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 strains to be tested for AST on Mueller-Hinton agar (MHA) plates for 16-18 h at 37 &#176;C3.</li>
		<li>Prepare the inoculum of each strain to match 0.5 McFarland turbidity standard based on the method specified in the CLSI standard3and then dilute 10 times.</li>
		<li>Place 200 &#181;L of sterile normal saline into the horizontal 1st well (A1) of the 96-well plate as the negative control, two suspensions of quality control organism into the horizontal 2nd and 3rd wells (A2 and A3) as the positive control, and quality control, respectively. Add 200 &#181;L of the diluted inoculum suspensions of each tested stain into the corresponding 93 wells in the 96-well seed block.</li>
	</ol>
	</li>
	<li>Preparation of antimicrobial agar plate
	<ol>
		<li>Set the concentration ranges of different antibacterial agents tested according to the calculation range of the Lar index (from 0.125R to 8R). The concentrations range from the quality control range or 0.0625R (subject to the lower range) to 8R. 
		NOTE: If the Lar index is not calculated, the range of antibiotic concentrations can be set according to the needs of AST.</li>
		<li>Execute a log2 doubling dilution scheme for antibiotic solution beginning with a suitable stock concentration based on the agar dilution method specified in the CLSI standard3.</li>
		<li>Sterilize 50 mL glass bottles containing 18 mL of Mueller-Hinton agar media. Add 2 mL of the appropriate dilutions of the antimicrobial solution to 18 mL of molten media cooled to 45-50 &#176;C, mix thoroughly, and pour into the plates in the biosafety cabinet.</li>
		<li>Allow the agar to solidify at room temperature (RT), leave a gap under the lid of incubated plates and blow to dry the agar surface before inoculating.</li>
		<li>Label the types of antimicrobial agents and concentrations on the reverse side of the incubated plates. Arrange the multiple incubated plates of each antimicrobial agent in a stack in log2-doubling dilution order.</li>
		<li>Prepare two drug-free agar plates as the controls for each antimicrobial agent.</li>
	</ol>
	</li>
	<li>Inoculation steps for 96-dot matrix inoculator
	<ol>
		<li>Install the autoclaved inoculation pin plate on the support of a 96-dot matrix inoculator in the biosafety cabinet.</li>
		<li>Place the prepared seed block with tested strains and an agar incubated plate on the mobile carrier, with the same positioning angle for the two plates.</li>
		<li>Push the mobile carrier so the seed block is directly below the inoculation pin plate.</li>
		<li>Press the operating handle, move the inoculation pin plate down, and direct the 96 pins to the inocula in 96 wells of the seed block.</li>
		<li>Release the operating handle with control, then reset the inoculation pin plate under the action of the spring.</li>
		<li>Press the operating handle 2-3 times to stir each inoculum well and dip. Push and move the carrier plate so the incubated plate is directly below the inoculation pin plate.</li>
		<li>Press the operating handle, move the inoculation pin plate down, and stop for 1-2 s to make the inoculation pins contact the surface of the incubated plate fully.</li>
		<li>Release the operating handle. This completes one inoculation. Replace another incubated plate and continue the cycle until one group of antimicrobial agar plates is finished.</li>
		<li>Replace another inoculation pin plate and seed block, and inoculate another group of tested strains. Cycle until all inoculations are completed. 
		NOTE: Inoculate a control agar plate (no antimicrobial agent) first, then the plate in order of drug concentration from low to high, and a second control agar plate last to ensure no contamination or antimicrobial agent carry-over. The inoculating volume relies on the volume of the natural deposition of each pin of approximately 2 &#181;L.</li>
	</ol>
	</li>
	<li>Incubating the antimicrobial agar plates
	<ol>
		<li>Incubate the inoculated antimicrobial agar plates at RT until the moisture in the inoculum spots is absorbed into the agar.</li>
		<li>Invert the plates and incubate them for 16-20 h at 37 &#176;C for the tested strains to ensure that the uninhibited bacteria form colonies.</li>
	</ol>
	</li>
	<li>Image acquisition and data statistics
	<ol>
		<li>Double-click on 96-dot matrix AST image acquisition system to open the program.</li>
		<li>Click on Test Information in the taskbar. Click on New to create a new test task, and fill in the information according to the prompts, including the code, name, source, bacteria, number of strains, antibiotics, and gradient.</li>
		<li>Click on Data Collection &#62; Photograph &#62; Test item to select the new task created. Click on Antibiotics to select the name of the antibiotic, and click on Gradient to select the initial concentration of this antibiotic.</li>
		<li>Click on Connect to connect with the image acquisition converter.</li>
		<li>Place the corresponding incubated plates on the detection plate base with the missing angle at the right front for orientation and push into the image acquisition converter.</li>
		<li>Click on Collection to obtain the images. The antibiotic gradient will automatically jump to the next gradient. Place the next plate in turn and continue to click on Collection until the plates for this antibiotic have been collected.</li>
		<li>Click on Antibiotics, and select the next set of incubated plates. Click on Gradient to select the starting gradient and proceed to the next round of image collection.</li>
		<li>After completing all collections, click on Submit. The program will automatically recognize the number of white pixels formatted at each inoculation point in the images, determine if there is colony formation and convert the images into MIC values.</li>
		<li>Click on Query to obtain all MIC results of the strains against the tested antibiotics. 
		NOTE: The intelligent high throughput AST system is suitable for determining MICs of large batches of bacterial strains. The testing process, including preparation, inoculation, incubation, and result reading, takes 3 days. The types of antibiotics and MIC detection ranges can be set according to respective needs, and the main consumables can be reused.</li>
	</ol>
	</li>
	<li>Calculation of the Lar index
	<ol>
		<li>Determine the Lar index accurately with the formula: <img alt="Equation 1" src="/files/ftp_upload/64785/64785eq01.jpg" style="margin: auto;" />, where: 
		MICi: minimum inhibitory concentration. 
		The range of MIC distributions from MIC-3 to MIC3 represents serial twofold concentrations centered on R: 0.125R, 0.25R, 0.5R, R, 2R, 4R, and 8R. 
		<img alt="Equation 2" src="/files/ftp_upload/64785/64785eq02.jpg" style="margin: auto;" /> is 2i, and the range of i is -3 to 3. 
		R: the breakpoints of resistance of bacteria against antimicrobial agents standardized by CLSI. 
		f: the MIC frequency distribution. 
		NOTE: The general Lar index is the arithmetic mean of all Lar indices. After the Lar index is calculated, round off the final value to two significant digits after the decimal point.</li>
	</ol>
	</li>
</ol>
2. Intelligent high-throughput phage screening system9
<ol>
	<li>Preparing the phage seed block and double-layer incubated plates containing bacteria.
	<ol>
		<li>Use the double-layer agar method10 or liquid culture method11 for making different phages. Dilute to a suitable parallel concentration with a titer of 1 x 104-5 pfu/mL, and add 200 &#181;L of the phage inoculum into the 96-well seed block.</li>
		<li>Make double-layered plates with bacteria (10 mL of bottom agar media [agar 12 g/L] and 6 mL of upper semi-agar media [6 g/L] with 100 &#181;L of bacteria [0.5 McFarland]) to be tested.</li>
		<li>Make a double-layer incubated plate for each strain to be tested. Leave a gap under the lid of the double-layered plate and blow to dry the agar surface in the biosafety cabinet.</li>
	</ol>
	</li>
	<li>Screening test
	<ol>
		<li>Place the prepared phage seed block and double-layer plate on the mobile carrier of the 96-dot matrix inoculator, and transfer all phage inocula to the semi-agar surface. Continue the cycles until all tested strains are completed.</li>
		<li>Let the inoculated double-layer plates remain at RT until the moisture in the inoculum spots is absorbed fully into the semi-agar.</li>
		<li>Invert the plates and incubate under suitable conditions for the tested strains for 4-6 h to ensure that clear lytic spots are formed.</li>
	</ol>
	</li>
	<li>Analyzing data
	<ol>
		<li>Obtain and save the image of the experimental result of each double-layer plate by the image acquisition converter (steps 1.5.4-1.5.6).</li>
		<li>Record the number and morphologies of the different shapes of spots into a spreadsheet based on the obtained images, and calculate the respective proportions of the different kinds of phages.</li>
	</ol>
	</li>
</ol>

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CLSI document M100. , (2022).</li><li>Yuqing, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antimicrobial Sensitivity Testing Standard of EUCAST. , (2017).</li><li>Barnini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rapid+method+for+direct+antimicrobial+susceptibility+testing+of+bacteria+from+positive+blood+cultures.">A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures.</a> BMC Microbiology. 16 (1), 185-192 (2016).</li><li>H&#246;ring, S., Massarani, A. S., L&#246;ffler, B., R&#246;del, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antimicrobial+susceptibility+testing+in+blood+culture+diagnostics+performed+by+direct+inoculation+using+the+VITEK&#174;-2+and+BD+PhoenixTM+platforms.">Rapid antimicrobial susceptibility testing in blood culture diagnostics performed by direct inoculation using the VITEK&#174;-2 and BD PhoenixTM platforms.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 38 (3), 471-478 (2019).</li><li>Dupuis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Cobasbact+automated+antimicrobial+susceptibility+testing+system.">Evaluation of the Cobasbact automated antimicrobial susceptibility testing system.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 4 (2), 119-122 (1985).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+of+bacterial+antimicrobial+resistance+detection+and+its+operation+method.">A system of bacterial antimicrobial resistance detection and its operation method.</a> China Patent. , (2019).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+test+plate+for+screening+bacteriophage+of+zoonotic+pathogens+and+its+application.">A high throughput test plate for screening bacteriophage of zoonotic pathogens and its application.</a> China Patent. , (2022).</li><li>Adams, M. 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Yuqing Liu et al. have filed Chinese patents for the 96-dot matrix inoculator and image acquisition converter and their applications (Patent number ZL 201610942866.3 and Patent number ZL 201910968255.X).

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><tbody><tr><td>96 well&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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 &lt;em&gt;Salmonella &lt;/em&gt;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 &amp;mu;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>&lt;em&gt;Salmonella &lt;/em&gt;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&#039;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...

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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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1.2K Views.  Key Laboratory of Livestock and Poultry Multi-omics of MARA. Dit protocol presenteert een methode op meerdere niveaus, zowel voor EST-detectie als voor de faagfiguur. Het introduceerde ook een grote index voor reddingen om EMR-niveaus van verschillende besturingstechnologie te vergelijken. Deze kosteneffectieve techniek is intelligent, high-throughput en multifunctioneel.Het heeft de oprichting van VAMS mogelijk gemaakt, een veterinair monitoringnetwerk dat 20.000 epidemische genen en analytische fagen heeft verzameld. Deze methode kan ook worde...

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

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Immunology and Infection

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioengineering

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Medicine

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

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

Samenvatting

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