Journal of Microbiology

Journal Article

Furan-based Chalcone Annihilates the Multi-Drug-Resistant Pseudomonas aeruginosa and Protects Zebra Fish Against its Infection: Santosh Pushpa Ramya Ranjan Nayak , Catharine Basty , Seenivasan Boopathi , Loganathan Sumathi Dhivya , Khaloud Mohammed Alarjani , Mohamed Ragab Abdel Gawwad , Raghda Hager , Muthu Kumaradoss Kathiravan , Jesu Arockiaraj; J. Microbiol. 2024;62(2):75-89. Published online February 21, 2024; DOI: https://doi.org/10.1007/s12275-024-00103-6

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The emergence of carbapenem-resistant Pseudomonas aeruginosa, a multi-drug-resistant bacteria, is becoming a serious public health concern. This bacterium infects immunocompromised patients and has a high fatality rate. Both naturally and synthetically produced chalcones are known to have a wide array of biological activities. The antibacterial properties of synthetically produced chalcone were studied against P. aeruginosa. In vitro, study of the compound (chalcone derivative named DKO1), also known as (2E)-1-(5-methylfuran-2-yl)-3-(4-nitrophenyl) prop-2-en-1-one, had substantial antibacterial and biofilm disruptive action. DKO1 effectively shielded against P. aeruginosa-induced inflammation, oxidative stress, lipid peroxidation, and apoptosis in zebrafish larvae. In adult zebrafish, the treatment enhanced the chances of survivability and reduced the sickness-like behaviors. Gene expression, biochemical analysis, and histopathology studies found that proinflammatory cytokines (TNF-α, IL-1β, IL-6, iNOS) were down regulated; antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) levels increased, and histoarchitecture was restored in zebrafish. The data indicate that DKO1 is an effective antibacterial agent against P. aeruginosa demonstrated both in vitro and in vivo.

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Chalcone derivative enhance poultry meat preservation through quorum sensing inhibition against Salmonella (Salmonella enterica serovar Typhi) contamination
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The Journal of Nutritional Biochemistry.2025; 140: 109887. CrossRef
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Review

Current status and perspectives on vaccine development against dengue virus infection: Jisang Park , Ju Kim , Yong-Suk Jang; J. Microbiol. 2022;60(3):247-254. Published online February 14, 2022; DOI: https://doi.org/10.1007/s12275-022-1625-y

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Abstract PDF

Dengue virus (DENV) consists of four serotypes in the family Flaviviridae and is a causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. DENV is transmitted by mosquitoes, Aedes aegypti and A. albopictus, and is mainly observed in areas where vector mosquitoes live. The number of dengue cases reported by the World Health Organization increased more than 8-fold over the last two decades from 505,430 in 2000 to over 2.4 million in 2010 to 5.2 million in 2019. Although vaccine is the most effective
method
against DENV, only one commercialized vaccine exists, and it cannot be administered to children under 9 years of age. Currently, many researchers are working to resolve the various problems hindering the development of effective dengue vaccines; understanding of the viral antigen configuration would provide insight into the development of effective vaccines against DENV infection. In this review, the current status and perspectives on effective vaccine development for DENV are examined. In addition, a plausible direction for effective vaccine development against DENV is suggested.

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Journal Article

Potent antibacterial and antibiofilm activities of TICbf-14, a peptide with increased stability against trypsin: Liping Wang , Xiaoyun Liu , Xinyue Ye , Chenyu Zhou , Wenxuan Zhao , Changlin Zhou , Lingman Ma; J. Microbiol. 2022;60(1):89-99. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1368-9

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Abstract PDF

The poor stability of peptides against trypsin largely limits their development as potential antibacterial agents. Here, to obtain a peptide with increased trypsin stability and potent antibacterial activity, TICbf-14 derived from the cationic peptide Cbf-14 was designed by the addition of disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop. Subsequently, the trypsin stability and antimicrobial and antibiofilm activities of this peptide were evaluated. The possible mechanisms underlying its mode of action were also clarified. The results showed that TICbf-14 exhibited elevated trypsin inhibitory activity and effectively mitigated lung histopathological damage in bacteria-infected mice by reducing the bacterial counts, further inhibiting the systemic dissemination of bacteria and host inflammation. Additionally, TICbf-14 significantly repressed bacterial swimming motility and notably inhibited biofilm formation. Considering the mode of action, we observed that TICbf-14 exhibited a potent membrane-disruptive mechanism, which was attributable to its destructive effect on ionic bridges between divalent cations and LPS of the bacterial membrane. Overall, TICbf-14, a bifunctional peptide with both antimicrobial and trypsin inhibitory activity, is highly likely to become an ideal candidate for drug development against bacteria.

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Reviews

MINIREVIEW] Importance of differential identification of Mycobacterium tuberculosis strains for understanding differences in their prevalence, treatment efficacy, and vaccine development: Hansong Chae , Sung Jae Shin; J. Microbiol. 2018;56(5):300-311. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-8041-3

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Abstract PDF

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem in the 21st century because of its high mortality. Mtb is an extremely successful human-adapted pathogen that displays a multifactorial ability to control the host immune response and to evade killing by drugs, resulting in the breakdown of BCG vaccine-conferred anti-TB immunity and development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Although genetic components of the genomes of the Mtb complex strains are highly conserved, showing over 99% similarity to other bacterial genera, recently accumulated evidence suggests that the genetic diversity of the Mtb complex strains has implications for treatment outcomes, development of MDR/XDR Mtb, BCG vaccine efficacy, transmissibility, and epidemiological outbreaks. Thus, new insights into the pathophysiological features of the Mtb complex strains are required for development of novel vaccines and for control of MDR/XDR Mtb infection, eventually leading to refinement of treatment regimens and the health care system. Many studies have focused on the differential identification of Mtb complex strains belonging to different lineages because of differences in their virulence and geographical dominance. In this review, we discuss the impact of differing genetic characteristics among Mtb complex strains on vaccine efficacy, treatment outcome, development of MDR/ XDR Mtb strains, and epidemiological outbreaks by focusing on the best-adapted human Mtb lineages. We further explore the rationale for differential identification of Mtb strains for more effective control of TB in clinical and laboratory settings by scrutinizing current diagnostic methods.

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MINIREVIEW] Cure of tuberculosis using nanotechnology: An overview: Rout George Kerry , Sushanto Gouda , Bikram Sil , Gitishree Das , Han-Seung Shin , Gajanan Ghodake , Jayanta Kumar Patra; J. Microbiol. 2018;56(5):287-299. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7414-y

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Abstract PDF

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health issue of the present era. The bacterium inhabits the host macrophage and other immune cells where it modulates the lysosome trafficking protein, hinders the formation of phagolysosome, and blocks the TNF receptor- dependent apoptosis of host macrophage/monocytes. Other limitations such as resistance to and low bioavailability and bio-distribution of conventional drugs aid to their high virulence and human mortality. This review highlights the use of nanotechnology-based approaches for drug formulation and delivery which could open new avenues to limit the pathogenicity of tuberculosis. Moreover phytochemicals, such as alkaloids, phenols, saponins, steroids, tannins, and terpenoids, extracted from terrestrial plants and mangroves seem promising against M. tuberculosis through different molecular mechanisms. Further understanding of the genomics and proteomics of this pathogenic microbe could also help overcome various research gaps in the path of developing a suitable therapy against tuberculosis.

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Journal Articles

Bedaquiline susceptibility test for totally drug-resistant tuberculosis Mycobacterium tuberculosis: Ji-Chan Jang , Yong-Gyun Jung , Jungil Choi , Hyunju Jung , Sungweon Ryoo; J. Microbiol. 2017;55(6):483-487. Published online April 20, 2017; DOI: https://doi.org/10.1007/s12275-017-6630-1

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This study aimed to provide information that bedaquilline is significantly effective for treatment of totally drug resistant (TDR) Mycobacterium tuberculosis that shows resistant to all first- and second-line drugs-using an innovative disc agarose channel (DAC) system. Time-lapse images of single bacterial cells under culture conditions with different concentrations of bedaquiline were analysed by image processing software to determine minimum inhibitory concentrations (MICs). Bedaquiline inhibited the growth of TDR M. tuberculosis strains, with MIC values ranging from 0.125 to 0.5 mg/L. The results of the present study demonstrate that bedaquiline, newly approved by the United States Food and Drug Admi-nistration (FDA), may offer therapeutic solutions for TDR -TB.

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Sudapyridine (WX-081), a Novel Compound against Mycobacterium tuberculosis
Rong Yao, Bin Wang, Lei Fu, Lei Li, Kejun You, Yong-Guo Li, Yu Lu, Luiz Pedro Sorio de Carvalho
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Recent developments, challenges and future prospects in advanced drug delivery systems in the management of tuberculosis
Nitin Verma, Vimal Arora, Rajendra Awasthi, Yinghan Chan, Niraj Kumar Jha, Komal Thapa, Talha Jawaid, Mehnaz Kamal, Gaurav Gupta, Gang Liu, Keshav Raj Paudel, Philip Michael Hansbro, Brian Gregory George Oliver, Sachin Kumar Singh, Dinesh Kumar Chellappan
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V. I. Litvinov, E. Yu. Nosova
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Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis: Pallavi Sinha , Pradyot Prakash , Shashikant C.U. Patne , Shampa Anupurba , Sweety Gupta , G. N. Srivastava; J. Microbiol. 2017;55(1):63-67. Published online December 30, 2016; DOI: https://doi.org/10.1007/s12275-017-6127-y

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The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.

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The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis: Hsin-Chih Lai , Yu-Tze Horng , Pen-Fang Yeh , Jann-Yuan Wang , Chin-Chung Shu , Jang-Jih Lu , Jen-Jyh Lee , Po-Chi Soo; J. Microbiol. 2016;54(11):761-767. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6201-x

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Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL- 17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase- 1 in sputum. Results of detection of IL-10, IFN-γ, perforin- 1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acidfast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.

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From simple to complex: Protein‐based biomarker discovery in tuberculosis
Zaynab Mousavian, Gunilla Källenius, Christopher Sundling
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Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation: Elisabetta Iona , Manuela Pardini , Alessandro Mustazzolu , Giovanni Piccaro , Roberto Nisini , Lanfranco Fattorini , Federico Giannoni; J. Microbiol. 2016;54(8):565-572. Published online August 2, 2016; DOI: https://doi.org/10.1007/s12275-016-6150-4

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The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

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Research Support, Non-U.S. Gov't

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis: Fei-yu Wang , Yu-qing Zhang , Xin-min Wang , Chan Wang , Xiao-fang Wang , Jiang-dong Wu , Fang Wu , Wan-jiang Zhang , Le Zhang; J. Microbiol. 2016;54(4):330-337. Published online April 1, 2016; DOI: https://doi.org/10.1007/s12275-016-5627-5

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Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.

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Tolerance of Listeria monocytogenes to biocides used in food processing environments
Sanelisiwe Thinasonke Duze, Musa Marimani, Mrudula Patel
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Regulatory role and mechanism of the inhibition of the Mcl-1 pathway during apoptosis and polarization of H37Rv-infected macrophages
Ling Han, Yang Lu, Xiaofang Wang, Shujun Zhang, Yingzi Wang, Fang Wu, Wanjiang Zhang, Xinmin Wang, Le Zhang
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Current and emerging therapies to combat persistent intracellular pathogens
Philip Arandjelovic, Marcel Doerflinger, Marc Pellegrini
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PPARγ is critical for Mycobacterium tuberculosis induction of Mcl-1 and limitation of human macrophage apoptosis
Eusondia Arnett, Ashlee M. Weaver, Kiersten C. Woodyard, Maria J. Montoya, Michael Li, Ky V. Hoang, Andrew Hayhurst, Abul K. Azad, Larry S. Schlesinger, Thomas R. Hawn
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Research Support, N.I.H., Extramural

The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition: Shaleen B. Korch , Vandana Malhotra , Heidi Contreras , Josephine E. Clark-Curtiss; J. Microbiol. 2015;53(11):783-795. Published online October 28, 2015; DOI: https://doi.org/10.1007/s12275-015-5333-8

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Abstract

Toxin-antitoxin (TA) genes are ubiquitous among bacteria and are associated with persistence and dormancy. Following exposure to unfavorable environmental stimuli, several species (Escherichia coli, Staphylococcus aureus, Myxococcus xanthus) employ toxin proteins such as RelE and MazF to downregulate growth or initiate cell death. Mycobacterium tuberculosis possesses three Rel TA modules (RelMtb): RelBEMtb, RelFGMtb and RelJKMtb (Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, respectively), which inhibit mycobacterial growth when the toxin gene (relE, relG, relK) is expressed independently of the antitoxin gene (relB, relF, relJ). In the present study, we examined the in vivo mechanism of the RelEMtb toxin protein, the impact of RelEMtb on M. tuberculosis physiology and the environmental conditions that regulate all three relMtb modules. RelEMtb negatively impacts growth and the structural integrity of the mycobacterial envelope, generating cells with aberrant forms that are prone to extensive aggregation. At a time coincident with growth defects, RelEMtb mediates mRNA degradation in vivo resulting in significant changes to the proteome. We establish that relMtb modules are stress responsive, as all three operons are transcriptionally activated following mycobacterial exposure to oxidative stress or nitrogen-limiting growth environments. Here we present evidence that the relMtb toxin:antitoxin family is stress-responsive and, through the degradation of mRNA, the RelEMtb toxin influences the growth, proteome and morphology of mycobacterial cells.

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Research Support, Non-U.S. Gov'ts

Performance of a real-time PCR assay for the rapid identification of Mycobacterium species: Hye-young Wang , Hyunjung Kim , Sunghyun Kim , Do-kyoon Kim , Sang-Nae Cho , Hyeyoung Lee; J. Microbiol. 2015;53(1):38-46. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4495-8

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Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM species are prevalent in the environment and because they have fastidious properties. In the present study, we evaluated 279 clinical isolates grown in liquid culture provided by The Catholic University of Korea, St. Vincent’s Hospital using real-time PCR based on mycobacterial rpoB gene sequences. The positive rate of real-time PCR assay accurately discriminated 100% (195/195) and 100% (84/84) between MTB and NTM species. Comparison of isolates identified using the MolecuTech REBA Myco-ID? and Real Myco-ID? were completely concordant except for two samples. Two cases that were identified as mixed infection (M. intracellulare-M. massiliense and M. avium-M. massiliense co-infection) by PCRREBA assay were only detected using M. abscessus-specific probes by Real Myco-ID?. Among a total of 84 cases, the most frequently identified NTM species were M. intracellulare (n=38, 45.2%), M. avium (n=18, 23.7%), M. massiliense (n=10, 13.2%), M. fortuitum (n=5, 6%), M. abscessus (n=3, 3.9%), M. gordonae (n=3, 3.9%), M. kansasii (n=2, 2.4%), M. mucogenicum (n=2, 2.4%), and M. chelonae (n= 1, 1.2%). Real Myco-ID? is an efficient tool for the rapid detection of NTM species as well as MTB and sensitive and specific and comparable to conventional methods.

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Phosphorylation Regulates Mycobacterial Proteasome: Tripti Anandan , Jaeil Han , Heather Baun , Seeta Nyayapathy , Jacob T. Brown , Rebekah L. Dial , Juan A. Moltalvo , Min-Seon Kim , Seung Hwan Yang , Donald R. Ronning , Robert N. Husson , Joowon Suh , Choong-Min Kang; J. Microbiol. 2014;52(9):743-754. Published online September 2, 2014; DOI: https://doi.org/10.1007/s12275-014-4416-2

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Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome- associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

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Characterization of a Novel Antigen of Mycobacterium tuberculosis K Strain and Its Use in Immunodiagnosis of Tuberculosis: Paul J. Park , Ah Reum Kim , Yangkyo P. Salch , Taeksun Song , Sung Jae Shin , Seung Jung Han , Sang-Nae Cho; J. Microbiol. 2014;52(10):871-878. Published online August 27, 2014; DOI: https://doi.org/10.1007/s12275-014-4235-5

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*For correspondence. (S.J. Han) E-mail: hansjung@yuhs.ac / (S.N. Cho) E-mail: raycho@yuhs.ac Paul J. Park, Ah Reum Kim, Yangkyo P. Salch, Taeksun Song, Sung Jae Shin, Seung Jung Han*, and Sang-Nae Cho* Department of Microbiology and Institute for Immunology and Immunological Diseases, Brain Korea 21 Plus Project for the Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea (Received Apr 16, 2014 / Revised Jul 14, 2014 / Accepted Jul 16, 2014) Journal of Microbiology (2014) Vol. 52, No. 10, pp. 871–878 Copyright 􎨰􀁇2014, The Microbiological Society of Korea DOI 10.1007/s12275-014-4235-5 Characterization of a Novel Antigen of Mycobacterium tuberculosis K strain and Its Use in Immunodiagnosis of Tuberculosis Mycobacterium tuberculosis-specific antigens would be of great value in developing immunodiagnostic tests for tuberculosis (TB), but regional differences in molecular types of the organism may result in antigenic variation, which in turn affects the outcome of the tests. For example, the Beijing strains of M. tuberculosis are prevalent in East Asia, and in particular, the K strain and related strains of the Beijing family, are most frequently isolated during school outbreaks of TB in South Korea. From comparison of genome sequences between M. tuberculosis K strain and the H37Rv strain, a non-Beijing type, we identified a K strain-specific gene, InsB, which has substantial homology with the ESAT-6-like proteins. This study was, therefore, initiated to characterize the InsB protein for its immunogenicity in mice and to confirm its expression in TB patients by detecting antibodies to the protein. The InsB gene was cloned from M. tuberculosis K strain and expressed in Escherichia coli. The recombinant InsB protein was used for immunization of mice. All mice showed strong antibody responses to the InsB protein, and splenocytes stimulated with InsB showed strong IFN-γ and IL-17 responses and a weak IL-2 response, all of which have been implicated in disease expression and used for the immunodiagnosis of TB. Serum samples from TB patients also showed significant antibody responses to the InsB protein as compared to healthy control samples. These results indicate that the InsB protein is an M. tuberculosis K-strain-specific antigen that could further improve the current immunodiagnostic
methods
, especially for the South Korean population.

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Detection of Inhibitors of Phenotypically Drug-tolerant Mycobacterium tuberculosis Using an In Vitro Bactericidal Screen: Ian M. Bassett , Shichun Lun , William R. Bishai , Haidan Guo , Joanna R. Kirman , Mudassar Altaf , Ronan F. O’Toole; J. Microbiol. 2013;51(5):651-658. Published online June 25, 2013; DOI: https://doi.org/10.1007/s12275-013-3099-4

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Abstract PDF

Many whole cell screens of chemical libraries currently in use are based on inhibition of bacterial growth. The goal of this study was to develop a chemical library screening model that enabled detection of compounds that are active against drug-tolerant non-growing cultures of Mycobacterium tuberculosis. An in vitro model of low metabolically active mycobacteria was established with 8 and 30 day old cultures of M. smegmatis and M. tuberculosis, respectively. Reduction of resazurin was used as a measure of viability and the assay was applied in screens of chemical libraries for bactericidal compounds. The model provided cells that were phenotypically-resilient to killing by first and second-line clinical drugs including rifampicin. Screening against chemical libraries identified proteasome inhibitors, NSC310551 and NSC321206, and a structurally-related series of thiosemicarbazones, as having potent killing activity towards aged cultures. The inhibitors were confirmed as active against virulent M. tuberculosis strains including multi- and extensively-drug resistant clinical isolates. Our library screen enabled detection of compounds with a potent level of bactericidal activity towards phenotypically drug-tolerant cultures of M. tuberculosis.

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