| The developed high-throughput detection equipment for antimicrobial susceptibility testing includes high-throughput susceptibility testing inoculation instrument,96 dot matrix agar detection box,portable bacterial incubator,and susceptibility test image acquisition conversion instrument,etc.,realizing the high throughput of antimicrobial susceptibility testing and performance of accuracy,stability and standardization meet with need.According to the microdilution method standards of antimicrobial susceptibility testing provided by Clinical and Laboratory Standards Institute(CLSI),the structure and operation stability of the inoculator,the repeatability of the amount of inoculated bacteria and the comparison of the CLSI standard method for antimicrobial susceptibility testing were studied.By using the inoculator method and broth microdilution method of the CLSI antimicrobial susceptibility testing standard,minimum inhibitory concentration value of eight antibiotics against 48 clinical isolates of Escherichia coli from chicken,were tested.The results show that the inoculator is convenient for sterilization,easy to operate.It has reasonable structure,stable components,good repeatability of the amount of bacteria inoculated on the agar plate,and the number of bacteria each inoculation needle inoculated on plate is in line with CLSI standard.The results of the inoculation method is consistent with the broth micro dilution method at 95%,and the batch test results of two methods coincides with more than 90%,and the inoculator method takes less 5 time than the broth microdilution method.Aiming at the current situation of domestic farms that do not have the conditions for bacterial culture due to clinical laboratory tests,this research has developed a portable bacterial incubator with length,width and height of 40-50 cm,20-30 cm,and20-30 cm,respectively.The heating power is 30 to 40 W.It is mainly composed of a foam box with an upper cover that can be sealed,heating tape,wire,temperature control switch and other components.By temperature stability,temperature difference,comparison and verification with conventional constant temperature incubator culture performance,field culture application,etc.,these results showed that the portable bacterial incubator is small in size,light in weight,easy to carry,and the temperature in the box is relatively constant with temperature ranging 30°C to 42°C,which meets the requirements of conventional bacteria culture,and reduces the cost by 90%compared with conventional culture equipment.It is suitable for bacteria cultivated at the grassroots site and in the laboratory.The image acquisition and conversion instrument for antimicrobial susceptibility testing developed in this research includes a housing,a display screen,an image acquisition room,a control board,and an image acquisition and conversion device.The display screen is installed on the housing,and the control board,the image acquisition and conversion device,and the image acquisition room are set inside the shell.The drug sensitivity detection box is placed in the image acquisition room,and the image acquisition conversion device corresponds to the image acquisition room,and the control board is provided with a controller,and the data output port of the image acquisition conversion device is connected to the data reception port of the controller,The display screen is connected to the controller.The image acquisition and conversion device includes a scanner and a light source.The scanner and the light source are respectively placed under the drug sensitivity detection box.Stability verification show that plate tray and the operating system of the image acquisition and conversion instrument for antimicrobial susceptibility testing work well.The readings of each drug plate are consistent,showing that its repeatibilities is good.Verification by comparison with visual inspection results show that the two methods have the consistency while the inspection efficiency is more than 5 times that of visual inspection.The results of the detection sensitivity study show that the image acquisition instrument can identify colonies larger than 0.5mm in diameter on the agar plate.The software is designed with a memory function.For small-diameter colonies that cannot be identified for the first time,they can be identified when they are scanned again after the software identification marks.In order to facilitate the operation,a manual for the 96-dot drug susceptibility testing computer acquisition system v1.0 has been written,The above results show that the image acquisition and conversion instrument for antimicrobial susceptibility testing has reasonable design,stable operation,good reproducibility of reading results,high sensitivity and high detection efficiency,and the detection efficiency is more than 10 times higher than that of conventional visual inspection and manual data entry,so it is suitable for antimicrobial susceptibility testing for batch bacterial.In order to verify the optimized and integrated high-throughput detection technology for antimicrobial susceptibility testing,the integrated technology was used to conduct antimicrobial susceptibility testing on the isolated and identified clinically isolated bacteria,and on this basis,ith high level drug resistance gene was tested.The total number of strains isolated and identified in this study was 10617,mainly including Escherichia coli,Salmonella,Staphylococcus aureus,Klebsiella,Proteus,Enterococcus,etc.The main bacteria were tested for drug resistance,and some bacteria with high level drug resistance were screened,their resistance genes were tested,and the MIC frequency distribution of more than 10 commonly used drugs on bacteria was counted.The results showed that Escherichia coli has a high resistance rate to florfenicol,enrofloxacin,ampicillin,compound trimethoprim polymyxin,cefotaxime,ceftiofur,doxycycline and ciprofloxacin,the drug resistance rate above40%.And it is relatively sensitive to gentamicin,amoxicillin-clavulanic acid,levofloxacin and amikacin.The whole resistance level of Salmonella is slightly lower than that of Escherichia coli.Staphylococcus aureus is more than 70% resistant to compound trimethoprim,erythromycin,ampicillin and amoxicillin-clavulanic acid,and more sensitive to other drugs,less than 20%.The resistance of Escherichia coli from healthy animals to doxycycline,florfenicol,gentamicin,ceftiofur,and polymyxin is significantly lower than that of disease-derived Escherichia coli.To a certain extent,Escherichia coli from healthy animal feces has the role of a background indicator of the resistance of breeding livestock species.Both Escherichia coli and pathogenic Escherichia coli with multiple high-resistance phenotypes contain multiple resistance genes as well as multiple virulence genes.From 720 fresh fecal samples from 8 large-scale intensive chicken farms in Shandong Province,697non-repetitive Escherichia coli strains were isolated and identified.The results showed that 83 strains were detected to carry the mcr-1 gene.A total of 5 ESBL genes and 4 PMQR genes were detected from mcr-1 positive strains.The antimicrobial susceptibility testing only took 1 month,which is 5 months less than the conventional method,and the test cost is reduced by 80%.In summary,the high-throughput detection technology of antimicrobial susceptibility testing for bacterial is accurate,stable,repeatable,efficient,low-cost,and highly adaptable.It is a domestic initiative and has obtained national invention patents.It is suitable for various laboratory research,teaching and production,providing technical support for bacterial resistance testing and research. |
PDF Full Text Request