Instruction Manual Version 7.7 October 2015

Transcription

1 Instruction Manual Version 7.7 October 05 Principle Antibiotic configuration Test procedure Materials and equipment Breakpoint interpretation Storage and stability Batch REF: Delivery charge: LOT 500P5700 Expiration Date:

2 Used for Microdilution Antimicrobial Susceptibility Tests for Bacteria Isolated from Poultry Flocks The AviPro PLATE is meant to facilitate the standardised differentiation of the S. Enteritidis strain (Sm/Rif/Ssq) and S. Typhimurium strain (Nal/Rif9/Rtt) contained in AviPro Salmonella live vaccines: CONTENTS. INTRODUCTION.... PRINCIPLE ANTIBIOTIC CONFIGURATION AND BREAKPOINT CONCENTRATIONS STORAGE AND STABILITY PACKAGING ADDITIONAL MATERIALS AND EQUIPMENT MEDIA AND REAGENTS TEST PROCEDURE PRECAUTIONARY MEASURES GRAM STAIN PREPARATION OF INOCULUM.... BROTH INOCULATION..... MUELLER-HINTON OR MODIFIED ISO-SENSITEST BROTH..... H-BROTH....5 BROTH MICRODILUTION....6 SEALING AND INCUBATION MUELLER-HINTON OR MODIFIED ISO-SENSITEST BROTH H-BROTH....7 MEASUREMENT TEST PERFORMANCE AND DATA INTERPRETATION DATA ACCEPTANCE CRITERIA BREAKPOINT INTERPRETATION QUALITY ASSURANCE SAFE DISPOSAL WARRANTY GENERAL TECHNICAL REMARKS SYMBOLS OF LABEL (ACCORDING TO EN 90 AND PREN 375) WASTE DISPOSAL REFERENCES APDIX.... TECHNICAL INFORMATION NOTE ON MICRONAUT-SYSTEM... 9 October 05 von

3 FACT SHEET This Instruction Manual is meant for use in combination with the AviPro PLATE, Batch REF , LOT 500P5700, only. This panel of antibiotics is considered most appropriate for microdilution broth testing procedure for both identification of Lohmann Salmonella live vaccine strains and overall analysis of antimicrobial susceptibility in bacterial isolates from poultry origin. The FACT SHEET provides a summary of the actual panel of antibiotics in tabular format. For customers using the MICRONAUT software, the Batch REF , LOT 500P5700 of AviPro PLATE will be delivered together with the corresponding updated MICRONAUT software on compact disc. If delayed, the customer should kindly inform our representative of Lohmann Animal Health directly. Actual panel of antimicrobial agents precoated to AviPro PLATE N o Class of antibiotics Agent Abbreviation Remarks β- lactam antibiotics Amoxicillin see * Oxacillin see ** 3 Penicillin G Ceftiofur CET 5 Cefpodoxime-Proxetil CPP 6 Cefotaxime CTX 7 Polypeptide antibiotics Colistin COL see *** Fluoroquinolones Enrofloxacin 9 Macrolides Erythromycin 0 ErythromycinD D Vaccine marker Tilmicosin TILM Tylosin TLS 3 Lincosamides Lincomycin LIN Aminoglycosides Neomycin NEO 5 Streptomycin STR Vaccine marker 6 Lincosamides/Aminocyclitole Lincomycin-Spectinomycin LIS s 7 Ansamycins Rifampicin RAM Vaccine marker Aminocyclitols Spectinomycin SPT 9 Tetracyclines Tetracycline Class representative for chlortetracycline-, oxytetracycline, doxycycline. 0 Pleuromutilins Tiamulin Folate pathway inhibitors Trimethoprim-sulfamethoxazole *The AviPro PLATE omits the recommended enterococcal specific penicillin G () breakpoint of µg/ml and the staphylococcal specific amoxicillin (Ampicilin / ) breakpoint of µg/ml (Table II-A). Instead, β -lactam-resistant enterococci will be tested using the enterococcal specific Ampicilin / breakpoint of µg/ml. The testing of amoxicillin instead of penicillin is preferred due to the fact as drug of choice for treatment of enterococcal infections and in order to avoid double testings. The penicillin-resistant staphylococci will be detected using the staphylococcal specific penicillin breakpoint of µg/ml. The preferred use of penicillin rather than aminopenicillins ensures testing of susceptibility of all staphylococci to all penicillinase-labile penicillins. Amoxicillin is a semisynthetic analogue of ampicillin. Both aminopenicillins are comparably in their in vitro activities and have a similar range of activity. Differences between both compounds rely on pharmacology which is based on a greater gastrointestinal absorption of amoxicillin. Amoxicillin can be used to predict the activity of aminopenicillins 9 October 05 3 von

4 **Oxacillin is used to test for susceptibility to methicillin, nafcillin, and cloxacillin. The breakpoint of µg/ml has to be used to detect methicillin resistance in coagulase positive veterinary staphylococci (MRSA) and the breakpoint of µg/ml has to be used to predict methicillin resistance in other Staphylococcus spp. ***The CLSI Document M00-S0, Vol. 30, No. (00) [] recommends breakpoint concentrations for colistin (Non- Enterobacteriaceae), which allows the following interpretation: µg/ml = susceptible, µg/ml = intermediate, µg/ml = resistant. There are no CLSI breakpoints for Enterobacteriaceae, but the EUCAST breakpoints are S, R. ****CPP and CTX are indicator Cephalosporins. Enterobacteriaceae with ESBLs (Extended-Spectrum Beta- Lactamases) are resistant in the presence of these Cephalosporins. ENQUIRIES Enquiries relating to distribution of AviPro PLATE and panel layout of relevant antibiotics should be addressed to: Lohmann Animal Health GmbH (an Ely Lilly company) Heinz-Lohmann-Str. 77 Cuxhaven Germany Tel +9 (0) Fax +9 (0) Enquiries relating to technical aspects of the microdilution assay procedure, data interpretation, MICRONAUT software and equipment should be addressed to: Gesellschaft für mikrobiologische Diagnostika mbh MERLIN Diagnostika GmbH Kleinstr Bornheim Hersel Germany Tel. +9 (0) Fax +9 (0) October 05 von

5 . Introduction The AviPro PLATE format is designed to establish an in-vitro microdilution antimicrobial susceptibility test method for global use in poultry veterinary diagnostic laboratories. The use of the AviPro PLATE allows determination of the activity of antimicrobial agents against gram-positive and gram-negative bacteria in poultry flocks. The breakpoint testing procedure is used for direct measure of antimicrobial activity revealing minimal inhibitory concentrations (MICs ). They provide information on quantitative changes in susceptibility and resistance of bacterial isolates to antimicrobial agents. The results are aimed at giving clear guidance for appropriate therapeutic intervention. The panel of antibiotics pre-coated to the PLATE comprises also those used as markers to allow discrimination of field Salmonella from Salmonella metabolic drift mutants contained in Lohmann Salmonella live vaccines. Thus the results aid to control take of Lohmann Salmonella live vaccines. In addition, the data generated by this method is designed to be objective and unbiased by the laboratory s interpretation of values. Prior to first use of a new batch of AviPro PLATE the user is highly advised to consult the FACT SHEET to assure awareness of any changes regarding selection and concentration of antibiotics in comparison to previous batches of AviPro PLATE. The panel of antibiotics are in line with a counsel of poultry diagnosticians highly experienced with the current situation in the field. The performance of the test procedure broadly follows the documentation of the standard performance criteria issued by the Clinical and Laboratory Standard Institute (CLSI) [, 3], formerly National Committee for Clinical Laboratory Standards (NCCLS), Wayne, Pennsylvania, USA. Thus the AviPro PLATE is meant to contribute to establishing internationally accepted standards on antimicrobial resistance in animals and in animal-derived food pursued by the Office International des Epizooties (OIE) [5].. Principle The AviPro PLATE quantitatively measures the in-vitro activity of an antimicrobial agent against a given bacterial culture isolated from poultry. The round-bottom wells of the 96-well test PLATE are pre-coated with various concentrations of antibiotics administered in poultry (see APDIX: Table I and the AviPro PLATE layout). The PLATE layout also considers antibiotics exploited as vaccine markers to identify the Lohmann Salmonella live vaccine strains, respectively (see APDIX: AviPro PLATE layout). The wells are filled with broth inoculum, containing a standardised number of colony forming units of the bacterial isolate grown on agar PLATEs. The antibiotics rehydrate and regain full activity. After incubation at 35 ºC for 6 0 hours, inhibition of bacterial growth is measured using either a multichannel spectrophotometer or visually. The in-vitro activity of an antimicrobial agent to a given bacterial isolate is determined by MIC breakpoints. The antimicrobial agents are examined at the specific breakpoint concentrations representing the MIC values of lower (LB) and upper breakpoint (UB) necessary for differentiating between the interpretive clinical categories of susceptible (S), intermediate (I) and resistant (R) rather than in the full range of doubling-dilution concentrations to determine MICs. Bacterial growth at both LB and UB concentrations indicates resistance, growth at only the LB signifies an intermediate result, and no growth at either concentration is interpreted as susceptible. Considering the limited range of drug concentrations, breakpoint testing allows a greater number and variety of antimicrobial agents to be incorporated into a broth microdilution set up than into trays designed for full range dilution testing. In contrast to agar diffusion method it avoids inherent errors associated with extrapolating disk diffusion zone sizes and MIC values. The MIC values are recorded automatically by the specified software programme (MICRONAUT) or manually on the data sheet (see APDIX: AviPro PLATE data sheet). 3. Antibiotic configuration and breakpoint concentrations The antibiotics rifampicin, streptomycin and erythromycin D are used in concentrations for reliable identification Lohmann Salmonella live vaccine strains (see appendix, Table I-B). The panel of antimicrobial agents pre-coated to Defined as the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in broth dilution susceptibility test [3]. 9 October 05 5 von

6 wells of the AviPro PLATE is broadly configured in concentrations to allow breakpoint interpretation according to the CLSI. Any changes of selection and concentration of antibiotics in comparison to previous batches of AviPro PLATE are highlighted on the FACT SHEET.. Storage and stability The sealed AviPro PLATE and auxiliaries have to be used up to the expiry date as indicated on the label and have to be stored at 5 ºC 5 ºC, except the H-broth has to be stored at ºC ºC. 5. Packaging The packaging contains 00 pieces of AviPro PLATE and adhesive PLATE sealers. The single AviPro PLATE is individually pouched in aluminium foil. The PLATE contains antibiotics at varying concentrations preferably used for poultry (see APDIX: AviPro PLATE layout). The PLATE layout considers two susceptibility tests per PLATE. Each test encompasses wells exploiting a panel of antimicrobial agents and one growth control. The wells of row A to D are reserved for test, wells of row E to H for test, accordingly. 6. Additional materials and equipment The AviPro PLATE is not supplied in a ready-to-use kit format. Items supplied by MERLIN to facilitate microdilution antimicrobial susceptibility test method are given in tabular format: Mueller-Hinton broth or ISO-Sensitest broth modified without salt (MERLIN #M/E-37-00) H-broth (MERLIN #M/E-3-00) -channel reservoir (MERLIN #M/R ) -channel reservoir (MERLIN #M/R ) MICRONAUT pipette (MERLIN #M/BH or L3-0-00) Disposables tips for MICRONAUT pipettes (MERLIN #M/BH or #L ) MICRONAUT Skan (MERLIN #M/L5-0-00) MICRONAUT Software (MERLIN #M/U ) NaCl 0.9 % (MERLIN #M/E-3-00) McFarland Standard Blood agar PLATE Sterile loop Incubator 37 ºC Marker pen The desiccant sachet accompanying each plate contains cobalt chloride. Colour change from blue to red may indicate presence of humidity preventing plate from further use for testing. 9 October 05 6 von

7 Products of MERLIN Diagnostika GmbH are distributed by Genzyme Virotech GmbH Löwenplatz 5 65 Rüsselsheim Germany Tel. +9 (0) Fax: +9 (0) info@virotech.de Website: 7. Media and reagents Table. Composition of Media and Reagents 3 Reagents NaCl 09 % l ISO-Sensitest bouillon modified (00 tubes each ml) H-bouillon (00 tubes each ml) Mueller-Hinton II bouillon Components Sodium chloride ISO-Sensitest bouillon di-sodiumphosphate Haematin, NaOH, Tween 0, Pyridoxal ß-nicotinamide, Adenindinucleotide, Columbia broth base, Glucose, Yeast extract, Neopeptone Agarose type II A Beef extract, Acid casein hydrolysate starch. Test procedure The test procedure for aerobic bacteria is illustrated on the flow chart of the APDIX. It is good laboratory practice to prewarm salt solutions and broths in a waterbath or incubator (37 C) to avoid deviation of bacterial growth caused by thermal gradient.. Precautionary Measures - In-vitro diagnostic for veterinary use only - Do not pipette the reagents by mouth - Only for proper use - Samples, bacteria cultures and the inoculated test PLATEs have to be considered as potentially infectious and must be treated properly and with respect to the corresponding precautionary measures by qualified staff. It is important to work aseptically during the whole test procedure. For information please refer to the HHS Publication entitled BioSafety in Microbiological and Biomedical Laboratories [], or to the corresponding national legal requirements. - Upon reading and evaluation of the tests, all samples, inoculated and contaminated products (pipette tips, reservoirs and test PLATEs) must be autoclaved, burnt or disinfected in a bactericidal solution before disposal. - It is important to follow the instructions carefully; each deviation may influence the quality of the results. - The test results should be interpreted by qualified staff with experience in microbiology. The case history, clinical signs, origin of samples, colony and microscopic morphology, serology and the identification of bacteria must be taken into consideration when interpreting the results.. Gram Stain Gram-positive bacteria are purple, whereas gram-negative bacteria are red to pink. It is recommended to follow carefully the respective standard microbiology protocol or instructions of use supplied from the manufacturer of gram-stain method kits. 3 For particular bacteria groups it is advisable to add supplement to the broth. 9 October 05 7 von

8 .3 Preparation of Inoculum Prepare a tube with 5 ml of NaCl 0.9 %. Pick several identical bacterial colonies grown on the agar PLATE without additives incubated for hours. Suspend the colonies in NaCl 0.9 % to a uniform solution matching the turbidity of a McFarland standard. Invert the tubes and the McFarland standard several times and vortex thoroughly. Using a spectrophotometer, compare absorbance of McFarland standard versus bacterial suspension measured at wavelength of 60 nm or 690 nm depending on the broth used. Alternatively, compare the McFarland standards versus bacterial suspension examining the fluid in the tubes against a white background with contrasting lines.. Broth Inoculation.. Mueller-Hinton or Modified ISO-Sensitest Broth For gram-negative and gram-positive bacteria, transfer 50 µl of bacteria suspension into 0- ml of Mueller-Hinton or modified ISO-Sensitest broth. Invert the tubes several times and vortex thoroughly... H-Broth For fastidious bacteria (e.g., Streptococcus, Coryneform bacteria, Haemophilus, Neisseria, Ornithobacterium Rhinotracheale, Riemerella) transfer 00 µl of the bacteria suspension into 0- ml of pre-heated H-broth. Invert the tubes several times and vortex thoroughly..5 Broth Microdilution Seal off the AviPro PLATE and leave it not longer than 30 minutes until used. Identify the test PLATE on the front, rather than the topside. Transfer the test bacterial suspension into the -channel reservoir as illustrated in the pipetting scheme of AviPro PLATE format. For two tests, fully mount the eight-channel pipette with properly fitted disposable tips. Each four tips correspond to one of two multi-channel reservoirs. Aspirate each tip with appropriate volume of bacteria suspension. Pipette 00 µl out of the channel into corresponding wells of the test PLATE..6 Sealing and Incubation.6. Mueller-Hinton or Modified ISO-Sensitest Broth To prevent drying, seal the PLATE with adhesive non-perforated PLATE sealer or cover the PLATE with a tightly fitting plastic cover (do not stack more than 5 PLATEs). Place the PLATE in an incubator at 35 ºC for 6 0 hours..6. H-Broth To prevent drying, cover the PLATE with a tight fitting plastic cover (do not stack more than 5 PLATEs). Do not use adhesive non-perforated PLATE sealer. Place the PLATE in an incubator with CO -gassing environment at 35 ºC for 0 hours..7 Measurement Remove the PLATE sealer or PLATE used as a lid from the test PLATE. Ensure that there are no bubbles in any of the wells, as this will cause optical aberrations. If necessary, rupture any bubbles with a clean pipette tip. For preparation of McFarland turbidity standard refer to paragraph 3..3 of the CLSI M3-A3, Vol. No. [3]. Using a spectrophotometer the range of absorbance of McFarland standard at 60 nm wavelength should be validated by the individual laboratory. 9 October 05 von

9 Ensure that there is no condensation or there are no smudges (e.g., fingerprints) on the bottom of the test PLATE. If necessary, wipe the bottom of the test PLATE clean with a soft lint-free paper tissue. For automated measurement, place the AviPro PLATE onto the PLATE carrier of the multichannel spectrophotometer and measure absorbance of test and growth control wells at 60 nm or 690 nm wavelength, depending on the broth used. For visual measurement, determine presence or absence of bacterial growth in test and growth control wells. 9. Test performance and data interpretation 9. Data Acceptance Criteria The intensity of bacterial growth is expressed in optical density (OD) values measured at wavelength of 60 nm or 690 nm, depending on broth used. The values are evaluated, interpreted and the plausibility is checked with the MICRONAUT Software. The well of the growth control must be fully covered by bacterial colonies (cloudy), otherwise the test must be repeated. The software programme will make a record automatically. For visual measurement, the test PLATE is accepted if growth control shows sufficient and visible bacterial growth. The test PLATE must be rejected if the growth control showed no growth. The results should be recorded on a separate data sheet (see APDIX: AviPro PLATE data sheet). 9. Breakpoint Interpretation For antibiotics of the AviPro PLATE, tested in four or fewer consecutive concentrations, or in non-consecutive concentrations, the interpretive S, I and R categories are determined on the basis of the lower and upper breakpoint MIC values (see APDIX: Tables I-A, I-B, II-A, II-B, II-C, II D, and III). The lower breakpoint (LB) is represented by the MIC value, which fully prevents from sufficient and visible bacterial growth, i.e. the bacterial isolate is susceptible to an antimicrobial agent. The upper breakpoint (UB) is represented by the MIC value, which partly prevents from sufficient and visible bacterial growth indicating intermediate activity of the antimicrobial agent. Measuring of MIC values above UB, therefore, demonstrates resistance of bacterial isolate to a particular antimicrobial agent. By means of MICRONAUT software MIC values are automatically analysed and interpreted for a given bacterial isolate and antimicrobial agent. Measurement results and corresponding interpretive clinical categories including MIC range may be reported to veterinarians for direct therapeutic use. Table. Breakpoint interpretation of an antimicrobial agent. Visual reading Automated reading (OD) Clinical interpretation LB UB LB UB Bacterial < 0. < 0. Susceptible (S) Growth + 0. < 0. Intermediate (I) Resistant (R) OD = optical density value analysed by MICRONAUT software programme; LB = lower breakpoint concentration; UB = upper breakpoint concentration; + symbol = well showing presence of bacteria; symbol = well showing absence of bacteria. Intermediate category has not been determined for all antibiotics yet. 0. Quality assurance Quality control testing and the interpretive criteria for AviPro PLATE are in compliance with the CLSI documents M00- S0 [] and M3-A3 [3]. For quality assurance of AviPro PLATE, the analysis of bacterial reference quality control strains is highly recommended, e.g. in weekly intervals. For culture collection numbers of reference strains refer to Table 3 of the CLSI document M3-A3 [3]; an excerpt is given in Table 3 of the instruction manual. Table 5 of the CLS document provides the corresponding acceptable quality control ranges of MICs (μg/ml). See also APDIX:Tables II-A, II-B, II-C, II D and III. 9 October 05 9 von

10 Table 3. Excerpt of bacterial reference quality control strains taken from the CLSI document M3-A3, Table 3 [3]. Strain ATCC No DSMZ No Staphylococcus aureus subsp. aureus ATCC 93 DSM 569 Escherichia coli ATCC 59 DSM 03 Pseudomonas aeruginosa ATCC 753 DSM 7 Enterococcus faecalis ATCC 9 DSM 570 ATCC = American Type Culture Collection DSMZ = Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Laboratories are encouraged to determine colony-forming units on inoculum suspensions periodically to ensure that the final inoculum concentration routinely obtained approximates 5 x 0 5 CFU/mL. This can be accomplished be removing a 0.0 ml aliquot from the growth-control well or tube immediately after inoculation and diluting it in 0 ml of 0.9 % saline. After mixing, a 0. ml aliquot is spread over the surface of a suitable agar medium. Following incubation, the presence of approximately 50 colonies would indicate an inoculum density of 5 x 0 5 CFU/mL as stated in paragraph 0. () of the CLSI M3-A3, Vol. No. [3].. Safe disposal The used AviPro PLATE and potentially contaminated materials must be disinfected, burnt or autoclaved.. Warranty The instruction manual should be strictly followed to accomplish validity of the AviPro PLATE. Alterations of the test procedure would seriously affect the quality of test performance and exclude any claim to compensation. 3. General technical remarks For standardised procedure, technical remarks should be strictly followed: - Use a single pure and healthy culture from blood agar without additives, which should be not older than hours. Select fastidious bacteria from blood agar (without additive) after incubation at 35 ºC 37 ºC for hours. - Subculture a purity control on blood agar from the reservoir suspension. - Culture gram-positive bacteria and gram negative bacteria on blood agar before inoculation. - For the correct McFarland adjustment, shake thoroughly both the bacterial suspension and the McFarland standard before each comparison. - Store H-broth at + ºC to + ºC, and pre-warm broth at 35 ºC 37 ºC for hour in the incubator before inoculation of bacteria. - In case of the insufficient growth of Haemophilus and lipophilic Corynebacteria add NAD supplement solution (Oxoid, cat. no. SR5). - For Mueller-Hinton broth, assure the concentration of bivalent cations for Ca ++ (0 5 mg/litre) and for Mg ++ (0.5 mg/litre). Adjust the correct ph of the Mueller-Hinton broth between ph 7. and ph 7. at room temperature (5 ºC). - If you do not use culture media ready for use follow the instructions for preparation carefully. - Use NaCl 0.9 %. - Follow carefully the incubation instructions and do not incubate bacteria less than 6 hours and fastidious bacteria less than 0 hours. - Incubate fastidious bacteria in H-broth at enriched CO -atmosphere if necessary. - For automated reading, check the respective panel type selection and enter H taking into account inoculation of fastidious bacteria in H-broth. - Best accuracy and precision of results is accomplished by stringent use of growth media and saline consistently purchased from any single source (e.g., MERLIN Diagnostika GmbH). 9 October 05 0 von

11 - For susceptibility testing of anaerobic bacteria you may ask for technical support from Lohmann Animal Health and MERLIN Diagnostika.. Symbols of label (according to EN 90 and pren 375) LOT IVD REF Number of tests per PLATE Conditions of storage Reference to test protocol Reference to safety measures Expiry date Logo of the assigned company according to packaging ordinance for material utilisation of the accumulated packaging (valid within Germany) Batch number In-vitro diagnostic for veterinary use only Product number 5. Waste disposal The packaging materials (cardboard packaging and multi layer films) can be thrown into the respective dustbins and can be disposed of with the household rubbish. 6. References [] Clinical and Laboratory Standards Institut (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, Twentieth Informational Supplement. CLSI document M00-S0, Vol. 30, No., Clinical and Laboratory Standards Institute, Wayne, PA, USA, 0. [] BioSafety in Microbiological and Biomedical Laboratories, HHS Publication No. (CDC) , 3 rd Edition, May 993. [3] Clinical Laboratory Standards Institutes (CLSI). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard - third edition. Document M3-A3, Vol. 3, No., Wayne, PA, USA, 0. [] Technical Information on LINCO-SPECTIN TM, Pharmacia & Upjohn, 00. [5] OIE International Standards on Antimicrobial Resistance. OIE (World Organsisation for Animal Health), Paris, France, 003. [6] Trolldenier H. Quantitative sensitivity of frequently isolated veterinary pathogens as certained by multicentric studies in comparison to chemotherapeutics used in veterinary medicine. Praktische Tierarzt 00;():5-6. German [7] Luhofer G, Böttner A, Hafez HM, Kaske M, Kehrenberg C, Kietzmann M, Klarmann D, Klein G, Krabisch P, Kühn T, Richter A, Sigge C, Traeder W, Waldmann K-H, Wallmann J, Werckenthin C, Schwarz S. Layout proposals of the working group antibiotic resistance for microtitre PLATEs to be used in routine antimicrobial susceptibility testing of bacterial pathogens from infections of large food-producing animals and from mastitis cases. Berl Munch Tierarztl Wochenschr 00;7(7-):5 5. German 9 October 05 von

12 7. Appendix - Flow chart of microdilution antimicrobial susceptibility testing procedure - AviPro PLATE layout of antimicrobial agents (µg/ml) - Table I-A. MIC breakpoints and interpretive standards of antimicrobial agents - Table I-B. Antimicrobial agents used as resistance marker to discriminate field strains of Salmonella Enteritidis (S.E.) and Salmonella Typhimurium (S.Tm.) from Salmonella metabolic drift mutants in Lohmann Salmonella live vaccines - Table II-A. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gram-positive reference strain Staphylococcus aureus ATCC 93 - Table II-B. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gram-positive reference strain Enterococcus faecalis ATCC 9 - Table II-C. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gram-negative reference strain Escherichia coli ATCC 59 - Table II-D. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gram-negative reference strain Pseudomonas aeruginosa ATCC Table III. Rationalisation of interpretive results listed in Tables II-A, II-B, II-C and II D - AviPro PLATE data sheet - Technical information note on MICRONAUT-System. 9 October 05 von

13 Flow chart of microdilution antimicrobial susceptibility testing procedure Table I. Registration of Antimicrobial Agents Used for Pre-coating of AviPro PLATE. Antimicrobial Agent Agent Abbreviation Reference Ampicillin AMP EU Ceftiofur CEF NCCLS Colistin CST EU Enrofloxacin Single pure bacteria O colony in NaCL 0.9% matching EU, NCCLS turbidity Erythromycin of a McFarland standard EU, NCCLS Gentamicin GEN NCCLS Lincomycin LIN EU, NCCLS Neomycin NEO EU, NCCLS Oxacillin EU Penicillin G EU, NCCLS Rifampicin RIF not registered for poultry Gram-negative bacteria Spectinomycin Gram-positive bacteria SPEC EU, NCCLS Streptomycin STRE NCCLS Tetracycline EU, NCCLS Tiamulin EU Trimethoprim-sulfamethoxazole EU, NCCLS EU: refer to Council Regulation (EEC) No5/003; NCCLS: refer to NCCLS M3-A (00). Inoculate 50 µl in 0- ml Mueller-Hinton broth or or in 0- Figure ml. AviPro ISO-Sensitest PLATE broth Layout Outlining Type and in 0- Dilution ml Range ISO-Sensitest (µg/ml) of broth Antimicrobial Agents T E S T A B AMP 6 AMP CEF CEF O O Inoculate 50 µl in 0- ml Mueller-Hinton broth GEN GEN /76 /3 Fastidious bacteria (Streptococci, coryneform bacteria, Haemophilus, Neisseria) 6 Inoculate 00 µl in 0- ml H-broth STRE RIF 50 SPEC 6 C AMP CEF O GEN /9 CST LIN 0 SPEC 3 D AMP CEF Dispense NEO 00 µl GEN in each well of an AviPro PLATE /9.5 O CST LIN 0 Growth Control T E S T E F AMP 6 AMP CEF CEF O O GEN GEN /76 /3 6 STRE RIF 50 SPEC 6 G H AMP AMP CEF CEF Seal O AviPro PLATE GEN without perforation CST or /9 Stack up maximum 5 plates and seal the upper plate O NEO GEN CST /9.5 LIN LIN 0 0 SPEC 3 Growth Control Incubate at 35 ºC 37 ºC for hours (Incubate fastidious bacteria in an enriched CO atmosphere for hours) Read plates visually or photometrically 9 October 05 3 von

14 AviPro PLATE layout of antimicrobial agents (µg/ml) T E S T A B C CET CPP CTX TILM 6 TILM NEO 6 LIS /3 /3 /9 0,5 COL STRE 00 RAM 50 D TLS TLS.0 NEO /9.5 COL LIN 0 Growth Control T E E 6 CET TILM 6 LIS /3 S T F CPP TILM /3 0, STRE 00 G CTX NEO 6 /9 COL 0 RAM 50 H TLS TLS.0 NEO /9.5 COL LIN 0 Growth Control D = Erythromycin used as resistance marker to discriminate Salmonella field strains from Salmonella metabolic drift mutants used in Lohmann Salmonella live vaccines. Wells exploiting resistance markers are emphasised by lined background. 9 October 05 von

15 Table I-A. MIC breakpoints and interpretive standards of antimicrobial agents Antimicrobial agent Agent Breakpoint (µg/ml) Reference Veterinary pathogen abbreviation LB UB Amoxicillin / Ampicilin [3] Enterobacteriaceae Staphylococci Enterococci Streptococci (not S. pneumoniae) Listeria monocytogenes 6 Ceftiofur CET [3] Cattle Respiratory Disease Mannheimia haemolytica Pasteurella multocida Histophilus somni Swine Respiratory Disease Actinobacillus pleuropneumoniae Pasteurella multocida Salmonella Choleraesuis Streptococcus suis Cefpodoxime-Proxetil CPP [3] for ESBL screening Cefotaxime CTX [3] for ESBL screening Colistin COL [] Pseudomonas aeruginosa Acinetobacter spp. Other non-enterobacteriaceae Doxycycline [] Enrofloxacin [3] Chicken and turkeys Pasteurella multocida Escherichia coli Erythromycin [3] Organisms other than Streptococci Streptococci Lincomycin LIN [6] Neomycin NEO [6, 7] Oxacillin [3] Staphylococcus aureus Staphylococci 0,5 Penicillin G Staphylococci Enterococci [3] Listeria monocytogenes Spectinomycin Bovine (Respiratory Disease) Mannheimia haemolytica Pasteurella multocida Histophilus somni Tetracycline Organisms other than streptococci Tiamulin Swine (Respiratory Disease) Actinobacillus pleuropneumoniae SPT [3] [3] [3] 9 October 05 5 von

16 Antimicrobial agent Agent Breakpoit (µg/ml) Reference Veterinary pathogen abbreviation LB UB Tilmicosin TILM [3] Bovine (Respiratory Disease) Mannheimia haemolytica Swine Respiratory Disease Pasteurella multocida Actinobacillus pleuropneumoniae 6 Trimethoprim-sulfamethoxazole [3] Staphylococcus spp. Enterobacteriaceae Streptococcus pneumoniae Haemophilus influenzae /3 /9.5 /9 /3 Tylosin TLS [6] MIC breakpoints and interpretive criteria are based on human rather than animal data. Minimal inhibitory concentration (MIC) at lower (LB) and upper breakpoint (UB), representing the susceptible and intermediate criteria, respectively, was validated on basis of veterinary indication and taxaspecific bacterial isolate; bacterial growth at MIC values above the UB indicates antimicrobial resistance. Table I-B. Antimicrobial agents used as resistance markers to discriminate field strains of Salmonella Enteritidis (S.E.) and Salmonella Typhimurium (S.Tm.) from Salmonella metabolic drift mutants used in Lohmann Salmonella live vaccines. Antimicrobial agent Veterinary pathogen Erythromycin Chicken S.E. Sm/Rif/Ssq (vaccine strain) S.E. Leipzig (field strain) S.Tm. Nal/Rif9/Rtt (vaccine strain) S.Tm. Moskau 5 (field strain) Rifampicin Chicken S.E. Sm/Rif/Ssq (vaccine strain) S.E. Leipzig (field strain) S.Tm. Nal /Rif9/Rtt (vaccine strain) S.Tm. Moskau 5 (field strain) Streptomycin Chicken S.E. Sm/Rif/Ssq (vaccine strain) S.E. Leipzig (field strain) Agent abbreviation RAM STR Breakpoint (µg/ml) LB MIC range < 0 30 < 0 30 > 50 < 50 > 50 < 50 Reference 00 > < 00 For each marker, the minimal inhibitory concentration (MIC) at lower breakpoint (LB) is shown in combination with the MIC range. MIC values were validated at Lohmann Animal Health. LAH LAH LAH 9 October 05 6 von

17 Table II-A. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gram-positive reference strain Staphylococcus aureus ATCC 93. Antimicrobial agent Abbreviation MIC range [µg/ml] Breakpoint LB + UB Interpretive result Amoxicillin /Ampicilin S Ceftiofur CET -.0 S Cefpodoxime CPP - I/R Colistin 3 CST + R Cefotaxime 3 CTX - I/R Doxycycline S Enrofloxacin S Erythromycin 0. + S/I Lincomycin LIN - S/I Neomycin NEO - S Oxacillin 0. S Penicillin G 0. R Spectinomycin SPT R Tetracycline 0. + S Tiamulin.0 6 S Tilmicosin TILM S Trimethoprim- /9.5 /3 S Sulfamethoxazole Tylosin TLS - S/R ATCC: American Type Culture Collection ( Taken from CLSI, M3-A3, 00. Data validated by MERLIN Diagnostika GmbH. Rationale of interpretive results is given in Table III. The methodology should be re-examined. 3 Taken from CLSI, M00-S0, 00. Taken from CLSI, M3-A3 Vol.. 5 Table II-B. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on grampositive reference strain Enterococcus faecalis ATCC 9. Antimicrobial agent Abbreviation MIC range [µg/ml] Breakpoint LB + UB Interpretive result Amoxicillin /Ampicilin S Doxycycline S Enrofloxacin 0. + S/I Erythromycin + I Lincomycin LIN - S/I Neomycin NEO 6 6 R Oxacillin 3 R Penicillin G S Spectinomycin SPT I/R Tetracycline 3 + I/R Tilmicosin TILM R Trimethoprim- /9.5 /3 S Sulfamethoxazole Tylosin TLS - S/R For further explanation see Table II-A. 9 October 05 7 von

18 Table II-C. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gramnegative reference strain Escherichia coli ATCC 59. Antimicrobial agent Abbreviation MIC range [µg/ml] Breakpoint LB + UB Interpretive result Amoxicillin /Ampicilin + 6 S Ceftiofur CET -.0 S Cefpodoxime CPP -.0 S Colistin 3 COL - + S Cefotaxime 3 CTX S Doxycycline S Enrofloxacin S Lincomycin LIN - S/I Neomycin NEO S Spectinomycin SPT S/I Tetracycline + S Trimethoprim- Sulfamethoxazole /9.5 /3 S For further explanation see Table II-A. Table II-D. Range of minimal inhibition concentration (MIC) and interpretive result for antibiotics tested on gramnegative reference strain Pseudomonas aeruginosa ATCC 753. Antimicrobial agent Abbreviation MIC range [µg/ml] Breakpoint LB + UB Interpretive result Ceftiofur CET 6-6 R Colistin 3 COL + S Cefotaxime 3 CTX -3 R Doxycycline S Enrofloxacin + I/R Lincomycin LIN - S/I Spectinomycin SPT 56 - > R Tetracycline 3 + I/R Trimethoprim- Sulfamethoxazole /5 3/60 /3 R For further explanation see Table II-A. 9 October 05 von

19 Table III. Rationale of interpretive results listed in Tables II-A, II-B, II-C and II D. Interpretive result S I R S/I I/R S/I/R Rationale The MIC range of the control strain is below or equivalent to the lower breakpoint concentration. The MIC range of the control strain is above the lower breakpoint concentration but below or equivalent to the upper breakpoint concentration. The MIC range of the control strain is above the upper breakpoint concentration. The MIC range of the control strain is below, equivalent or above the lower breakpoint concentration but below or equivalent to the upper breakpoint concentration. The MIC range of the control strain is above the lower breakpoint concentration but below or equivalent or above the upper breakpoint concentration. The MIC range of the control strain is below or equivalent to the lower breakpoint concentration, or the MIC range of the control strain is above the lower breakpoint concentration but below or equivalent to the upper breakpoint concentration, or the MIC range of the control strain is above the upper breakpoint concentration. 9 October 05 9 von

20 AviPro PLATE data sheet ID PLATE:.... Batch REF: Delivery charge: LOT 500P5700 Expiration Date: Test (wells A - D), ID isolate : Test (wells E H), ID isolate : A 6 CET TILM 6 LIS /3 B CPP TILM /3 0, STRE 00 C CTX NEO 6 /9 CST 0 RAM 50 D TLS TLS NEO /9.5 CST LIN 0 Growth Control E 6 CET TILM 6 LIS /3 F CPP TILM /3 0, STRE 00 G CTX NEO 6 /9 CST 0 RAM 50 H TLS TLS NEO /9.5 CST LIN 0 Growth Control Amoxicillin (), Ceftiofur (CET), Cefpodoxim-Proxetil (CPP), Cefotaxim (CTX), Colistin (CST), Enrofloxacin (), Erythromycin (), Erythromycin () 5, Lincomycin (LIN), Lincospectin (LIS), Neomycin (NEO), Oxacillin (), Penicillin G (), Rifampicin (RAM) 7, Doxycycline (), Streptomycin (STR) 7, Tetracyclin (), Tiamulin (), Tilmicosin (TILM), Trimethoprim-sulfamethoxazole () ), Tylosin (TLS) Wells exploiting resistance markers are emphasised by lined background. 5 Antibiotics used to discriminate Salmonella field strains from Salmonella metabolic drift mutants used in Lohmann Salmonella live vaccines. 9 October 05 0 von

21 . Technical information note on MICRONAUT-System Gesellschaft für mikrobiologische Diagnostika mbh Photometric measurement and data interpretation of AviPro PLATE using MICRONAUT-System For microdilution antimicrobial susceptibility testing of bacteria isolated from poultry flocks, the photometric analysis of the AviPro PLATE format is based on measuring the turbidity of broth used to grow bacteria. The absorbance is measured at wavelength of 60 nm and 690 nm, respectively, depending on the broth exploited. The intensity of bacterial growth is determined by MICRONAUT Software using default threshold values. Their definition takes into account both the absorbance of bacterial growth characteristics und absorbance of the broth itself. The MICRONAUT-Software facilitates transfer of default threshold values to the MICRONAUT-SKAN Photometer for evaluation of absorbance units. The standardised AviPro PLATE format includes a positive control (growth control). Due to absence of a negative control, the reading procedure does not require any readjustment of zero level for absorbance measurements ( blanking procedure ), which is e.g. common practice for analysis of absorbance units using enzymelinked immonosorbent assay methods. Taking into account that the absorbance spectrum of photometers may vary between manufacturers, which can t be compensated by default threshold settings of the MICRONAUT-Software, the complementary use of the MICRONAUT-SKAN Photometer for analysis of the AviPro PLATE format is imperative! In summary, the use of MICRONAUT-System consisting of two components, namely, the MICRONAUT- Software and MICRONAUT-SKAN Photometer provides an automated and standardised analysis tool based on default threshold settings to assure validity of data obtained from AviPro PLATE. 9 October 05 von