. Validation of the tetracycline post-screening assay 19

4. CONCLUSIONS 23

5. REFERENCES 25

6. TABLES 26

7. FIGURES 34

5

LIST OF TABLES

Table 1. Table showing the effect of a fixed mass of MMP-8, on the Premi®Test

response to a range of antimicrobial compounds, at different residue

concentrations, in fortified chicken muscle.

Table 2. Table showing the effect of a fixed mass of MMP-8, on dilutions of

doxycycline fortified tissue extracts on the Premi®Test response.

Table 3. Table showing the effect of TCN buffer and nutrient broth on the

Premi®Test response to a range of antimicrobial compounds at different

residue concentrations in fortified chicken muscle.

Table 4. Validation data showing repeatability measurements, false positive and

negative rates and CCβ determination for the TCN Assay in fortified

chicken muscle.

Table 5. Table showing the results from the investigation of analyte specificity of

the TCN assay using fortified chicken muscle.

Table 6. Ruggedness – Investigation of the effect of TCN buffer on the

Premi®Test responses to a wider range of tetracycline compounds in

fortified chicken muscle.

Table 7. Ruggedness – honey.

Table 8. Blind Trial – Results generated from the blind trial using the integrated

AMS procedure.

Table 9. Inter-Laboratory Validation Study.

6

LIST OF FIGURES

Figure 1. Outline for the TCN tetracycline post-screening assay.

Figure 2. Integrated AMS strategy for penicillins, sulphonamides and tetracyclines.

Figure 3. Graphical representation - Results from the Blind Trial of the Integrated

AMS procedure.

Figure 4. Photographic image - Effect of a fixed mass of MMP-8 on the Premi®Test

response to a range of antimicrobial compounds at different concentrations

in fortified chicken muscle.

Figure 5. Photographic image - Effect of a fixed mass of MMP-8 on doxycycline

fortified tissue extract dilutions on th Premi®Test response.

Figure 6. Photographic image - Effect of TCN buffer and nutrient broth on the

Premi®Test response to a range of antimicrobial compounds at different

residue concentrations in fortified chicken muscle.

Figure 7. Photographic image - Results of analyte specificity of the TCN assay

using fortified chicken muscle.

Figure 8. Photographic image - Results from the investigation of analyte specificity

of the TCN assay using fortified chicken muscle.

Figure 9. Photographic image - Ruggedness – investigation of the effect of TCN

buffer on the Premi®Test responses to a wider range of tetracycline

compounds in fortified chicken muscle.

Figure 10. Photographic image - Ruggedness - honey.

Figure 11. Photographic image - Blind Trial using the Integrated AMS procedure.

7

INTRODUCTION

1.1 Under EU legislation, members of the tetracycline class of antibacterial agents are

permitted for use in the treatment and prophylaxis of bacterial diseases in food-

producing animal species. Maximum Residue Limits (MRLs) have been set

under 2377/90 (as amended) ranging from 100 – 600 µg kg-1

dependent on the

species and tissue type. As part of the Defra-funded Statutory and Non-Statutory

surveillance schemes, there is a requirement to monitor for tetracyclines in food

of animal origin. From, and including the surveys undertaken in, 2002 until

September 2004 residues of tetracyclines have been found in pig, poultry and calf

kidney in the range of 780 – 4020 µg kg-1

[3]. Honey has been previously found

to contain high residue levels of tetracyclines. Currently oxytetracycline is the

only registered antibiotic for the control of the foulbroud diseases, European Foul

Brood (EFB) and American Foul Brood (AFB) in bee colonies in some countries

[4].

1.2 Tetracyclines are antibiotics that inhibit protein synthesis by preventing the

attachment of aminoacyl-tRNA to the ribosomal acceptor (A). Tetracycline

molecules comprise a tetra cyclic nucleus to which various groups are attached:

Chemical Structure

H

N(CH3)2

OH

CONH2

O

OH

OHOOH

R1 R2 R3 R4

H

Functional Groups

R1 R2 R3 R4 Origin

Tetracycline H CH3 OH H S. texas

Chlortetracycline Cl CH3 OH H S. aureofasciens

Oxytetracycline H CH3 OH H S. rimosus

Doxycycline H CH3 H OH Derivative

8

1.3 Tetracyclines are broad-spectrum agents that exhibit activity against a wide range

of Gram-positive and Gram-negative bacteria, they have been widely used in both

human and veterinary medicine. In some countries their use has been expanded to

include addition, at sub therapeutic levels, to animal feed to act as growth

promoters. Bacterial resistance to the tetracycline class of antibiotics has

followed the introduction of these agents for clinical, veterinary and agricultural

use. This bacterial resistance has major implications to human health thus it is

important to monitor the levels of these antibiotics in the animal production sector

to provide consumers with confidence that the food products are antibiotic free

before entering the food chain.

1.4 To interact with their target, tetracyclines must first cross one or more membrane

systems depending on whether the organism is Gram-negative or Gram-positive,

and then interact with the 30S subunit. Bacterial resistance to tetracyclines can

occur during either membrane transport or at binding. It is intended that by using

one of these resistance mechanisms a method for the rapid and class selective

identification of tetracyclines following a primary anitimicrobial screen will be

developed.

1.5 Initial studies (FD 02/12) carried out at the Central Science Laboratory, as part of

the Defra funded project VM0291, identified matrix metalloproteinase 8 (MMP-

8) as having high binding affinity to the tetracycline class of antibiotics. The

MMP-8 is seen to selectively reverse the tetracycline response - it binds to the

tetracycline thus rendering it unrecognisable to the transport protein and hence

halts its entry into the cell. Using this binding mechanism a rapid and class

selective method for tetracyclines was previously evaluated although proving to

be very expensive in terms of the cost of the protein.

1.6 As part of the current research project other potential binding targets and

mechanisms for the inhibiting the tetracycline class were identified by way of a

9

literature search. The Tet class of proteins were identified as having a high

binding affinity for tetracyclines. The Tet proteins have been characterised

depending on their mechanism of resistance, for example, Tet(M) convers

resistance based on ribosomal binding where-as Tet(A) is related to efflux

through the cell membrane [5]. Okerman et al [6] recently reported on a rapid

tetracycline test using the Tet(R) protein. The Tet(R) protein plays a part in

bacterial tetracycline resistance by disrupting the prokaryotic efflux pump

mechanism. This tetracycline assay has been marketed as a commercial product

called the ‘Tetrasensor’. The Tetrasensor is a dip-stick based technology, the dip-

stick end-point can be read visually or using a optical reader device [7].

1.7 Metal ion chelation using a calcium containing buffer was also investigated as a

mechanism for disrupting the antimicrobial activity of the tetracyclines and thus

selectively reversing the primary Premi®Test positive response into negative

response.

1.8 This report documents the evaluation of the three approaches for selective

reversal of the tetracycline response on the Premi®Test described above and the

subsequent validation of the optimum assay format.

10

EXPERIMENTAL

2.1. Chemicals and Reagents

All solvents and reagents were of analytical grade and obtained where possible

from Sigma-Aldrich (Poole, Dorset) with the exceptions of; Lab Lemco broth

(Lot Ch-B.: 291574) and �-lactamase (Lot Ch-B.: 310608), which were

purchased from Oxoid (Basingstoke, Hampshire), acetonitrile, acetone and HPLC

water were purchased from Fisher Scientific (Loughborough, Leicestershire),

MMP-8 (CLF034021) was purchased from R&D Systems Inc. (Minneapolis,

USA). The Premi®Test kits were purchased from DSM (The Netherlands).

2.2. Equipment – The scanner system

The Premi®Test end-point was measured using the Premi®Scan (custom version)

software, purchased from DSM (The Netherlands). The Premi®Scan software

was installed on a PC running Windows 2000 professional operating system. A

Hewlet Packard Scanjet 7400 series was used for the measurement.

2.3. Preparation of Solutions and Buffers

2.3.1 TCN buffer

(50mM TRIS, pH 7.5, 20 mM CaCl2 and 150mM NaCl)

7.88 g TRIS, 2.19 g CaCl2, 8.76 g NaCl dissolved in 1 litre of water and adjusted

to pH 7.5 with 1 M NaOH

note: Light sensitive solution, storage conditions in the dark between 4 - 8 oC for

1 month.

2.3.2 MMP-8 Solution

1 ml of TCN buffer was added to the 10 µg lypophilized MMP-8 to give a

solution concentration of 10 µg kg-1

. The reconstituted enzyme was vortexed for

11

30 seconds and allowed to stand for 1 hour before use. The solution was stored at

–20 oC for up to 6 months in 300 µl aliquots in order to limit the number of

freeze-thaw cycles.

2.3.3. Lab Lemco Broth

8 g (+/- 0.05 g) of powdered media dissolved in 1 litre of HPLC water.

The broth solution was stored in the dark between 4 – 8 oC for up to 1 week.

2.4. Standards and Spiking Solutions

Stock standard solutions of the tetracycline, sulphonamide and penicillin

compounds were prepared on a monthly basis as detailed in the table below.

Compound Supplier

Reference

Purity

%

Purity

corrected

(g)

Dissolution

solvent

Volume

(ml)

Conc.

(mg ml-1

)

Tetracycline T3258 100 0.0100 Methanol 10 1

Doxycycline D9891 99 0.0099 Methanol 10 1

Oxytetracycline O5875 87.6 0.0124 Methanol 10 1

Chlortetracycline C4881 80 0.0120 Methanol 10 1

Penicillin G 13752 98 0.00102 PBS pH 6.0 10 1

Sulfadiazine S8626 99 0.0101 Methanol 10 1

Sulfadimethoxine 86026 99 0.0101 Acetone 10 1

Sulfamethazine S6256 100 0.0100 Acetone 10 1

Sulfamethoxypyridazine S7257 99.6 0.0101 Acetone 10 1

Sulfaquinoxaline S7382 98 0.0102 Acetone 10 1

Amoxicillin 26787-78-0 99 0.0101 PBS pH 6.0 10 1

Tylosin T-6134 90 0.01106 Methanol 10 1

Cloxacillin 27555 97 0.0103 Water 10 10

Intermediate standard solutions were prepared by dilution of the stock standards

using an appropriate solvent (either water or methanol).

12

For example, 100 µl of a 1 mg ml-1

solution was adjusted to 10 ml using water to

give a concentration of 10 µg kg-1

.

Spiking solutions were prepared in water, the concentrations prepared related to

the MRL of the individual compounds.

For example, 1000 µl of a 10 µg kg-1

intermediate solution was adjusted to 10 ml

using water to give a concentration of 1 µg kg-1

. 400 µl of the 1 µg kg-1

spiking

solution was used to fortify a 4 g tissue sample to give a tissue concentration

equivalent to 100 µg kg-1

.

2.5. Fortified Samples

Fortified samples were prepared for each analyte by the addition of a fixed

volume of spiking solution to known, homogenised blank tissue. The fortified

samples were then allowed to stand for 10 minutes prior to analysis.

2.6. Development and Optimisation of Post-Antimicrobial Screening, Tetracycline

Selective Assay

The rapid and class selective method previously reported for tetracyclines –

Report FD 02/12 was further investigated. The overall aim was to fully integrate

the optimised tetracycline assay with the pre-existing antimicrobial screening

strategy. Experiments were undertaken using known chicken muscle blank,

generated as part of a controlled animal study at CSL (Organic Meat Project, FD

03/24). A fixed mass of metal ion chelate was selected for the following

members of the tetracycline class, chlortetracycline, oxytetracycline, tetracycline

and doxycycline. The effect of the differing antimicrobial potencies within the

tetracycline class and the potential for wide ranging residue levels has been

accounted for by inclusion of both a direct and diluted measurement of the tissue

extract. Figure 1, shows the outline of the TCN tetracycline post-screening assay.

The tetracycline selective TCN assay was integrated with the existing

sulphonamide and penicillin antimicrobial screening strategy outlined in Figure 2.

13

2.7. Validation Study

Validation experiments were carried out in accordance with the 2002/657/EC

guidelines for validation of qualitative analytical methods [2]. The individual

experiments undertaken include: -

2.7.1 Repeatability, false positive and negative rates and determination of the CCβ

In order to determine the following performance parameters of the TCN assay, a

total of 21 different known blank chicken samples were analysed using the

Premi®Test with and without the addition of the TCN buffer. A replicate set of

the blank samples were fortified with chlortetracycline at 100 µg kg-1

. In

addition, samples of blank chicken muscle and chicken muscle fortified with

penicillin G and a sulphonamide mixture were included for quality control

purposes.

2.7.2 Analyte specificity

Samples of chicken muscle were fortified with a range of known Gram-positive

inhibitors – pencillin G, cloxacillin, sulphadiazine and tylosin at 2 times the

respective MRL’s in chicken muscle (600 µg kg-1

, 200 µg kg-1

and 200 µg kg-1

).

The fortified samples were analysed with and without the addition of the TCN

buffer to demonstrate the class selectivity of the tetracycline inhibition response.

2.7.3 Ruggedness (small method changes)

Ruggedness experiments were undertaken, including a change in matrix (chicken

muscle to honey) and inclusion of additional tetracycline compounds,

oxytetracyline and tetracycline.

14

2.7.4 The Blind Trial

Six samples (a mixture of spikes and blanks) were prepared and coded by one

analyst and analysed by a second analyst, (with no knowledge of the identity of

the samples) in accordance with the integrated procedure. The ultimate aim was

for the second analyst to correctly determine the class specific identities of

residues present within the sample batch.

2.7.5 Inter-laboratory Validation of the TCN assay following the fluid expression

procedure

An inter-laboratory study was conducted at a second laboratory DSM, The

Netherlands. The matrix selected was chicken muscle to be fortified with

oxytetracycline at 200 µg kg-1

. The fortified samples (n = 10), blanks and

appropriate quality control samples were extracted following the DSM tissue fluid

expression procedure (previously reported FD 02/12) and applied to the

Premi®Test ampoules with and without the addition of TCN buffer.

15

RESULTS AND DISCUSSION

3.1. Evaluation of the different approaches for the tetracycline selective reversal assay

in combination with the Premi®Test

3.1.1. Matrix Metalloproteinases (MMPs) Approach

It is widely known that the tetracyclines bind to many proteins preferentially, if

not exclusively as complexes with metal ions. [8]

The matrix metalloproteinases are a family of zinc and calcium dependent

endopeptidases with the combined ability to degrade all the components of the

extracellular matrix and have been found to play a major role in both

physiological and pathological conditions including embryonic development,

tissue remodelling and arthritis, cancers and cardiovascular disease. [9]

Biomedical research has demonstrated the tetracyclines to be potent MMP

inhibitors and to be effective in the treatment of various disease states, for

example, rheumatoid arthritis. [10]

In order to exert their inhibitory effect on the protein structure the tetracyclines

have a binding region located within the MMP structure. The inhibition of the

MMP enzymes by the tetracyclines is reported to occur via a chelation of zinc and

or calcium ions which are a requirement for the maintenance of the normal

structural hydrolytic activity and conformation of MMPs. [10]

The hypothesis for the tetracycline selective reversal observed following the

addition of MMP-8 to a sample prior to incubation on the Premi®Test, is that the

tetracycline-MMP-8 complex is unable to penetrate the Bacillus cell membrane

via the cell’s active transport mechanism.

As the starting point for this development study, the extraction protocol and

MMP-8 assay previously reported in report FD 02/12 was investigated. The

original conditions for the tetracycline selective post-screening assay were set up

and initial validation experiments were conducted to determine the reproducibility

of the MMP-8 assay with a different analyst, reagents and equipment.

16

The original data reported in FD 02/12 were successfully replicated [Table 1]. In

presence of MMP-8 the chlortetracycline (CTC) positive response at 100 µg kg-1

is selectively reversed. The differences within the antimicrobial potency between

members of the tetracycline class, chlortetracycline and doxycycline (DOX),

previously reported are also evident. The mass of MMP-8 applied to extracts was

fixed at 50 µg, this mass is sufficient to reverse the CTC response at 100 µg kg-1

but not sufficient to reverse the effect of DOX at the same concentration or that of

CTC at a concentration equivalent to the MRL in porcine kidney, 600 µg kg-1

.

Additional experiments were conducted to standardise the mass of protein at

50 µg and vary the extract dilution factor in order to compensate for the observed

differences in inhibitory potency within the tetracycline class and potential

residue levels [Table 2].

Although the initial experiments demonstrate that the MMP-8 assay conditions

described in FD 02/12 were reproducible the cost of the human recombinant

(hrMMP-8) protein is very high and the commercial supply is limited to a

specialist company within the U.K. The cost for the MMP-8 component per

single assay is greater than £10 making the cost per sample questionable for a

qualitative screening assay. Therefore, additional mechanisms were investigated,

including the Tet proteins, section 3.1.2. and metal ion chelation, section 3.1.3.

3.1.2. Tet Efflux Protein Approach

A literature search for alternative binding proteins was conducted as part of the

project to determine the feasibility of developing the knockout assay using a less

expensive binding protein.

The Tet class of proteins, including the Tet(R), Tet(A), Tet(M) and Tet(L) were

identified as having the correct binding characteristics required for the

tetracycline selective inhibition assay. The Tet class of efflux proteins are

comprised of a wide range of prokaryotic proteins, the majority of which fall

within structurally related families of the major facilitator superfamily (MFS) of

17

transporters that have either 12 or 14 transmembrane segments (TMS). The 12

TMS Tet proteins include most of the tetracycline efflux proteins found in Gram-

negative bacteria and the 14 TMS efflux proteins are predominately found in

Gram-positive bacteria. The 2 groups of Tet proteins are structurally similar to a

larger group of drug and multi-drug efflux proteins and function by a secondary

antiporter mechanism [11].

The Tet proteins are membrane linked, multi-meric complex proteins. Obtaining a

commercial supply of the purified proteins was not feasible. For this reason it was

not possible to determine the experimental effectiveness of this class of proteins.

3.1.3. Metal Ion Chelation Approach - (TCN Assay)

The ability of the tetracyclines to chelate polyvalent metal cations is a well-

established property of the class of compounds. There are many tetracycline

extraction, clean up and analysis methods reported in the literature based on

transition metal ion chelation columns, for example, Chelating Sepharose Fast

Flow columns [12].

In order to investigate metal ion chelation as a potential tetracycline inhibition

strategy a TRIS based buffer containing additional calcium and sodium ions

(TCN buffer) was investigated. Calcium forms a 2:1 metal-ion to ligand complex.

Formation of the calcium complex involves the addition of one metal ion to the C-

10, C-11 site with subsequent addition of a second metal ion at the C-12 and C-1

site [13]. Initial experiments showed that the TCN buffer selectively reversed the

tetracycline positive response observed on the Premi®Test. Based on the

negligible cost of the buffer reagents and simplicity of this approach the metal ion

chelation strategy was selected for further optimisation and 2002/657/EC

validation as a qualitative screening assay.

18

3.2. Optimisation of the chelation tetracycline selective reversal assay

3.2.1. TCN Assay Optimisation

Initial experiments with the addition of 50 µl of TCN buffer at pH 7.5 (TRIS

based buffer with the addition of 10 mM CaCl and 150 mM NaCl) to 100 µl of

extract showed that the Premi®Test positive response elicited by tetracycline

compounds could be selectively reversed. Throughout the optimisation and

validation experiments the Premi®Test ampoules were measured using the

Premi®Scan system following the incubation period. A scanner value (Z-score)

of greater than zero is classified as a positive result and a scanner value of zero or

lower is classified as a negative result.

The data presented in Table 3 indicates that the TCN buffer alone is capable of

selectively reversing the positive Premi®Test response caused by tetracyclines.

The mode of action of the TCN buffer (metal ion chelation) appears to be

selective for the tetracyclines. The test response to penicillin G and mixed

sulphonamide fortified samples is not reversed in the presence of the TCN

treatment. Treatment with 50 µl of a nutrient broth solution (not containing

calcium) does not exhibit the same tetracycline specific reversal affect as the TCN

buffer.

The dilution approach developed for the MMP-8 assay was employed for the

TCN assay whereby a fixed volume of the reagent was applied to both the

undiluted tissue extract and also a diluted portion of the tissue extract. The

method is detailed in Figure 2. It was found that a 1:16 dilution of the final

extract whilst maintaining a fixed volume of TCN buffer was sufficient to reverse

the positive response elicited by the strongest potency tetracycline, doxycycline at

a concentration equivalent to twice the MRL for porcine kidney, 1200 µg kg-1

.

By performing both a direct and diluted measurement on the tissue extract it is

possible to distinguish between a weak potency / low concentration tetracycline

residue and a high potency / high concentration tetracycline residue.

19

3.3. Validation of the tetracycline post-screening assay

3.3.1. Repeatability and determination of the CCβ

The results presented in Table 4 show that out of the 21 known blank chicken

samples analysed with and without the addition of TCN buffer, both sets of 21

results show a negative response, i.e. no false positive results were recorded. The

assay is not adversely affected by naturally occurring interferences within the

matrix type, chicken muscle.

The results for the replicate set of samples fortified with CTC at 100 µg kg-1

show

that 20 out of 21 samples (without the addition of TCN buffer) are positive. The

Premi®Test qualitative CCβ (β error of 5 %) for CTC in chicken muscle is less

than or equal to 100 µg kg-1

. The calculated CCβ value indicates that the

Premi®Test is reliably detecting CTC at the MRL concentration in chicken

muscle.

Following treatment with TCN buffer the CTC fortified set of samples show 21

results out of 21 give a negative response on the Premi®Test. The data shows

that the TCN assay reliably identified all 21 CTC fortified samples as being

positive for tetracyclines at the MRL concentration.

3.3.2. Analyte Specificity

The results presented in Table 5 show that the TCN assay is selective for the

tetracycline class of antimicrobial compounds. The Premi®Test response to a

range of compounds from the other antimicrobial classes including the β-lactams,

sulphonamides and macrolides is not reversed from a positive to a negative result

following treatment with TCN buffer.

20

3.3.3. Ruggedness- Small changes to the Assay Procedure

3.3.3.1.Additional tetracycline compounds

The Premi®Test response to a wider selection of the tetracycline class of

compounds was investigated following treatment with TCN buffer. Table 6 shows

results for oxytetracycline, doxycycline and tetracycline fortified at 100 µg kg-1

in

chicken muscle, n=6. The results show that the TCN assay effectively reverses

the control Premi®Test positive response to a negative response for all

tetracyclines tested, at 100 µg kg-1

chicken muscle tissue.

3.3.3.2 Additional matrices – honey

The TCN assay methodology was investigated in combination with honey

extracts. Table 7 shows the results for honey fortified with chlortetracycline and

oxytetracycline following treatment with TCN. The TCN assay was found to be

transferable to a different matrix showing no false positive or negative results

both with and without the TCN treatment.

3.3.4. The Blind Sample Trial

The results for the blind trial of the integrated AMS procedure are shown in Table

8 and Figures 3 and 11. In this test the second analyst was able to determine the

identity of six unknown samples of muscle correctly by following the combined

integrated strategy flow diagram (Figure 2).

Sample Code 44: is chicken muscle fortified with doxycycline at 1000 µg kg-1

.

Treatment with TCN caused a small downward shift in response in the undiluted

extract compared to the untreated and the p-ABA (sulphonamide specific

treatment) and β-lactamase (pencillin specific treatment) controls. The Z-score

21

for the TCN control is slightly below zero, indicating that the positive response

was by caused by a higher concentration or a more potent tetracycline compound.

Sample Code 26: is chicken muscle fortified with amoxicillin at 10 µg kg-1

.

Treatment with β-lactamase caused a downward shift in the scanner response

compared to that of the three other treatment controls. The Z-score for the β-

lactamase control is below zero, indicating that the positive was caused by

penicillin.

Sample Code 64: is chicken muscle fortified with chlortetracycline at 100 µg kg-1

.

Treatment with TCN caused a downward shift in the scanner response compared

to that of the three other treatment controls. The Z-score for the TCN control is

significantly below zero, indicating the positive was caused by a lower

concentration or a less potent tetracycline.

Sample Code 80: is unfortified chicken muscle. The scanner responses for all

four control treatments remain in the lower negative region of the plot.

Sample Code 84: is chicken muscle fortified with oxytetracycline at 200 µg kg-1

.

Treatment with TCN caused a downward shift in the scanner response compared

to that of the three other treatment controls. The Z-score for the TCN control is

below zero, indicating the positive was caused by a tetracycline compound.

Sample Code 12: is chicken muscle fortified with sulphadiazine at 200 µg kg-1

and chlortetracycline at 100 µg kg-1

(to mimic combinatorial drug therapy).

Following the p-ABA and TCN treatment the scanner responses remain in the

positive region. The untreated control shows a stronger positive response,

however, despite the loss of one component, a “weaker” positive response is still

observed. A positive sample showing this pattern of responses is indicative of a

higher-level combinatorial dose or a positive elicited by a different class of

antibiotics.

22

3.3.5. The Inter-Laboratory Validation

The inter-laboratory validation was conducted using chicken muscle fortified with

OTC and extracted following the DSM fluid expression procedure. Table 9

shows the experimental results. At 200 µg kg-1

all ten replicates show a positive

response without the addition of TCN. Following treatment with TCN, the

control response is reversed to a negative response in eight out of the ten

replicates (80 % response reversal). The TCN assay was confirmed to be

selective for tetracycline compounds, the quality control sample responses; blank

extract and extract fortified with pencillin G and mixed sulphonamides (n=6)

were not significantly affected by the TCN treatment. However, the presence of

TCN buffer was seen to decrease the incubation time of the Premi®Test by

between five and ten minutes.

In order for the TCN assay to be used in combination with the fluid expression

extraction procedure, further optimization is required to ensure that a positive

response elicited by a tetracycline is inhibited in 100 % of cases.

23

CONCLUSIONS

4.1. The developed procedure is a tetracycline selective post-screening assay for use

as part of the integrated antimicrobial screening strategy. A variety of approaches

were investigated for class selective inhibition of the tetracycline response on the

Premi®Test as described in section 3.1. The metal ion chelation (TCN buffer)

approach was considered to be optimal assay format based on the negligible cost

of the buffer components and simplicity of the assay.

4.2. Due to the differential antimicrobial potencies within the tetracycline class and

the potential for very high residue levels, the developed procedure incorporates

both direct analysis of the extract and of a dilution of the extract. The volume of

TCN buffer is fixed in both cases. The results generated following this approach

gives the analyst an indication of the tetracycline level present in the sample.

4.3. The TCN assay has been successfully validated in accordance with the

2002/657/EC guidelines for qualitative screening methods. The CCβ of the

integrated AMS for CTC in chicken muscle was confirmed as 100 µg kg-1

(β error

equals 5 %) at this concentration the false negative rate is 5 % or less indicating

that the screening assay is capable of reliable detection of CTC at the MRL

concentration in chicken muscle. The assay performance was investigated using

21 different known blank chicken muscle samples, no false positive results were

recorded with or without the addition of the TCN buffer showing the assay is not

affected by naturally occurring interferences within the matrix. A replicate set of

the 21 blank muscle samples were fortified with CTC at the 100 µg kg-1

, in all 21

samples the positive response was reversed following TCN treatment. The TCN

assay has been demonstrated to be selective for the tetracycline class and the

conditions are transferable to another matrix type, honey. An inter-laboratory

validation experiment was conducted at DSM using the alternative, field-test fluid

expression. The results show that the TCN assay is compatible with this

extraction technique. The positive responses in eight out of the ten chicken

24

muscle samples fortified with OTC at 200 µg kg-1

were inhibited by TCN buffer.

Further optimization, for example, of the chelator ion concentration is required to

increase the inhibition rate above 80 %.

4.4. The TCN buffer assay is complementary to the existing integrated AMS

procedure previously developed and reported with FD 02/12. The TCN assay has

been incorporated within the AMS post-screening procedure, as detailed in the

flow diagram, Figure 2 . In order to ensure the reliable operation of the procedure

a blind coded trial was conducted. The second analyst was able to correctly

identify the class-specific identity of all six unknown samples.

4.5. The integrated AMS procedure is suitable for integration with other screening

assays, for example biosensor assays for chloramphenicol and streptomycin in

order to deliver a wider screen for a range of Gram-positive inhibitory

antimicrobial compounds.

25

REFERENCES

1. S.Stead, M.Sharman, J.A.Tarbin, E.Gibson, S.Richmond, J.Stark, E.Geijp, Meeting

the Maximum Residue Limits; an improved screening technique for the rapid

detection of antimicrobial residues in animal food products, 2004, Food Additives

and Contaminants, (21) 216-221

2. Commission Decision 2002/657/EC, Official Journal of the European Communities,

17.8.2002, L221/8

3. http://www.vmd.gov.uk/mavis/mavis.htm

4. P.J.Elzen, D.Westervelt, D.Causey, J.Ellis, H.R.Hepburn, P.Neumann, Method of

Application of Tylosin, an Antibiotic for American Foulbrood Control, with Effects

on Small Hive Beetle (Coleoptera:Nitidulidea) Populations, 2002, Journal of

Economic Entomology, Vol. 95 (6): 1119-1122

5. I.Chopra, M.Roberts, Tetracycline Antibiotics: Mode of Action, Applications,

Molecular Biology, and Epidemiology of Bacterial Resistance, 2001, Microbiology

and Molecular Biology Reviews 232-260

6. L.Okerman, S.Croubels, M.Cherlet, K.De Wasch, P. De Backer and J. Van Hoof,

Evaluation and establishing the performance of different screening tests for

tetracycline residues in animal tissues, 2004, Vol. 21 (2): 145-153

7. http://www.diffchamb.com/website/Archive/Templates/Item/product.asp?iSecId=58

6

8. S.Schneider, G.Brehm, C.Leypold, M.Reiher, Kerr gated resonance Raman study of

tetracyclines and their complexes with divalent metal ions, 2001/2002, Central

Laser Facility Annual Report 100-102

9. R&D Systems website, http://www.rndsystems.com/asp/molecule.asp?

Name=MMP%2D8

10. H.W. Macartney, H, Tschesche, The metal ion requirement for activation of latent

collagenase from human polymorphonuclear leucocytes, 1981, Physiological

Chemistry, 362(11): 1523-1531

11. Jie Jin, A.A.Guffanti, C.Beck, T.A.Krulwich, Twelve-Transmembrane-segment

(TMS) Version (delta TMS VII-VIII) of the 14-TMS Tet(L) antibiotic resistance

protein retains monovalent cation transport modes but lacks tetracycline efflux

capacity, 2001, Journal of Bacteriology, Vol. 183, No. 8 :2667-2671

12. M.C.Carson, Simultaneous determination of multiple tetracycline residues in milk

using metal chelate affinity chromatography, 1993, J AOAC Int. 76(2): 329-34

13. E.C.Newman, C.W.Frank, Circular dichroism spectra of tetracycline complexes

with Mg+2

and Ca+2

, 1976, Journal of Pharmaceutical Science, 65(12): 1782-32

26

TABLES

Table 1. Showing the effect of a fixed mass of MMP-8 on the Premi®Test response to a

range of antimicrobial compounds at different residue concentrations in fortified chicken

muscle.

Sample Identity Fortified Set Result Fortified Set + MMP-8 Result

(µµµµg kg-1

) Scanner Z Score Scanner Z Score

Spike 1a CTC 100 3.5 Positive -6.2 Negative

Spike 1b CTC 100 3.6 Positive -6.8 Negative

Spike 2a CTC 100 6.2 Positive -9.3 Negative

Spike 2b CTC 100 5.8 Positive -7.4 Negative

Spike 3a CTC 600 6.0 Positive 6.5 Positive

Spike 3b CTC 600 6.6 Positive 7.1 Positive

Spike 4a DOX 100 7.3 Positive 4.8 Positive

Spike 4b DOX 100 6.8 Positive 5.2 Positive

Spike 5a DOX 600 7.8 Positive 7.9 Positive

Spike 5b DOX 600 8.5 Positive 8.3 Positive

Quality Control

Samples (µµµµg kg-1

)

Fortified Set

Scanner Z Score

Result Fortified Set + MMP-8

Scanner Z Score

Result

Blank 1 -3.8 Negative -16.4 Negative


PenG 10 7.9 Positive 7.4 Positive


Table 2. Showing the effect of a fixed mass of MMP-8 on doxycycline fortified tissue

extract dilutions on the Premi®Test response.

Sample Identity Fortified Set Result Fortified Set + MMP-8 Result

(µµµµg kg-1


Spike 1 CTC 100 2.3 Positive -13.5 Negative

Spike 2 DOX 600 6.0 Positive 7.6 Positive

Spike 3 DOX 1000 3.7 Positive 5.4 Positive

Spike 2 DOX 600 7.0 Positive -11.4 Negative

1:8 extract dilution

Spike 3 DOX 1000 8.0 Positive -14.6 Negative

1:16 extract dilution

27

Quality Control


)

Fortified Set

Scanner Z Score

Result Fortified Set + MMP8

Scanner Z Score

Result

Blank -2.0 Negative -17.4 Negative


CTC = chlortetracycline, DOX = doxycycline, PenG = penicillin G

Table 3. Showing the effect of TCN buffer and nutrient broth on the Premi®Test response to a

range of antimicrobial compounds at different residue concentrations in fortified chicken muscle.

Sample Identity

(µµµµg kg-1

)

Fortified Set Result Fortified Set

+TCN

Result Fortified

Set +Broth

Result

Scanner

Z Score

Scanner

Z Score

Scanner

Z Score

CTC 100 5.1 Positive -0.5 Negative 4.5 Positive

DOX 1000

1:16 extract dilution 6.8 Positive -1.3 Negative 7.9 Positive

Quality Control


)

Fortified Set

Scanner

Z Score

Result Fortified Set

+TCN

Scanner

Z Score

Result Fortified Set

+broth

Scanner

Z Score

Result

Blank -5.9 Negative -15.1 Negative -10.9 Negative

PenG 10 5.3 Positive 6.6 Positive 6.7 Positive

Sulfamixture 200 1.4 Positive 0.7 Positive 4.6 Positive

CTC = chlortetracycline, DOX = doxycycline, PenG = penicillin G, Sulfamixture = mixture

of five sulphonamides; sulfamethazine, sulfamethoxypyridazine, sulfaquinoxaline,

sulfadiazine & sulfadimethoxine.

28

29

Table 4. Validation data showing repeatability measurements, false positive and negative rates and CCβ

determination for the TCN Assay in fortified chicken muscle.

Blank Sample Blank Set Blank Set + TCN CTC Fortified

Blank Sample

Fortified

Set

Fortified Set

+ TCN

LIMS Reference Result Result (µµµµg kg-1

) Result Result

54378 Negative Negative 100 Positive Negative



54382 Negative Negative 100 Negative Negative


















Quality Control

Samples

(µµµµg kg-1

)

Fortified Set Fortified Set

+ TCN

Blanks (n=4) Negative Negative

PenG 10 (n=3) Positive Positive

Sulfamixture 200

(n=2)

Positive Positive

PenG = penicillin G, Sulfamixture = mixture of five sulphonamides; sulfamethazine,

sulfamethoxypyridazine, sulfaquinoxaline, sulfadiazine & sulfadimethoxine.

30

Table 5. Showing the results from the investigation of analyte specificity of the TCN assay using

fortified chicken muscle.

Sample Identity

(µµµµg kg-1)

Fortified Set Result Fortified Set +TCN Result

Scanner Z Score Scanner Z Score

Spike 1 SDZ 200 8.7 Positive 7.0 Positive






Spike 7 Clox 600 9.0 Positive 8.2 Positive






Spike 13 TYL 100 8.8 Positive 7.1 Positive






Quality Control


)

Fortified Set

Scanner Z Score

Result Fortified Set+TCN

Scanner Z Score

Result




CTC 100 1.6 Positive -12.6 Negative

CTC 100 0.3 Positive -13.6 Negative

Blank 3 excluded due to extract contamination

CTC = chlortetracycline, SDZ = sulfadiazine, Clox = cloxacillin, TYL = tylosin.

31

Table 6. Ruggedness – investigation of the effect of TCN buffer on the Premi®Test responses to a

wider range of tetracycline compounds in fortified chicken muscle.

Sample Identity Fortified Set Result Fortified Set + TCN Result

(µµµµg kg-1


DOX 1 100 2.27 Positive -17.78 Negative






TC 1 100 5.51 Positive -11.54 Negative




TC 5 100 6.50 Positive *6.59 Positive


OTC 1 100 6.84 Positive -8.22 Negative






Quality Control Samples

(µµµµg kg-1

)

Fortified Set

Scanner Z Score


Scanner Z Score

Result




SDZ 200 7.57 Positive 6.87 Positive

*Spike 5 DOX excluded due to sample preparation error.

DOX = doxycycline, TC = tetracycline, OTC = oxytetracycline, SDZ = sulfadiazine

33

Table 7. Ruggedness – different matrix (HONEY).

Sample Identity Fortified Set Result Fortified Set + TCN Result

(µµµµg kg-1


CTC 1 100 3.71 Positive -11.76 Negative












Quality Control Samples

(µµµµg kg-1

)

Fortified Set

Scanner Z Score


Scanner Z Score

Result



Blank 3 -6.88 Positive -14.89 Positive

Blank 4 -7.25 Positive -15.93 Positive


PenG 10

SM 200

SM 200

8.14

8.68

8.58

Positive

Positive

Positive

8.09

7.51

7.49

Positive

Positive

Positive

CTC = chlortetracycline, OTC = oxytetracycline, PenG = penicillin G, Sulfamixture = mixture

of five sulphonamides; sulfamethazine, sulfamethoxypyridazine, sulfaquinoxaline, sulfadiazine

& sulfadimethoxine.

34

Table 8. Showing the results for the blind trial of the integrated AMS procedure.

Sample Identity Fortified Set

Scanner Z Score

Result Fortified Set +

TCN

Scanner Z Score


�-lactamase

Scanner Z Score


p-ABA

Scanner Z Score

Result

44 4.66 Positive -0.38 Negative 2.13 Positive 3.34 Positive

26 2.56 Positive 0.44 Positive -5.74 Negative 12.64 Positive


80 -7.67 Negative -13.97 Negative -7.24 Negative -3.17 Negative


12 7.49 Positive 5.46 Positive 5.00 Positive 4.92 Positive

Quality Control


)

Fortified Set

Scanner Z Score


TCN

Scanner Z Score


�-lactamase

Scanner Z Score


p-ABA

Scanner Z Score

Result

Blank1 -8.03 Negative -15.20 Negative -7.75 Negative -3.49 Negative

Blank 2 -7.22 Negative -14.90 Negative -4.84 Negative -2.92 Negative

PenG Spike 10 6.60 Positive 7.29 Positive -7.15 Negative 5.95 Positive

CTC Spike 100 4.91 Positive -1.75 Negative 3.27 Positive 4.32 Positive

SDZ spike 200 5.39 Positive 2.89 Positive 2.11 Positive -4.10 Negative

PenG = penicillin G, CTC = chlortetracycline, SDZ = sulfadiazine.

35

Table 9. Showing the results from the inter-laboratory validation conducting using OTC fortified

chicken muscle fluid.

Replicate Without TCN With TCN

Visual Scanner Z-Value Visual Scanner Z-Value

1 PPY Positive 3.54 Y Negative -10.63






7 PPY Positive 4.53 YYP Negative -5.05

8 PPY Positive 5.20 YYP Negative -3.34

9 PPY Positive 5.27 PY Positive 3.41

10 PPY Positive 6.06 P Positive 15.06

Quality Control Without TCN With TCN


) Visual Scanner Z-Value Visual Scanner Z-Value

Mean Blank

(n=6)

Y Negative -0.79 Y Negative -13.64

Mean Penicillin G

10 (n=6)

P Positive 13.61 P Positive 9.34

Mean Sulfamixture

200 (n=6)

P Positive 9.65 P Positive 13.93

Sulfamixture = mixture of five sulphonamides; sulfamethazine, sulfamethoxypyridazine,

sulfaquinoxaline, sulfadiazine & sulfadimethoxine.

36

FIGURES

Figure 1 Outline for the TCN / tetracycline post-screening assay.

Unknown sample extract

dilute with broth

(1:16)

incubate extract

with 50ul broth and

apply to

Premi�Test

[Control]

incubate extract

with 50ul TCN and

apply to

Premi�Test

incubate extract

with 50ul broth and

apply to

Premi�Test

[Control]

incubate extract

with 50ul TCN and

apply to

Premi�Test

100ul 100ul

100ul100ul

test produces a

PURPLE colour

test produces a

PURPLE colour

test produces a

YELLOW colour

test produces a

YELLOW colour

sample contains a tetracycline

(high potency / high

concentaration)

sample contains a tetracycline

(low potency / low

concentration)

37

Figure 2 The integrated AMS strategy for penicillins, sulphonamides and tetracyclines.

Sample (4g) Na2SO

4 (6g)

acetonitrile/

acetone,

70/30, v/v

(10 ml)

� homogenise (45 seconds)

� centrifuge (4500rpm, 4 oC, 10 mins)

� evaporate supernatant to near dry @ 40-45 oC

� resuspend in Lab Lemco broth (600 ul) in duplicate

sample stored @

4oC until required

100 ul of sample

applied to

Premi�Test

incubate @ 64 oC

until yellow end

point visible in

controls

sample does NOT contain

beta-lactam, sulphonamide or

tetracycline compound

sample positive for

an antimicrobial

compound

test produces a

PURPLE colour

test produces a

YELLOW colour

confirmation of class

identity required

post-screening

identification of

sulphonamides

post screening

identification of

beta-lactams

post-screening

identification of

tetracyclines

200ul

230ul

200ul

incubate extract

with 50 ul p-ABA

and apply to

Premi�Test

test produces a

YELLOW colour

test produces a

YELLOW colour

sample contains a

sulphonamide residue

incubate extract

with 20 ul beta-

lactamase and

apply to

Premi�Test

sample contains a

penicillin or cephalosporin

incubate extract

with 50 ul TCN

and apply to

Premi�Test

dilute extract with

broth and incubate

with 50 ul TCN

and apply to

Premi�Test

100ul

100ul

test produces a

YELLOW coloursample contains a tetracycline

Existing Procedure

test produces a

PURPLE colour

test produces a

PURPLE colour

test produces a

PURPLE colour

sample contains a non-

sulphonamide antibiotic


beta-lactam antibiotic


tetracycline antibiotic

New TC procedure

test produces a

YELLOW coloursample contains a tetracycline

38

Figure 3. Graphical representation –Blind Trial of the Integrated AMS procedure.

-20

-15

-10

-5

0

5

10

0 1 2 3 4 5 6 7 8 9 10

Sc

an

ne

r V

alu

e (

Z S

co

re)

Blank No Treatment Blank +p-ABA Blank +TCN Blank +b-lactamase

CTC No treatment CTC +p-ABA CTC +TCN CTC +b-lactamase

Sulphonamide No treatment Sulphonamide +p-ABA Sulphonamide +TCN Sulphonamide +b-lactamase

Unknown 44 No Treatment Unknown 44 +p=ABA Unknown 44 +TCN Unknown 44 +b-lactamase

Unknown 26 No Treatment Unknown 26 +p-ABA Unknown 26 +TCN Unknown 26 +b-lactamase





QC Samples Unknown Samples

Positive Region >0

Negative Region <0

Key

39

Figure 4. Showing the effect of a fixed

mass of MMP8 on the Premi®Test

response to a range of antimicrobial

compounds at different residue

concentrations in fortified chicken

muscle.

Figure 5. Showing the effect of a

fixed mass of MMP8 on

doxycycline fortified tissue extract

dilutions on the Premi®Test

response.

CTC = chlortetracycline, DOX = doxycycline, PenG = penicillin G

Figure 6. Showing the effect of TCN

buffer and nutrient broth on the

Premi®Test response to a range of

antimicrobial compounds at different

residue concentrations in fortified

chicken muscle.

40

Figure 7. Results of analyte specificity of the TCN assay using fortified chicken muscle.

PenG = penicillin G, SM = sulfamixture

41

Figure 8. Showing the results from the

investigation of analyte specificity of the

TCN assay using fortified chicken

muscle.

Figure 9. Ruggedness – investigation of the

effect of TCN buffer on the Premi®Test

responses to a wider range of tetracycline

compounds in fortified chicken muscle.

OTC = oxytetracycline, DOX = doxycycline, PenG = penicillin G, SM = sulfamixture, TC = tetracycline,

CTC = chlortetracyclne, SDZ = sulfadiazine, Clox = cloxacillin, TYL = tylosin.

42

Figure 11. Blind Trial using Integrated

AMS procedure.

PenG = penicillin G, CTC = chlortetracycline, SDZ = sulfadiazine, OTC = oxytetracycline

Figure 10. Ruggedness – HONEY

43 Post-Screening Classification of Tetracycline …randd.defra.gov.uk/Document.aspx?Document=vm02142_7196_FRA.pdf1 Post-Screening Classification of Tetracycline Positives Detected

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