A Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain and its preparation and application

Abstract

The invention discloses a Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain and its preparation and application. The Rv3444c-Rv3445c double gene deletion strain simultaneously lacks Rv3444c and Rv3445c genes relative to wild-type Mycobacterium tuberculosis. Compared with wild-type Mycobacterium tuberculosis, the Rv3444c-Rv3445c double gene deletion strain shows obvious virulence reduction and the ability to change host epigenetic inheritance at the cell and mouse levels, and the shown traits are related to the deletion of Rv3444c and Rv3445c genes. The successful construction of the Rv3444c-Rv3445c double gene deletion strain of the present invention and the research on the related functions of the genes Rv3444c and Rv3445c involved in it have important reference value for clarifying the relevant pathogenic mechanisms of members of the genus Mycobacterium and developing safer and more effective vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of zoonotic infectious diseases, and specifically relates to a Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain and its preparation method and application; the Rv3444c-Rv3445c double gene deletion strain simultaneously lacks the Rv3444c and Rv3445c genes. Compared with the wild-type strain, it exhibits low pathogenicity in cell and mouse models, and also exhibits the characteristics of entering the host cell nucleus and inducing host epigenetic reprogramming. Background Art

[0002] Tuberculosis is a zoonotic disease caused by the Mycobacterium tuberculosis complex, which can be transmitted between a variety of animals, including cattle and sheep, and humans. It is estimated that approximately one-quarter of the global population is infected with tuberculosis. According to the World Health Organization, in 2021, approximately 10.6 million people worldwide were infected with tuberculosis, and 1.6 million people died from the disease (including 187,000 people living with HIV).

[0003] Tuberculosis is a respiratory disease transmitted by humans or animals. When aerosols containing Mycobacterium tuberculosis are inhaled by humans or animals, the bacteria are recognized and ingested by alveolar macrophages. Macrophages kill and eliminate infected Mycobacterium tuberculosis through innate immune pathways, such as cytokine secretion, reactive oxygen species production, phagolysosome engulfment, and autophagy. However, the bacteria have also evolved a series of strategies to evade host cell killing, such as inhibiting phagolysosomal maturation, inhibiting autophagy, and secreting effector proteins that interfere with host cell epigenetic regulation.

[0004] Effector proteins are crucial for pathogens to resist host immune clearance and are associated with pathogen virulence. Studies have shown that effector proteins secreted by Mycobacterium tuberculosis function within the cell nucleus, modulating host epigenetic regulation and altering immune responses, thereby promoting their own survival (Bhat et al., 2017, Duan et al., 2016, Jose et al., 2016). The ESX secretion system is a key secretion system of Mycobacterium tuberculosis. Many proteins in this system are associated with bacterial virulence, but the functions of many proteins in this family are not fully understood. Rv3444c and Rv3445c are members of the ESX protein family of Mycobacterium tuberculosis. Studies have shown that Rv3444c forms dimers with EsxT and EsxU, encoded by Rv3445c, to promote the upregulation of tumor necrosis factor and interleukin-6 (Pandey et al., 2018). However, whether EsxT and EsxU, encoded by genes Rv3444c and Rv3445c, enter the host cell nucleus and directly regulate host epigenetic regulation has not been reported. Summary of the Invention

[0005] The present invention aims to discover Mycobacterium tuberculosis with lower virulence and provide a reference for the development of safer and more effective attenuated vaccines for the prevention and treatment of tuberculosis.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain, which is simultaneously deleted in the Rv3444c and Rv3445c genes relative to the wild-type Mycobacterium tuberculosis (ie, the parent strain).

[0008] Compared with the parent strain, the Rv3444c-Rv3445c double gene deletion strain provided by the present invention exhibits significantly reduced virulence and the ability to change host epigenetics at the cellular and mouse levels, and the shown traits are related to the deletion of the Rv3444c and Rv3445c genes.

[0009] In a specific embodiment of the present invention, the parent strain is Mycobacterium tuberculosis H37Rv, and after the Rv3444c and Rv3445c genes are deleted at the same time, the nucleotide sequence of the resulting Rv3444c-Rv3445c double gene deletion strain is shown in the sequence table SEQ ID NO.1; further, the Rv3444c-Rv3445c double gene deletion strain is specifically Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) Ra3444c-5c-K0, deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2024121 and the deposit date January 15, 2024.

[0010] Specifically, the experimental data of the present invention show that the Rv3444c-Rv3445c double gene deletion strain has at least the following characteristics relative to the parent strain and the complemented strain:

[0011] a) has lower toxicity to cells and / or mammals; wherein cells include human monocytes and macrophages, and mammals include non-human mammals such as mice. The Rv3444c-Rv3445c double gene deletion strain of the present invention, its complemented strain, and the parental strain were used to infect human monocytes and macrophages and mice, respectively. The results showed that the survival of the Rv3444c-Rv3445c double gene deletion strain in human monocytes and macrophages, as well as in the lungs and spleens of mice, was significantly lower than that of the parental strain and the complemented strain.

[0012] b) Inducing epigenetic changes in host cells. The Rv3444c-Rv3445c double gene deletion strain of the present invention and its parent strain were infected with human monocytes and macrophages, and DLO-HiC library sequencing revealed that the Rv3444c-Rv3445c double gene deletion strain caused epigenetic changes.

[0013] The second aspect of the present invention provides a method for preparing an Rv3444c-Rv3445c double gene deletion strain, specifically: by packaging a temperature-sensitive phage carrying homology arms, infecting the parent strain of Mycobacterium tuberculosis, and using resistance screening to obtain the Rv3444c-Rv3445c double gene deletion strain.

[0014] In a specific embodiment of the present invention, the parent strain is Mycobacterium tuberculosis H37Rv, and hygromycin B is used as a resistance screening marker during resistance screening.

[0015] A third aspect of the present invention provides a method for reducing the virulence of Mycobacterium tuberculosis, specifically by simultaneously deleting the Mycobacterium tuberculosis genes Rv3444c and Rv3445c. Experimental data from the present invention demonstrate that EsxT and EsxU are secreted effector proteins that can enter the nuclei of human monocytes and macrophages and induce epigenetic changes in the host. Compared to the parental strain, the strain resulting from the Rv3444c-Rv3445c double gene deletion significantly reduces virulence.

[0016] A fourth aspect of the present invention provides the use of an Rv3444c-Rv3445c double-gene deletion strain in the preparation of a low-virulence Mycobacterium tuberculosis inoculum. Compared to the parent strain, the Rv3444c-Rv3445c double-gene deletion strain exhibited significantly reduced survival in human mononuclear macrophages, mouse lungs, and spleens, and significantly reduced pathogenicity in mice. This indicates that the Rv3444c-Rv3445c double-gene deletion strain of the present invention can be prepared into a low-virulence inoculum and potentially serve as a live attenuated vaccine for the prevention and treatment of tuberculosis.

[0017] The fifth aspect of the present invention provides a biological preparation, which uses the Rv3444c-Rv3445c double gene deletion strain of the present invention as one of the active ingredients.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention simultaneously deletes the Rv3444c and Rv3445c genes to obtain a Mycobacterium tuberculosis Rv3444c-Rv3445c double-gene deletion strain. This strain exhibits significantly reduced virulence against THP1 cell lines and mice compared to the parent strain. The present invention also confirms that both EsxT and EsxU are secreted effector proteins that can enter the host cell nucleus and exhibit a phenotype that alters the host's epigenetic genes. The successful construction of the Rv3444c-Rv3445c double-gene deletion strain and the study of the related functions of the genes involved, Rv3444c and Rv3445c, in the present invention are of great reference value for clarifying the pathogenic mechanisms of members of the genus Mycobacterium and developing safer and more effective vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following table shows the construction and verification results of the Rv3444c-Rv3445c double gene deletion strain of Mycobacterium tuberculosis of the present invention. A shows the agarose gel analysis of the PCR amplification products of the left and right homology arms; B shows the agarose gel analysis of the PCR amplification products of the recombinant plasmid p3444-3445; C shows the agarose gel analysis of the PCR amplification products of the recombinant phagemid 3444-3445; D shows the agarose gel analysis of the PCR amplification products of the Rv3444c-Rv3445c double gene deletion strain (K / O); E shows the sequencing alignment results of the Rv3444c-Rv3445c double gene deletion strain (K / O); and F shows the in vitro growth curves of the Rv3444c-Rv3445c double gene deletion strain (K / O), the parental strain (WT), and the complemented strain (Comp.). In the figure, L represents the left homology arm, R represents the right homology arm, WT represents the wild-type Mycobacterium tuberculosis genome, K / O represents the Rv3444c-Rv3445c double gene deletion strain genome, Comp. represents the complemented strain, and P represents the plasmid.

[0021] Figure 2 The results of the virulence test of the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain of the present invention are shown. A is the survival of the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain (K / O), the complemented strain (Comp.) and the parent strain (WT) in human mononuclear macrophage THP1 cells 48 hours after infection; B is the bacterial load of the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain (K / O), the complemented strain (Comp.) and the parent strain (WT) in the mouse lungs (Lung) on ​​the 28th day after infection; C is the bacterial load of the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain (K / O), the complemented strain (Comp.) and the parent strain (WT) in the mouse lungs (Lung) on ​​the 28th day after infection. Figure 28 shows the bacterial load in the spleens of mice infected with the Rv3444c-Rv3445c double-gene deletion strain (K / O), the complemented strain (Comp.), and the parental strain (WT). D shows damage to the lungs and spleens of mice infected with the Rv3444c-Rv3445c double-gene deletion strain (K / O), the complemented strain (Comp.), and the parental strain (WT) on day 28 after infection, as shown by HE staining (bar, 100 μm). In the figure, ns indicates no significant difference, * indicates P < 0.05, and **** indicates P < 0.00005.

[0022] Figure 3This is a verification of the fact that Rv3444c-Rv3445c in the present invention enters the host cell nucleus and induces epigenetic changes. Among them, A is the laser confocal microscopy observation result of Rv3444c and Rv3445c entering the nucleus of human mononuclear macrophage THP1 cells, with green for Rv3444c, red for Rv3445c, and blue for the nucleus; B is the statistical situation of the topological domains (TADs) changes in the interaction matrix of the human mononuclear macrophage THP1 genome caused by the Rv3444c-Rv3445c double gene deletion strain (K / O) of Mycobacterium tuberculosis; C is the topological changes of the same site in the interaction matrix of the human mononuclear macrophage THP1 genome caused by the Rv3444c-Rv3445c double gene deletion strain (K / O) of Mycobacterium tuberculosis. Splitting of the structural domain; D is the statistical situation of the changes in chromatin loops (Loops) of the THP1 genome in human monocytes and macrophages caused by the Rv3444c-Rv3445c double gene deletion strain (K / O) of Mycobacterium tuberculosis; E is the complete superposition of the changes in chromatin loops (Loops) of the THP1 genome in human monocytes and macrophages caused by the Rv3444c-Rv3445c double gene deletion strain (K / O) of Mycobacterium tuberculosis; F is the disappearance of loops (Loops) at the same chromatin site of the THP1 gene in human monocytes and macrophages caused by the Rv3444c-Rv3445c double gene deletion strain (K / O) of Mycobacterium tuberculosis. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art.

[0024] The wild-type Mycobacterium tuberculosis (i.e., the parent strain) used in the following examples was Mycobacterium tuberculosis H37Rv (deposit number ATCC 27294, the deposited strain was resuscitated and cultured before use); the following experiments were all conducted in the Animal Biosafety Level 3 Laboratory of Huazhong Agricultural University; the complemented strain (Comp.) used in the following examples was obtained by electroporating the pMV261 plasmid carrying the Rv3444c and Rv3445c genes into the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain.

[0025] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0026] Example 1 Preparation of Mycobacterium tuberculosis Rv3444c-Rv3445c Double Gene Deletion Strain

[0027] Based on the Mycobacterium tuberculosis genome sequence from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and the principle of homologous recombination, primers for the left and right homologous arms of the Rv3444c-Rv3445c gene were designed. High-fidelity enzymes were used to amplify the left and right homologous arms by PCR. The amplified length of the left and right homologous arms was 800 bp ( Figure 1 A).

[0028] The left and right homology arms and the p0004s plasmid were purified by Van91I digestion and then ligated with T4 DNA ligase. After transformation, single clones were identified by PCR. The amplified length was 800 bp, and the recombinant plasmid p3444-3445 ( Figure 1 B).

[0029] The phAE159 plasmid and the recombinant plasmid p3444-3445 were digested and dephosphorylated with Pac I, then ligated with T4 DNA ligase. After packaging using a packaging kit, single clones were identified by PCR (primers LF / R and RF / R). PCR results showed a band of approximately 800 bp, consistent with theoretical results, and were named phagemid 3444-3445 ( Figure 1 C).

[0030] Electrotransformation of phagemid 3444-3445 into Mycobacterium smegmatis MC 2 -155, 30 ℃ culture to obtain plaques, after expansion culture to obtain high titer phage liquid, infected with wild-type Mycobacterium tuberculosis, after hygromycin B resistance screening, two pairs of primers F / R1 and F / R2 were used to perform PCR identification of the Rv3444c-Rv3445c double gene deletion strain (K / O). F is a specific sequence within the left homology arm, and R1 is a specific sequence within the genes Rv3444c-Rv3445c. Using F / R1 amplification, the wild-type strain can amplify a fragment of approximately 498bp, while the gene deletion strain cannot amplify a gene fragment of the corresponding size. R2 is a specific sequence within the right homology arm. Because the Rv3444c and Rv3445c genes of the gene deletion strain are replaced by the hygromycin gene and the SacB gene, when using F / R2 amplification, the wild-type strain amplifies a short fragment of 1143bp in length, while the gene deletion strain amplifies a long fragment of 4185bp in length ( Figure 1 D).

[0031] Table 1 Sequence information of primers used in this example

[0032]

[0033] To further verify whether the target gene was knocked out, the correctly identified long-fragment PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the PCR product gene fragment consisted of homology arms, hygromycin gene, and SacB gene, and no target gene sequence was detected, indicating that the target gene was knocked out ( Figure 1 E).

[0034] It can be seen that this example obtained a Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain, whose nucleotide sequence is shown in SEQ ID NO.1.

[0035] The Rv3444c-Rv3445c double gene deletion strain (K / O), complemented strain (Comp.) and parent strain (WT) were cultured in 7H9 medium for 28 consecutive days. The absorbance at OD600nm was measured every 4 days. It was found that the Rv3444c-Rv3445c double gene deletion did not affect the growth rate of Mycobacterium tuberculosis ( Figure 1 F).

[0036] The Rv3444c-Rv3445c double gene deletion strain prepared in this embodiment was named Mycobacterium tuberculosis Ra3444c-5c-K0 and deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2024121.

[0037] Example 2 Detection of the Rv3444-Rv3445 double gene deletion strain of the present invention on host cells and mice

[0038] (1) Detection of the ability to survive in host cells.

[0039] To investigate the survival ability of the Rv3444c-Rv3445c double gene deletion strain of Mycobacterium tuberculosis in host cells, bacterial OD was measured one day before infection. 600 The bacterial OD was measured again on the day of infection. 600 The corresponding bacterial solution was collected and centrifuged at 3000r / min for 10min, resuspended in 1640 culture medium, and repeatedly pipetted with an insulin syringe to single bacteria. Human mononuclear macrophage THP1 cells were cultured at 2.5×10 5Cells were seeded in 24-well plates, with triplicate wells per group. The plates were gently shaken to evenly distribute the cells. After 24 hours of PMA-induced attachment, cells were infected at an MOI of 10 for 4 hours and washed three times with prewarmed PBS. This was designated Day 0. For samples on Day 0, 500 µL of Tween-80 was added to each well and incubated at 37°C for 10 minutes to lyse the cells. For the remaining samples, medium 1640 containing amikacin (50 µg / mL) was replaced and cells were lysed in the same manner on Day 2. The cell lysate was diluted tenfold, and 50 µL was plated on 7H11 plates in six replicates. After incubation at 37°C for 3 weeks, CFU were counted. The number of bacteria per mL = number of colonies × dilution factor × 20. Data are presented as the number of viable intracellular bacteria on Day 2 divided by the number of viable intracellular bacteria on Day 0. The results showed that the number of intracellular viable cells in the Rv3444c-Rv3445c double gene deletion strain (K / O) was significantly lower than that in the parent strain (WT), while there was no significant difference between the complemented strain (Comp.) and the parent strain (WT). Figure 2 A).

[0040] (2) Pathogenicity testing in mice.

[0041] To investigate the pathogenicity of the Rv3444c-Rv3445c double gene deletion strain of Mycobacterium tuberculosis to mice, 10 SPF-grade C57BL / 6 female mice aged 7 weeks were selected and inoculated with PBS, the parental strain (WT), the double gene deletion strain (K / O), and the complemented strain (Comp.) at a dose of 5×10 6 CFU was injected into the tail vein of mice for infection. After 28 days, the mice were euthanized, and the lungs and spleens were taken out and placed in a sterilized 2mL EP tube for weighing. The remaining organs were fixed with 4% paraformaldehyde. 1mL PBS and steel beads were added for grinding, and the samples were spread on 7H11 plates after ten-fold dilution. The samples were cultured in a 37°C incubator for 3 weeks and the CFU were counted. In addition, the lungs and spleens fixed with 4% paraformaldehyde were embedded in paraffin, sliced, and stained with HE. The lesions were observed and images were collected under an ordinary light microscope. The pathological changes of inflammatory infiltration, necrosis, and granulomas in the tissues were evaluated and analyzed. The results showed that the bacterial load in the lungs and spleen of the Rv3444c-Rv3445c double gene deletion strain (K / O) was significantly lower than that of the parent strain (WT) and the complemented strain (Comp.), indicating that Rv3444c-Rv3445c is a virulence factor of Mycobacterium tuberculosis ( Figure 2 B. Figure 2C). Pathological results showed that in the parental strain (WT) and complemented strain (Comp.) infection groups, most alveolar cavities disappeared, alveolar septa thickened, and a small amount of lymphocytes proliferated. The red and white pulp of the spleen were not clearly demarcated, and multinucleated macrophages increased slightly. Compared with the parental strain (WT) infection group, the Rv3444c-Rv3445c double gene deletion strain (K / O) infection group had a milder lesion and a significantly lower bacterial load ( Figure 2 D).

[0042] Example 3 Detection of Host Cell Epigenetics by Mycobacterium tuberculosis Rv3444c-Rv3445c Double Gene Deletion Strain

[0043] Based on the amino acid sequence, the inventors conducted bioinformatics predictions and found that the proteins encoded by Rv3444c and Rv3445c may be nuclear import proteins. To confirm this conclusion, in this case, Rv3444c-Rv3445c-overexpressing Mycobacterium tuberculosis was infected with human mononuclear macrophage THP1 cells. Indirect immunofluorescence detection showed that the proteins encoded by genes Rv3444c and Rv3445c (including EsxT, EsxU, and the dimer formed by the two) entered the host cell nucleus ( Figure 3 A).

[0044] Subsequently, the Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain (K / O) and the parental strain (WT) were used to infect human mononuclear macrophage THP1, and a DLO-HiC library was constructed and high-throughput sequencing was performed. The nextflow framework process packaged by nf-core was used to perform quality control, filtering, alignment and quantitative analysis of differential genes for GO pathway enrichment. DLO-HiC-Tools was used to organize and analyze the three-dimensional genome data, and the interaction matrix within and between chromosomes at the three-dimensional genome level was output. The topological domains (TADs) and chromatin loops (Loops) in the interaction matrix were detected and identified based on the interaction matrix. Combined with the transcriptome data, the analysis found that the Rv3444c-Rv3445c double gene deletion strain (K / O) caused changes in the TAD interactions of the three-dimensional genome of human mononuclear macrophage THP1 ( Figure 3 B). Comparative analysis revealed that the deletion of Rv3444c and Rv3445c genes resulted in TAD splitting at the same site ( Figure 3 C). Analyze the difference results of Loops and calculate the proportion ( Figure 3 D and 3E), it was found that the deletion of Rv3444c and Rv3445c genes led to the disappearance of the loop at the same site ( Figure 3 F).

[0045] The above results indicate that the proteins encoded by genes Rv3444c and Rv3445c enter the host cell nucleus and regulate epigenetic changes.

[0046] In summary, the present invention successfully constructed a Mycobacterium tuberculosis Rv3444c-Rv3445c double-gene deletion strain. Compared with the parent strain, the double-gene deletion strain showed obvious weakening of virulence and the ability to change the host epigenetic at the cellular and mouse levels, and the shown traits were related to the deletion of the Rv3444c and Rv3445c genes.

[0047] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solutions of the present invention rather than to limit them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. Use of a Mycobacterium tuberculosis Rv3444c-Rv3445c double gene deletion strain in the preparation of a low-toxic Mycobacterium tuberculosis inoculant, characterized in that: The Rv3444c-Rv3445c double gene deletion strain lacks both the Rv3444c and Rv3445c genes relative to the wild-type Mycobacterium tuberculosis, and has at least the following characteristics: have lower toxicity to cells and / or mammals; Resulting in epigenetic changes in host cells.

2. The use according to claim 1, characterized in that: The wild-type Mycobacterium tuberculosis is Mycobacterium tuberculosis H37Rv.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the Rv3444c-Rv3445c double gene deletion strain is shown in SEQ ID NO.

1.

4. The use according to claim 2, characterized in that: The Rv3444c-Rv3445c double gene deletion strain is Mycobacterium tuberculosis ( Mycobacterium tuberculosis )Ra3444c-5c-K0, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2024121.

5. A method for reducing the virulence of Mycobacterium tuberculosis, characterized in that: The Rv3444c and Rv3445 genes of Mycobacterium tuberculosis are deleted at the same time to obtain the Rv3444c-Rv3445c double gene deletion strain, and the Rv3444c-Rv3445c double gene deletion strain has lower toxicity to cells and / or mammals than wild-type Mycobacterium tuberculosis.

6. The method according to claim 5, characterized in that The Rv3444c-Rv3445c double gene deletion strain was obtained by packaging a bacteriophage carrying homologous arms, infecting wild-type Mycobacterium tuberculosis, and using resistance screening.

7. The method according to claim 6, characterized in that The wild-type Mycobacterium tuberculosis is Mycobacterium tuberculosis H37Rv, and the resistance screening uses hygromycin B as a resistance screening marker.

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