Name: application of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system and lar index of antimicrobial resistance
Uploaded: 2023-07-21T14:45:00
Description: Watch this Scientific Journal Video about Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance at JoVE.com

This work was supported by the National Key Research and Development Project (2019YFA0904003); Modern Agricultural Industrial System in Shandong Province (SDAIT-011-09); International Cooperation Platform Optimization Project (CXGC2023G15); Major Innovation tasks of agricultural Science and technology innovation project of Academy of Agricultural Sciences Shandong, China (CXGC2023G03).

The Salmonella strains used in this study were collected from poultry in Shandong, China, after obtaining approval from the Biosafety Committee of the Institute of Animal Sciences and Veterinary Medicine, Shandong Academy of Agricultural Sciences, China.
1. Application of the intelligent high-throughput AST system8
<ol>
	<li>Inoculum preparation
	<ol>
		<li>Incubate the quality control organism Escherichia coli and 93 Salmonella<...

<ol>
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The agar dilution method has been well-established and used widely. The principle of the high-throughput AST system was that of the agar dilution method. One of the critical steps within the protocol was the accurate high throughput transfer of 96 inocula at one time, which was performed multiple times in a row. To complete this critical step, the pins of the 96-dot matrix inoculator were uniform and very smooth. The natural deposition of each pin was a volume of approximately 2 &#181;L, which aggregated into small dropl...

As antimicrobial agents have been widely used to prevent bacterial infectious diseases, antimicrobial resistance (AMR) has become a global public health problem1. Combating AMR is the current main mission of monitoring AMR of epidemiological pathogens and synergistic therapy of sensitive antimicrobial agents and lytic bacteriophages2.
In vitro antimicrobial sensitivity testing (AST) is the mainstay for monitoring therapy and detecting the level of AMR. It is an important part of antimicrobial pharmacology and the critical basis for clinical medication. The Clinical and Laboratory Standards Institute (CLSI) of the United States and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have formulated and revised international criteria of AST and continuously modified and supplemented AST methods and the breakpoints to determine the MIC of one certain &#34;organism-antimicrobial agent&#34; combination as sensitive (S), resistant (R) or intermediate (I)3,4.
From the 1980s to the 1990s, automatic micro broth dilution instruments were rapidly developed and applied to clinical practice, with examples including Alfred 60AST, VITEK System, PHOENIXTM, and Cobasbact5,6,7. However, these instruments were expensive, required high-cost consumables, and their detection ranges were designed for clinical patient medication5,6,7. For these reasons, they are not suitable for veterinary clinical examination and detection of large quantities of highly resistant strains. In this study, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator (Figure 1), image acquisition converter (Figure 2), and corresponding software8, was developed to conduct AST for a batch of bacteria strains against multiple antimicrobial agents at one time by the agar dilution method. Moreover, the system was also used to detect and analyze the lysis patterns of phages against antimicrobial-resistant bacteria9, and lytic phages were selected efficiently from the phage library. This system was found to be efficient, affordable, and easy to operate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64785/64785fig01.jpg" /> 
Figure 1: Structural diagram of the 96-dot matrix inoculator. 1: Inoculation pin plate; 2: Mobile carrier; 3: Seed block; 4: Incubated plate; 5: Base; 6: Operating handle; 7: Limit pin. <a href="https://www.jove.com/files/ftp_upload/64785/64785fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64785/64785fig02.jpg" /> 
Figure 2: Structural diagram of the image acquisition converter. 1: Shell; 2: Display screen; 3: Image acquisition room; 4: Detection board base; 5: Detection board in and out of warehouse; 6: Control board; 7: Image acquisition conversion device; 8: Light source; 9: Image scanner. <a href="https://www.jove.com/files/ftp_upload/64785/64785fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well&#160; culture plate</td>
			<td>Beijing lanjieke Technology Co., Ltd</td>
			<td>11510</td>
			<td></td>
		</tr>
		<tr>
			<td>96-dot matrix AST image acquisition system</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>96-dot matrix inoculator&#160;</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>N/A</td>
			<td>Patented product</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Qingdao hi tech Industrial Park Haibo Biotechnology Co., Ltd</td>
			<td>HB8274-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Amikacin&#160;</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>A857053</td>
			<td></td>
		</tr>
		<tr>
			<td>Amoxicillin</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>A822839</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>A830931</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>BSA224S</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated calculation software for Lar index of AMR</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria Salmonella strains</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>N/A</td>
			<td>Animal origin</td>
		</tr>
		<tr>
			<td>Bacterial resistance Lar index certification management system V1.0</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftiofur</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>C873619</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>C824343</td>
			<td></td>
		</tr>
		<tr>
			<td>Clavulanic acid</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>C824181</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean worktable</td>
			<td>Suzhou purification equipment Co., Ltd</td>
			<td>SW-CJ-2D</td>
			<td></td>
		</tr>
		<tr>
			<td>Colistin sulfate</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>C805491</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plate</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>N/A</td>
			<td>Patented product</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>D832390</td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>E809130</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Florfenicol</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>F809685</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>G810322</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle 50 mL</td>
			<td>Xuzhou Qianxing Glass Technology Co., Ltd</td>
			<td>QX-7</td>
			<td></td>
		</tr>
		<tr>
			<td>High-throughput resistance detection system V1.0</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition converter</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>N/A</td>
			<td>Patented product</td>
		</tr>
		<tr>
			<td>Meropenem</td>
			<td>Shanghai McLean Biochemical Technology Co., Ltd</td>
			<td>M861173</td>
			<td></td>
		</tr>
		<tr>
			<td>Mueller-Hinton agar</td>
			<td>Qingdao hi tech Industrial Park Haibo Biotechnology Co., Ltd</td>
			<td>HB6232</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 mm x 15 mm</td>
			<td>Qingdao Jindian biochemical equipment Co., Ltd</td>
			<td>16021-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 90 mm x 15 mm</td>
			<td>Qingdao Jindian biochemical equipment Co., Ltd</td>
			<td>16001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella phages</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker incubator</td>
			<td>Shanghai Minquan Instrument Co., Ltd</td>
			<td>MQD-S2R</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbidimeter</td>
			<td>Shanghai XingBai Biotechnology Co., Ltd</td>
			<td>F-TC2015</td>
			<td></td>
		</tr>
		<tr>
			<td>Varms base type library system V1.0</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical high-pressure steam sterilizer</td>
			<td>Shanghai Shen&#39;an medical instrument factory</td>
			<td>LDZX-75L</td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary pathogen resistance testing management system</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary resistance cloud monitoring and phage control platform V1.0</td>
			<td>Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences</td>
			<td>In-house software copyright</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system and lar index of antimicrobial resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical &amp; Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

Following the protocol of the intelligent high-throughput AST system, its application was illustrated by Salmonella from poultry in Shandong, China, as an example.
The growth of Salmonella strains on agar plates with ampicillin (R of 32 &#181;g/mL) at concentrations from 2 to 256 &#181;g/mL determined by the image acquisition converter is shown in Figure 3. The horizontal 1st well A1 was the negative control and showed no colony growth...

Watch this Scientific Journal Video about Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance at JoVE.com

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

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