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Microbiological Assay

MD0101 / MD0102 / MD0103

10 tests / 20 tests / 50 tests

Non-fermenting bacteria AST

The Non-fermenting bacteria AST (antimicrobial susceptibility testing) is intended for in vitro determination of
antimicrobial susceptibility of Non-fermenting bacteria.

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OBELIS S.A.
Bd. Général Wahis, 53
1030 Brussels
Belgium

AUTOBIO DIAGNOSTICS CO., LTD.

No.87 Jingbei Yi Road
National Eco & Tech Development Area
Zhengzhou
China
450016
For any technical assistance please contact us in English at:Email: [email protected]
Contact your local dealers for all product-related questions in your local language

April 20, 2020/ Autobio Diagnostics

Introduction Components

The primary objective of susceptibility testing in the clinical setting is to 1. Reagent pack
predict the outcome of treatment of an infected individual with the
chosen antimicrobial agents. Results must be derived in a timely manner
10 20 50
with a high degree of accuracy and reproducibility. Antimicrobial agent
susceptibility testing (AST) is usually carried out to determine which AST Plate 10 plates 20 plates 50 plates
antimicrobial agent will be most successful in treating a bacterial infection
in vivo. Clinical laboratories can choose to determine the susceptibility of Broth 16.0 mL*10 16.0 mL*20 16.0 mL*50
bacteria by a number of methods mainly including disc diffusion, dilution
(broth microdilution method, broth macrodilution method and agar Indicator
3.0 mL*1 3.0 mL*1 3.0 mL*1
dilution), serial dilution (E-test) minimum inhibitory concentration (MIC) Solution
determination 1,2, of which can be performed manually or with Note: The volume of the Broth and Indicator Solution indicated in above
automation. table is the minimum dispensing volume.
 AST Plate
The MIC of an organism is defined as the minimum amount of The AST Plate is coated with varying concentrations of antimicrobial
antimicrobial agent required to inhibit the growth of the test organism agents. The distribution of antimicrobial agents on AST Plate is shown on
over a specified time interval (which is related to the growth rate of the Table 1.
bacteria). The rapid and accurate determination of the MIC value for an  Broth
antimicrobial agent/organism combination can significantly improve The MH Broth is used to enrich for targeted organism for detection.
patient management and prognosis since it enables the prompt  Indicator Solution
administration of appropriate antimicrobial agent therapy. The Indicator Solution contains the colorimetric redox.

Measurement Principle 2. 1 sheet of result reading card

This test is based on broth microdilution method and redox method 3,4. Automated Microbiology System on which the kit can
The AST Plate is coated with varying concentrations of antimicrobial be use
agents at appropriate well locations. The Broth with the targeted
organism (density of 0.5 McFarland) is added with the colorimetric redox  AutoMic-i600
indicator (colorimetric oxidation-reduction) to make an inoculum
suspension. The suspension and the AST Plate are used for inoculation Materials Required but not Provided
and incubation. After incubation, the minimum inhibitory concentration
(MIC) is read according to the changes to the Indicator as well as bacterial 1. Micropipettor
turbidity. Organism determination is used in the interpretation of the MIC 2. Vibrator
values of each antimicrobial agent producing Susceptible, Intermediate or 3. Sterile tips or swabs
Resistant (SIR) result classifications. 4. Nephelometer
5. Turbidity tubes
6. Incubator
7. Sterilized saline water
8. Mineral oil
Table 1: Distribution of antimicrobial agents

ATM 1 ATM 2 ATM 3 ATM 4 ATM 5 TZP 1 TZP 2 TZP 3 TZP 4 TZP 5 TZP 6 TZP 7

CRO 1 CRO 2 CRO 3 CRO 4 CSL1 CSL2 CSL3 CSL4 CSL 5 CZA 1 CZA 2 CZA 3

CAZ 1 CAZ 2 CAZ 3 FEP 1 FEP 2 FEP 3 FEP 4 FEP 5 FEP 6 FEP 7 FEP 8 FEP 9

CAZ 4 CAZ 5 CAZ 6 MEM 1 MEM 2 MEM 3 MEM 4 MEM 5 MEM 6 MEM 7 MEM 8 MEM 9

SAM 1 SAM 2 SAM 3 IPM 1 IPM 2 IPM 3 IPM 4 IPM 5 IPM 6 IPM 7 IPM 8 CON

AMK 1 AMK 2 AMK 3 AMK 4 AMK 5 DOR 1 DOR 2 DOR 3 DOR 4 DOR 5 DOR 6 DOR 7

GEN 1 GEN 2 GEN 3 GEN 4 POL 1 POL 2 POL 3 POL 4 POL 5 POL 6 POL 7 POL 8

TOB 1 TOB 2 TOB 3 TOB 4 TOB 5 CHL 1 CHL 2 CHL 3 CIP 1 CIP 2 CIP 3 CIP 4

TGC 1 TGC 2 TGC 3 TGC 4 TGC 5 TGC 6 TGC 7 TGC 8 LVX 1 LVX 2 LVX 3 LVX 4

TCY 1 TCY 2 TCY 3 TCY 4 MNO 1 MNO 2 MNO 3 SXT 1 SXT 2 SXT 3 SXT 4 SXT 5

Abbreviation: ATM-Aztreonam, TZP-Piperacillin, CRO-Ceftriaxone, CSL-Cefoperazone/Sulbactam, CZA- Ceftazidime/avibatan, CAZ-Ceftazidime,

FEP-Cefepime, MEM-Meropenem, IPM-Imipenem, SAM-Ampicillin/Sulbactam, AMK-Amikacin, DOR-Doripenem, GEN- Gentamicin, POL-Polymyxin B,
TOB-Tobramycin, CHL-Chloramphenicol, CIP-Ciprofloxacin, TGC-Tigecycline, LVX-Levofloxacin, TCY-Tetracycline, MNO-Minocyline, SXT-Selectrin,
CON-Positive Control.
No.1-No.9: the number of serial concentrations of antimicrobial agents.

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Non-fermenting bacteria AST
Warnings and Precautions 3. Read results
For instrument
1. For professional use only. For in vitro diagnostic use only. The system could report MIC results and Interpretive Categorical Results
2. Follow the instruction for use carefully. Reliability of assay results (SIR). Some results may obtains only MIC without SIR when there is no
cannot be guaranteed if there are any deviations from the instructions breakpoint explanation.
in this package insert. For manually
3. Wear protective clothing and disposable gloves when dealing with  Well MEM, Well IPM, Well DOR: The MIC results could be read for
strips. Wash hands after operations. Handle the potentially the well with no turbidity appearance comparing to the Well CON
contaminated materials safely according to local requirement. (positive control), and its corresponding lowest value of the
4. Standard guidelines for the safe handling and disposal of infectious antimicrobial agent in the reading card are their MIC results.
organisms should be observed throughout all procedures.  Well SXT: MIC results could be read by comparing to the Well CON,
5. Do not smoke, drink, eat or use cosmetics in the working area. the first well that showed slight color change and significantly
6. Do not use if the plate or packaging appears to be damaged or reduced turbidity compared with the Well CON (more than 80%
turbidity precipitation appears in the broth. reduction compared with the positive control). Its corresponding
7. Do not mix or use components from kits with different batch codes. values of these antimicrobial agents in the reading card are their MIC
8. Micropipettor tips cannot be mixed to avoid contamination. results.
9. Avoid testing in harsh environments (such as those containing 84  MIC results of other wells could be read by comparing to the Well
disinfectants, sodium hypochlorite, acid, alkaline, or acetaldehyde CON, the colors stay the same as blue, and its corresponding lowest
and other high concentration corrosive gases and dust). value of these antimicrobial agent in the reading card are their MIC
10. The AST Plate cannot be reused. results.

Storage Interpretation of Results

1. Store all components at 2-8℃. When stored as directed, all reagents 1. For the same antimicrobial agent, if there is a blue well among a
are stable until the expiration date. serial of red wells or there is a red well among a serial of blue wells,
2. The AST Plate should be used within 8 hours once opened; the Broth then this well is a drift. The drift should be ignored when report
should be used immediately once open; store the Indicator Solution the MIC. But if drift is ≥2, the AST should be repeated.
sealed at 2-8℃ after opened. It cannot be used beyond the expiration 2. It might be caused by bacterial contamination if the whole plate
date. turns red, so the AST should be repeated.
3. It is invalid if there is no growth of bacterial or it remains blue of
Sample the Well CON, so the AST should be repeated.

1. Collect and prepare samples in accordance with the local Interpretive Category
requirement.
2. The test isolate must be a pure culture. After inoculum preparation, The Interpretive Categorical Results (SIR) should be interpreted by
the inoculation should be completed within 20 minutes and sampled referring to the latest MIC breakpoint interpretation standard published
within 2 hours. by CLSI4 or EUCAST5.

Measurement Procedure Limitations of the Procedure

1. Prepare the materials 1. A Gram stain test is required for the selection of the appropriate
 Allow the inoculated samples to balance at room temperature for at AST Plate types. Accurate AST results may not be made without
least 30 minutes. this test.
 Remove the kit from the refrigerated environment and return to 2. Use only well-isolated bacterial colonies from one of the
room temperature before opening the package. recommended primary isolation media. Use of mixed colonies
could result in inaccurate AST interpretations.
2. Order tests 3. A suspension equivalent of 0.5-0.6 McFarland standard must be
1) Add 1mL sterilized saline water into turbidity tube, pick several met a nephelometer. Use of alternate methods for suspension
pure culture isolates, mix it well in the turbidity tube, and prepare preparation may cause erroneous AST results.
0.5 McFarland bacterial suspension by using Nephelometer. 4. After the addition of the Indicator Solution to the broth tubes, mix
2) Add 100μL bacterial suspension into 1 vial Broth and mix well. by inversion. DO NOT VORTEX. Vortexing may cause air bubbles to
3) Add one drop of Indicator Solution into the Broth. form in the broth, which can result in inappropriate filling of the
For instrument AST Plate wells during inoculation.
4) Place this Broth and the AST plate in the instrument, 100μL 5. The results of this test indicate the Interpretive Categorical Results
inoculum is transferred into each well and the AST plate is (SIR) of in vitro microorganisms. The sensitivity of some
incubated automatically microorganisms to certain antimicrobial agents may be
Note: please refer to the AutoMic-i600’s operation manual. inconsistent in vitro and in vivo.
6. The test results of this product are used for clinical reference, and
For manually should not be used as only evidence for clinical diagnosis and
4) Transfer 100μL abovementioned Broth into each well and mix well. treatment. It should be combined the Information such as
5) Add one drop Mineral Oil into each well and cover the plate with symptoms, medical history, other laboratory tests, and treatment
lid. response.
6) Incubate the AST Plate at 35± 2℃ for 18-24 hours.

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Non-fermenting bacteria AST
Control Procedure 3. Between-batch precision: test the control statin Pseudomonas
aeruginosa (ATCC27853) with 3 batches of Non-fermenting bacteria
The recommended control requirement for this assay involves using Well AST(10 plates for each batch), the MICs results of at least 21 out of
CON (positive control）of the plate and reference strain to verify assay 22 antimicrobial agents should have a ≥85% consistency with the
performance. The result is valid if the following assigned specification for MIC requirements range. The results were met the requirements.
the controls is met:
Well CON: there is growth of bacterial or the color changes of the Well Literature References
CON.
Reference strain: conduct the operations described in Measurement 1. Miae Lee, Hae-Sun Chung. Different antimicrobial susceptibility testing
Procedure on recommended control Pseudomonas aeruginosa methods to detect ertapenem resistance in enterobacteriaceae: VITEK2,
(ATCC27853), MICs results of at least 21 out of 22 antimicrobial agents MicroScan, Etest, disk diffusion, and broth microdilution, J Microbiol
should fall into the acceptance range of MIC as in Table 2. Methods, 2015, 112(197):87-91.

Performance Characteristics 2. F.C. Tenover, Antibiotic Susceptibility Testing, Encyclopedia of

Microbiology (Third Edition), 2009: 67–77.
1. Pseudomonas aeruginosa (ATCC27853) is used as the control
strain, MICs results of at least 21 out of 22 antimicrobial agents 3. Clinical and Laboratory Standards Institute. 2018. Methods for Dilution
should meet the following criteria (shown in table 2 below). Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically:
approved standard. Document M07-A11, Clinical and Laboratory
Table 2: the MIC requirements range for 22 antibacterial drugs. Standards Institute.

Pseudomonas aeruginosa 4. Clinical and Laboratory Standards Institute. 2019. Performance

Antibacterial agents standards for antimicrobial susceptibility testing: approved standard.
(ATCC27853) (µg/mL)
Document M100-S29, Clinical and Laboratory Standards Institute.
Ceftriaxone ≤64
Cefrazidime ≤4 5. European Committee on Antimicrobial Susceptibility Testing. 2019.
Breakpoint tables for interpretation of MICs and zone diameters, Version
Cefepime ≤4
9.0, European Committee on Antimicrobial Susceptibility Testing.
Cefoperazone/sulbactam ≤16/8

Meropenem ≤1
Imipenem 1-4

Aztreonam ≤8
Ampicillin/Sulbactam >32/16

Piperacillin/Tazobactam ≤8/4

Ciprofloxacin ≤1

Levofloxacin ≤4
Gentamicin ≤2
Amikacin ≤4

Tobramycin ≤1

Chloramphenicol >32

Polymyxin B ≤2
Selectrin 8/152-32/608
Tetracycline 8-32

Minocyline ≥16

Tigecycline 4-16
Doripenem ≤0.5

Ceftazidime/Avibatan ≤4/4

Determine MIC for 3 batches of Non-fermenting bacteria AST, the results

were met the requirements.

2. Repeatability: test the same control strain Pseudomonas aeruginosa

(ATCC27853) with 10 plates from 1 batch of Non-fermenting
bacteria AST, the MICs results of at least 21 out of 22 antimicrobial
agents should have a ≥90% consistency with the MIC requirements
range. The results were met the requirements.

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Non-fermenting bacteria AST