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Background: Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole‐genome sequencing is a molecular method that allows for the rapid and cost‐effective detection of mutations in multiple genes associated with anti‐tuberculosis drug resistance. This sequencing approach addresses the limitations of culture‐based methods, which may not apply to certain anti‐TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. Methods: Thirty‐four M. tuberculosis isolates were subcultured on a Lowenstein–Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. Results: The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in‐depth study to understand their relationship to the phenotypic profile. Conclusions: Compared to the conventional culture method, the whole‐genome sequencing method has advantages in determining anti‐tuberculosis resistance profiles, especially in reduced time and bias. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant E. coli is a growing concern in various settings, but environmental studies are rare compared to clinical research on human and animal health. This study aimed to identify the prevalence of E. coli bacteria resistant to different antibiotics in the environment by examining E. coli resistant to cefotaxime isolated from ruminant stool, feed, and compost. The phenotyping test was conducted through antibiotic susceptibility test using Kirby-Bauer disk-diffusion method, followed by the One-Way variance (ANOVA) analysis of the antibiotic susceptibility test results. Of the 12 isolates exposed to cefotaxime, six showed resistance to this antibiotic, and all isolates, including those resistant to cefotaxime, were resistant to eight out of ten types of antibiotics. All isolates had resistance to at least two to five types of antibiotics. The phenotypic pattern between fecal isolates and non-fecal isolates did not differ significantly, except for the antibiotics amoxicillin (p≤0.05) and ampicillin (p≤0.05). The overlapping resistance patterns observed in animal feed, animal stool, and compost suggest a potential link between their microbiological compositions.

The global spread of antibiotic resistance poses a significant threat to public health and is one of the main causes of this problem. Livestock farming plays a significant role in the horizontal and vertical transmission of treatment-resistant genes and bacteria. These processes involve contact with agricultural products and the environment, raising concerns for public health, and farming communities. The farming community is composed of a staggering 608 million farms worldwide, and their livelihood depends heavily on livestock farming. To address this issue, a multidisciplinary One Health approach focusing on integrated monitoring and intervention for humans, animals, and the environment is essential. Water, sanitation, and hygiene (WaSH) programs have the potential to significantly reduce the risk of exposure to antibiotic-resistant bacteria, particularly extended spectrum beta-lactamase (ESBL) Escherichia coli, by obstructing the transmission route between humans and animals. Additional risk reduction measures for ESBL E. coli infection in animals include vaccination and biosecurity program implementation. Water, sanitation, and hygiene and biosecurity measures must be combined to maximize the effectiveness of the One Health program. Therefore, this study aimed to describe recent advances in biosecurity and WaSH interventions in the livestock environment, analyze the effects of these interventions on human and animal health, and investigate potential future scenarios within the quantitative microbial risk assessment framework. This study used an integrative literature review through searches of four databases, a review of World Health Organization documents through websites, and an examination of relevant texts from previously obtained reference lists. Although hygiene and sanitation are often combined, there is still a lack of quantitative evaluation of the efficacy of integrating WaSH with biosecurity in livestock. In addition, the integration of the WaSH program with biosecurity has potential as a One Health intervention in the coming years. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) due to infection of the Mycobacterium tuberculosis has become a concern since this disease has been suffered by most of the world's population and causes death in large numbers. The increasing number of TB patients has increased our vigilance to reduce the spread of this disease. One of the efforts to provide effective treatment for cases of infection by this bacterium is to determine the proper anti-tuberculosis drug. Methods: This study tested M. tuberculosis isolates against several tuberculosis drugs, such as isoniazid, quinolone (ofloxacin and moxifloxacin), kanamycin, capreomycin, bedaquiline, linezolid, and clofazimine in MGIT liquid medium. The isolates used were bacterial stock belonging to the Department of Microbiology, Medical Faculty, Universitas Indonesia. All isolates used had a resistance phenotype to the anti-tuberculosis drug rifampicin determined by the GeneXpert MTB/Rif method. Results: From all the isolates tested, the percentage of anti-tuberculosis resistance was as follows: low-dose isoniazid 70.4%, high-dose isoniazid 66.7%, ofloxacin 9.9%, low-dose moxifloxacin 9.9%, high-dose moxifloxacin 2.5%, and bedaquiline 5.1%. There was no resistance to linezolid and clofazimine among the tested isolates. This study also found that 57 isolates were multi-drug resistant (MDR) strains, six isolates were pre-extensively drug resistance (XDR), and one isolate was an XDR strain. Conclusion: This study presented an overview of the resistance profile of M. tuberculosis to several first-, second-line and new tuberculosis drugs in vitro. The results of this study can be used by stakeholders in the health sector to develop policies for better management of tuberculosis. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). Bacille Calmette-Guérin (BCG) is the only licensed vaccine against TB, and it is effective in children but not in adults. The Vaccine Research Team, Department of Microbiology FKUI has developed a DNA-based TB vaccine candidate pcDNA3.1-rpfB. This candidate induces immune responses in mice, but its potency is unknown. The gold standard for potency testing of TB vaccine is the challenge method. The BSL3 animal laboratory for the challenge method is currently unavailable at FKUI. Therefore, mycobacterial growth inhibition assay (MGIA) was used as a preliminary test before the in vivo challenge test was conducted. The principle of MGIA is to reculture Mtb in a Mycobacteria Growth Indicator Tube (MGITTM) from co-cultured Mtb with mammalian cells that have been previously treated with pcDNA3.1-rpfB, pcDNA3.1 (negative control), and BCG (positive control). MGITTM shows the time to positivity, which is the time that has lapsed until a positive growth of Mtb is detected. In addition, measurements of interferon (IFN)? levels by enzyme-linked immunosorbent assay were carried out. This study concluded that pcDNA3.1-rpfB can inhibit the growth of Mtb in vitro and showed no statistical difference from BCG. The IFN? levels from co-culturing did not correlate with the level of inhibition of the growth of Mtb in vitro. [ABSTRACT FROM AUTHOR]

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. It is a persistent global health problem with a high mortality rate. Currently, TB is controlled by administering the Bacillus Calmette-Guerin (BCG) vaccine, but the effectiveness of its protection varies among individuals in a population. The pe/ppe gene family comprises a typical group of genes that play a role in avoiding the host immune response and inducing persistent TB infection. Based on in silico analysis, the pe11 gene has estimated immunogenicity and potential as a TB seed vaccine candidate. The pe11 gene from an Indonesian isolate of an M. tuberculosis Beijing strain was amplified by polymerase chain reaction (PCR) and inserted into the mammalian expression vector pcDNA3.1. The recombinant vector pcDNA3.1-pe11 was used to transform Top10 competent Escherichia coli. Clones from the transformation were subjected to colony PCR to confirm the direction of the insert. Sequencing was performed to confirm the correctness of the insert sequence. In this study, the pe11 gene was successfully cloned into the pcDNA3.1 vector in the correct direction to assure PE11 expression. No mutations were found in the pe11 gene insert, compared with the M. tuberculosis H37Rv sequence as the standard. A pcDNA3.1 vector containing the pe11 gene derived from an M. tuberculosis Beijing strain was successfully constructed. [ABSTRACT FROM AUTHOR]

BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guérin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro. METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg/ ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays. RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10. CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine. [ABSTRACT FROM AUTHOR]

Antigen sub units for vaccine studies are typically isolated from recombinant proteins in an expression system. However, not all protein expression systems are used to express the specific protein. In this study, we optimized the pGEX system combined with the modified protein refolding to express and isolate M. tuberculosis proteins, especially proteins that are expressed as an inclusion body. Resuscitation promoting factor B (RpfB) protein is one of the Resuscitation promoting factor (Rpf) family of proteins that has been studied for its ability to induce cellular immunity in animal tests. Silico analyses demonstrate how RpfB is included in cell wall and cell processes. The Rpf family proteins are promising antigens that can be used as a TB vaccine candidate. The polymerase chain reaction was briefly performed using specific primers to amplify the full length of the rpfB. PCR amplification products were then purified, cut by restriction endonucleases, and cloned in to pGEX 6-P1. Protein expression was done in the Escherichia coli BL21 strain, and expressed protein was isolated using the modified protein refolding and solubilization method. The complex protein expression that appeared as inclusion bodies were successfully isolated and can be detected as complex GST-RpfB through the western blotting process. Our study results indicate that this system and our modified method are suitable for M. tuberculosis inclusion body protein expression and isolation. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf) protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNArpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile, recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral immune response in mice model. [ABSTRACT FROM AUTHOR]

There was a concern on Mycobacterium tuberculosis spreading to the bone marrow, when it was applied on tuberculous spine infection. This research aimed to study the probability of using autologous bone marrow as a source of mesenchymal stem cell for patients with tuberculous spondylitis. As many as nine patients with tuberculous spondylitis were used as samples. During the procedure, the vertebral lesion material and iliac bone marrow aspirates were obtained for acid fast staining, bacteria culture, and PCR (polymerase chain reaction) tests for Mycobacterium tuberculosis at the Clinical Microbiology Laboratory of Faculty of Medicine Universitas Indonesia. This research showed that there was a relationship between diagnostic confirmation of tuberculous spondylitis based on the PCR test and bacterial culture on the solid vertebral lesion material with the PCR test and bacterial culture from the bone marrow aspirates. If the diagnostic confirmation concluded positive results, then there was a higher probability that there would be a positive result for the bone marrow aspirates, so that it was not recommended to use autologous bone marrow as a source of mesenchymal stem cell for patients with tuberculous spondylitis unless the PCR and culture examination of the bone marrow showed a negative result. [ABSTRACT FROM AUTHOR]

The cell envelope is the target for many antibiotics. In Gram-positive bacteria, membrane alterations and dysfunction caused by antibiotics are sensed mainly by two classes of signal transduction systems: the ECF sigma factors and the two-component signal transduction systems (TCSs). Enduracidin is an antibiotic that inhibits the transglycosylation step of peptidoglycan biosynthesis, and is an attractive target for further antibiotic development studies. We assessed transcriptional responses to enduracidin in Bacillus subtilis cells using a high-density tiling chip, and compared the results with responses to bacitracin, which inhibits the lipid II cycle of peptidoglycan synthesis. We exploited the quantitative advantage of the tiling chip to introduce a new criterion, an increase in transcriptional level, in addition to the conventional induction ratio, in order to distinguish genes of biological significance from those with lower induction ratios. Our results indicate that introduction of the new criterion led to unambiguous identification of core transcriptional responses to antibiotics, with a reduction in the number of possible background genes, compared to previous results obtained using gene arrays. We identified 129 genes that were significantly upregulated by enduracidin and/or bacitracin. Notably, we found that inactivation of the LiaRS TCS, which was the system most strongly induced by the two antibiotics, resulted in increased sensitivity to enduracidin, probably through a failure to induce LialH proteins. We noted that 33 genes belonging to the SigM regulon were induced by both antibiotics. Consistent with stronger induction of the SigM regulon in enduracidin-treated cells, inactivation of sigM resulted in increased sensitivity to enduracidin. In addition, and for the first time, we found that the Spx regulon was induced in cells challenged by enduracidin and bacitracin, suggesting that thiol-oxidative stress occurred in cells treated with antibiotics. These findings contribute to further our understanding of the molecular nature of genetic systems involved in antibiotic resistance. [ABSTRACT FROM AUTHOR]

The cell envelope is the target for many antibiotics. In Gram-positive bacteria, membrane alterations and dysfunction caused by antibiotics are sensed mainly by two classes of signal transduction systems: the ECF sigma factors and the two-component signal transduction systems (TCSs). Enduracidin is an antibiotic that inhibits the transglycosylation step of peptidoglycan biosynthesis, and is an attractive target for further antibiotic development studies. We assessed transcriptional responses to enduracidin in Bacillus subtilis cells using a high-density tiling chip, and compared the results with responses to bacitracin, which inhibits the lipid II cycle of peptidoglycan synthesis. We exploited the quantitative advantage of the tiling chip to introduce a new criterion, an increase in transcriptional level, in addition to the conventional induction ratio, in order to distinguish genes of biological significance from those with lower induction ratios. Our results indicate that introduction of the new criterion led to unambiguous identification of core transcriptional responses to antibiotics, with a reduction in the number of possible background genes, compared to previous results obtained using gene arrays. We identified 129 genes that were significantly upregulated by enduracidin and/or bacitracin. Notably, we found that inactivation of the LiaRS TCS, which was the system most strongly induced by the two antibiotics, resulted in increased sensitivity to enduracidin, probably through a failure to induce LiaIH proteins. We noted that 33 genes belonging to the SigM regulon were induced by both antibiotics. Consistent with stronger induction of the SigM regulon in enduracidin-treated cells, inactivation of sigM resulted in increased sensitivity to enduracidin. In addition, and for the first time, we found that the Spx regulon was induced in cells challenged by enduracidin and bacitracin, suggesting that thiol-oxidative stress occurred in cells treated with antibiotics. These findings contribute to further our understanding of the molecular nature of genetic systems involved in antibiotic resistance.