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Fresh milk was collected from a cow with mastitis by a technician of a dairy cattle breeding company in Shaanxi Province, China. The sample was not sterilized.
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Cation-adjusted Mueller-Hinton broth (CAMHB) was purchased from Difco (Detroit, MI, USA). Alamar blue was purchased from AbD Serotec (Oxford, UK). Twenty-seven types of antibiotics were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Gatifloxacin and rifabutin were procured from Toronto Research Chemicals Inc. (Toronto, Canada). The details of the 27 antibiotics are shown in Table 1.
No Antibiotics Concentration range (μg/mL) 95080(μg/mL) ShaanX15001 (μg/mL) RGM Dividing Value (μg/mL) S I R 1 Ofloxacin 64-0.031 0.062-0.125 0.125-0.25 - - 2 2 Isoniazid 256-0.125 2-4 4-8 5 3 Rifampicin 256-0.125 0.125-0.25 < 0.125 - - 1 4 Amikacin 16-0.008 0.125-0.25 0.125-0.25 16 32 64 5 Capreomycin 16-0.008 0.25-0.5 0.25-0.5 - - 2.5 6 Moxifloxacin 16-0.008 0.008-0.015 0.015-0.031 1 2 4 7 Kanamycin 64-0.031 0.25-0.5 0.125-0.25 - - 4 8 Gatifloxacin 16-0.008 0.015-0.031 0.031-0.062 - - - 9 Levofloxacin 16-0.008 0.031-0.062 0.062-0.125 2 4 8 10 Cycloserine 128-0.063 16-32 16-32 - - 50 11 P-aminosalicylic Acid 16-0.008 > 16 > 16 - - 2 12 Ethambutol 128-0.063 < 0.062 < 0.062 5 - 10 13 Streptomycin 128-0.063 0.125-0.25 0.062-0.125 5 - 10 14 Tobramycin 64-0.031 0.125-0.25 0.125-0.25 2 4 8 15 Rifapentine 32-0.016 4-8 4-8 - - - 16 Rifabutin 32-0.016 0.5-1 0.5-1 - - 2 17 Ciprofloxacin 32-0.016 0.031-0.062 0.062-0.125 1 2 4 18 Linezolid 64-0.031 0.25-0.5 0.125-0.25 8 16 32 19 Sparfloxacin 16-0.008 0.008-0.015 0.031-0.062 - - - 20 Pasiniazid 64-0.031 0.5-1 0.25-0.5 - - - 21 Clofazimine 16-0.008 0.125-0.25 0.125-0.25 - - - 22 Cefoxitin 64-0.031 16-32 8-16 16 32-64 128 23 Meropenem 64-0.031 32-64 8-16 4 8-16 32 24 Clarithromycin 32-0.016 0.25-0.5 0.125-0.25 2 4 8 25 Minocycline 32-0.016 0.015-0.031 < 0.015 1 2-4 16 26 Trimesulf 256-0.125 128-256 > 256 38 - 76 27 Sulfamethoxazole 256-0.125 > 256 > 256 - - - Note. '95080' is the reference M. elephantis strain, 'ShaanX15001' is the isolate, '-' represents a missing value. The reference values for the drug susceptibility test were reported in references[15, 17-19]. RGM, rapid growing mycobacteria. S, sensitive. I, intermediated. R, resistant. Table 1. Drug Sensitivity Test Results of the Isolate from Milk Samples
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The reference strain NTM95080 (originally purchased from National Institutes for Food and Drug Control) and H37Rv were provided by and stored at the Tuberculosis Laboratory, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.
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A 30-mL milk sample was placed in a 50-mL centrifuge tube and centrifuged at 12, 000 rpm for 15 min to obtain a precipitate. The precipitate was preprocessed with an equal volume of 4% NaOH (1:1) for 15 min to eliminate contaminants. Then, 45 mL of PBS (0.01 mol/L pH 7.2) was added to the sample to dilute NaOH. The sample was centrifuged again at 12, 000 rpm for 15 min to obtain a precipitate. The precipitate was resuspended in 1.0 mL PBS. To culture mycobacteria, L-J medium was inoculated with 0.5 mL of the suspension.
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After three generations of culture on L-J culture, individual colonies were selected and placed in 1.5-mL screw-capped centrifuge tubes with 0.5 mL water. Bacteria were inactivated by incubating the tubes in a water bath at 80 ℃ for 30 min. Bacterial cells were collected by centrifuging the incubated tubes at 12, 000 rpm for 5 min. The precipitate was resuspended in 300 μL water. To release genomic DNA from the bacterial suspension, the tubes were placed in a metal bath at 100 ℃ for 20 min, then centrifuged at 12, 000 rpm for 5 min. The supernatant was then collected for PCR amplification. Multiple loci PCR with seven target genes, including 16S rRNA, Rv0577, IS1561, Rv1510, Rv1970, Rv3877, and Rv3120, was used to identify the strain as Mycobacterium tuberculosis complex or NTM. Refer to reference[11] for the identification criteria and the PCR primers used to amplify the target gene loci from the chromosomal DNA of the isolate.
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Multi-locus sequence analysis (MLSA) was used to identify the Mycobacterium species of the isolate based on the sequences of the 16S rRNA, SodA, hsp65, and ITS genes. hsp65 was amplified using the hsp65F and hsp65R primers reported by Huard Richard C, et al.[11]. SodA was amplified using the SodlgF and SodlgF primers reported by Adékambi Toïdi, et al.[12]. 16s-23s ITS was amplified using the 16sF and 23sR primers reported by Roth A, et al.[13]. A total of 2 μL of DNA, 1 μL of primers, and 8.5 μL distilled water were added to 12.5 μL 2 × Taq PCR MasterMix to a final volume of 25 μL. PCR products were sent to Tsingke Company (Beijing, China) for two-way sequencing and sequence splicing to obtain the complete sequence of the PCR products. The sequencing results of 16SrRNA/SodA/hsp65/ITS were submitted to the National Center for Biotechnology Information (NCBI) website for homology comparison. Those that displayed a similarity of over 98% were accepted as species[14]. MEGA 6.01 software was used for cluster analysis. Alignment and evolutionary tree construction were conducted using MUSCLE with Maximum Likelihood method.
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We performed the alamar blue assay to test the sensitivity of the isolate ShaanX15001 and the reference strain NTM95080 to 27 antibacterial drugs. Minimum inhibitory concentration (MIC) testing was performed in accordance with the guidelines established and approved by Clinical and Laboratory Standards Institute[15]. We combined the existing MIC thresholds of the tested drugs to estimate the susceptibility of the isolate to different antibacterial drugs.
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Colonies that were growing well in L-J medium were scraped and placed in normal saline, suspended to 0.5 McIntosh concentration (approximately 0.5 mg/mL), and diluted with liquid medium at a ratio of 1:200 to a final concentration of approximately 105 CFU/mL of inoculant suspension.
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A total of 100 μL liquid CAMHB was added to each well of a 96-well plate except to border wells. Two-fold diluted drugs were added to the plate to establish the corresponding drug test range, as shown in Table 1. Finally, 100 μL of bacterial suspension was added to each well. Three negative controls were set. The drug-free control well (CAMHB + inoculum) was used as a reference for the addition of alamar blue. The well containing CAMHB without inoculum was used as reference to decide the interference of CAMHB with alamar blue. A series of control wells containing different concentration gradients of each drug and drug-CAMHB mixture were used as a reference for the interference of CAMHB mixture with alamar blue. The plates were sealed in individual Ziploc bags and then incubated at 37 ℃.
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After in-cubation at 37 ℃ for 24 h, the first drug-free control wells were examined using an indicator (20 μL alamar blue and 50 μL sterile 5% Tween-80). The plates were then re-incubated for 24 h. If the control well turned pink, all of the other wells containing drugs received the indicator. After an additional 24 h of incubation, the colors of all wells were recorded. If the color of the first drug-free growth control well did not change to pink, the second drug-free control well received the indicator and the above steps were repeated. MIC was based on the range between the non-discolored hole that corresponded to the lowest drug concentration and discolored hole that corresponded to the largest drug concentration[16]. The MIC value reflected the bacteriostatic activity of the drug. A low value indicated high bacteriostatic activity. The final MIC of each drug was calculated as the mean of two or three tests. The MIC thresholds indicating sensitivity, moderate susceptibility, and resistance were interpreted in references[15, 17-19].
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Experimental animals were scientifically and rationally treated in accordance with the '3R' principle: 'Reduction, Replacement, and Refinement.' This study was performed in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The company that provided the milk for this study also provided written informed consent.