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The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion. Agency for Science, Technology and Research (A*STAR) Ministry of Health (MOH) National Medical Research Council (NMRC) National Research Foundation (NRF) Following supports are acknowledged: N.K.V. to LeeKong Chian School of Medicine Strategic Academic In-itiative Grant (L0494003); H.C. to A*STAR Grant(C210812044); P.H., B.C.G., and W.M. to Singapore Na-tional Research Foundation (NRF)’s Intra-Campus for Re-search Excellence and Technological Enterprise (CREATE)Seed Collaboration Grant; P.H. and B.C.G. to Singapore-MIT Alliance for Research and Technology (SMART) An-timicrobial Resistance Interdisciplinary Group funded by theNRF under its CREATE program; T.A.A. to NUSMedpostdoctoral fellowship (NUHSRO/2019/046/PDF/19); J.P.B.to National University Hospital System Seed Fund (NUHSRO/2019/050/T1/Seed-Mar/03) and SMART (ING-001014 BIO);W.M. to Singapore Ministry of Health’s National MedicalResearch Council (OFIRG21jun-0038); M.A.M.V.S. to Na-tional Health Innovation Centre Singapore Innovation to De-velop Grant (NHIC-I2D-2104625).

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Bacteriophages (or simply ‘phages’) are viruses that replicate themselves within a host bacterial cell following infection of the host. For this project, environmental soil and water samples from around Singapore were collected to screen for phages, which have a myriad of purposes, due to their high specificity to their hosts. For instance, phages, coupled with genetic engineering of phage DNA, may provide an alternative to treating bacterial infections due to rapid replication times, and potential for non-intrusive therapy. Phages may also be used for diagnostics purposes, which allows scientists to potentially confirm the presence of a species of bacteria, and thus determine the need for the use of specific antibiotics. This research focuses on Staphylococcus aureus and Carbapanem-resistant Enterobacteriaceae, two of the species of the ESKAPE pathogens (Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The research aims to screen and isolate phages specific to these bacterial species (as they have high potential to achieve antibiotic resistance) and study them via purification, restriction enzyme digestion analysis and phenotypic characterisation. As a short summary, six distinct phages were found through the use of phenotypic and genotypic characterisation. Three of these phages targeted the Gram-positive S. aureus and Streptococcus pneumoniae, whereas the other three targeted Gram-negative K. pneumoniae. In particular, three of six studied phages targeted Carbapanem-resistant K. pneumoniae. The results of this study could provide insight into the species of phages present in Singapore, and whether they could be used for diagnostics and therapy Aslam (A Treatise on Electricity and Magnetism. Oxford, Clarendon, pp. 68–73, 2018 [1]), Dennehy (The Quarterly Review of Biology 85:109–109, 2010 [2])

The SARS-CoV-2 outbreak originated in China in late 2019 and has since spread to pandemic proportions. Diagnostics, therapeutics and vaccines are urgently needed. We model the trimeric Spike protein, including flexible loops and all N-glycosylation sites, in order to elucidate accessible epitopes for antibody-based diagnostics, therapeutics and vaccine development. Based on published experimental data, six homogeneous glycosylation patterns and two heterogeneous ones were used for the analysis. The glycan chains alter the accessible surface areas on the S-protein, impeding antibody-antigen recognition. In presence of glycan, epitopes on the S1 subunit, that notably contains the receptor binding domain, remain mostly accessible to antibodies while those present on the S2 subunit are predominantly inaccessible. We identify 28 B-cell epitopes in the Spike structure and group them as non-affected by the glycan cloud versus those which are strongly masked by the glycan cloud, resulting in a list of favourable epitopes as targets for vaccine development, antibody-based therapy and diagnostics., SCOPUS: ar.j, info:eu-repo/semantics/published

Treating M. abscessus infection is challenging due to the potent β-lactamase BlaMab (Beta-lactamase of M. abscessus ). Avibactam is a non-β-lactam, β-lactamase inhibitor shown to inhibit BlaMab. We tested whether avibactem can render piperacillin effective against M. Abscessus. In-vitro, avibactam enhanced the activity of piperacillin by 16–32 fold, with no significant effect on meropenem. In an in-vivo Galleria mellonella model, meropenem and piperacillin/avibactam significantly decreased infection burden compared to untreated controls. Neither piperacillin nor avibactam alone had a significant effect.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts., Present understanding of Plasmodium vivax biology is hampered by its inability to grow in vitro. Here, the authors developed an in vitro culture of its simian counterpart, P. cynomolgi, which shares morphological and phenotypic similarities with P. vivax, initiating a new phase in vivax research.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

Tuberculosis (TB) remains the second leading cause of death from an infectious disease globally, despite the incessant efforts to control it. Research and development into new TB medicines is imperative for effective TB control; however, new strategies for the rational use of existing drugs, such as through the identification of new drug targets, could also significantly enhance this process. Key enzymes involved in the essential metabolic and regulatory pathways are usually sought in the pursuit of potential drug targets. Uracil phosphoribosyltransferase (UPRT) is a key salvage pathway enzyme in the synthesis of uridine 5'-monophosphate (UMP) and a probable target of 5-fluorouracil (5-FU) in Mycobacterium tuberculosis (Mtb). To date, there is no structure available for UPRT from Mtb (MtUPRT) that would assist in the identification of appropriate inhibitors for the enzyme. Here we report the structure of MtUPRT along with its spontaneous mutational studies in the presence of 5-FU. We further mapped these four single nucleotide polymorphisms (SNPs) onto the MtUPRT structure, with two residues found to be conserved among the MtUPRT homologs. Notably, none of these SNPs are located in the 5-FU binding pocket. However, the mutants harboring these mutations showed increased MICs (minimum inhibitory concentration) as compared to wild type strains. The present study will aid in the screening of inhibitors of MtUPRT and thus assist in TB drug design and development.

Alleviating the burden of tuberculosis (TB) requires an understanding of the genetic basis that determines the emergence of drug-resistant mutants. PA-824 (pretomanid) is a bicyclic nitroimidazole class compound presently undergoing the phase III STAND clinical trial, despite lacking identifiable genetic markers for drug-specific resistant Mycobacterium tuberculosis . In the present study, we aimed to characterize the genetic polymorphisms of spontaneously generated PA-824-resistant mutant strains by surveying drug metabolism genes for potential mutations. Of the 183 independently selected PA-824-resistant M. tuberculosis mutants, 83% harbored a single mutation in one of five nonessential genes associated with either PA-824 prodrug activation ( ddn , 29%; fgd1 , 7%) or the tangential F 420 biosynthetic pathway ( fbiA , 19%; fbiB , 2%; fbiC , 26%). Crystal structure analysis indicated that identified mutations were specifically located within the protein catalytic domain that would hinder the activity of the enzymes required for prodrug activation. This systematic analysis conducted of genotypes resistant to PA-824 may contribute to future efforts in monitoring clinical strain susceptibility with this new drug therapy.

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Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007–2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community.

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi–infected human reticulocytes that are strikingly similar to those observed for P vivax. These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long-term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host-parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR-BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

Background Whole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, where conventional genotyping is often uninformative and field epidemiology often difficult. This study applies four candidate strategies for WGS-based identification of emerging M. tuberculosis subpopulations, employing both phylogenomic and population genetics methods. Results M. tuberculosis subpopulations in New York City and New Jersey can be distinguished via phylogenomic reconstruction, evidence of demographic expansion and subpopulation-specific signatures of selection, and by determination of subgroup-defining nucleotide substitutions. These methods identified known historical outbreak clusters and previously unidentified subpopulations within relatively monomorphic M. tuberculosis endemic clone groups. Neutrality statistics based on the site frequency spectrum were less useful for identifying M. tuberculosis subpopulations, likely due to the low levels of informative genetic variation in recently diverged isolate groups. In addition, we observed that isolates from New York City endemic clone groups have acquired multiple non-synonymous SNPs in virulence- and growth-associated pathways, and relatively few mutations in drug resistance-associated genes, suggesting that overall pathoadaptive fitness, rather than the acquisition of drug resistance mutations, has played a central role in the evolutionary history and epidemiology of M. tuberculosis subpopulations in New York City. Conclusions Our results demonstrate that some but not all WGS-based methods are useful for detection of emerging M. tuberculosis clone groups, and support the use of phylogenomic reconstruction in routine tuberculosis laboratory surveillance, particularly in areas with relatively less diverse M. tuberculosis populations. Our study also supports the use of wider-reaching phylogenomic and population genomic methods in tuberculosis public health practice, which can support tuberculosis control activities by identifying genetic polymorphisms contributing to epidemiological success in local M. tuberculosis populations and possibly explain why certain isolate groups are apparently more successful in specific host populations. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3298-6) contains supplementary material, which is available to authorized users.

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites., IMPORTANCE Several previous in vitro evolution studies have implicated the Plasmodium falciparum cyclic amine resistance locus (PfCARL) as a potential target of imidazolopiperazines, potent antimalarial compounds with broad activity against different parasite life cycle stages. Given that the imidazolopiperazines are currently being tested in clinical trials, understanding their mechanism of resistance and the cellular processes involved will allow more effective clinical usage.

The emergence of resistant Mycobacterium tuberculosis ( Mtb ) infection and the dearth of drugs against tuberculosis have made it imperative to identify and validate novel targets and classes of drugs for treatment. The pyrimidine operon regulatory protein (PyrR), a regulator of de novo pyrimidine synthesis, is an essential enzyme and a probable 5-fluorouracil (5-FU) target in Mtb , with mutations in PyrR attributable to 5-FU resistance. Here we report, for the first time, the co–crystal structure of the PyrR–5-FU complex along with mapping of spontaneous mutational sites of PyrR. A cluster of mutations in the presence of the drug usually indicates a plausible region of drug–target interaction. Notably, we observed that three of the mutated PyrR residues lie in close proximity to the 5-FU binding site, including the amino acid Val178, which is involved in water mediated hydrogen bonding contact with 5-FU. Computational modeling of the PyrR–5'-phosphoribosyl-α-1'-pyrophosphate (PRPP) complex revealed the location of several other mutations at the PRPP binding site of PyrR, indicating their probable role in resistance. Indeed, 5-FU-resistant strains harboring these mutations exhibited decreased susceptibility to 5-FU. Considering that pyrimidine analogs are predominantly regarded to inhibit PyrR, the present studies will be beneficial for the screening of appropriate inhibitors of PyrR and help provide insight into future TB drug design and development.

International audience; SETTING: Patients with suspected pulmonary tuberculosis (TB) visiting government TB diagnostic and treatment centres in Makassar City, South Sulawesi Province, Indonesia, from February to October 2008 were included in the study. OBJECTIVE: To determine the distribution of Mycobacterium tuberculosis genotypes in Makassar. DESIGN: Cross-sectional study. Spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and principal genetic grouping (PGG) were used to genotype the M. tuberculosis clinical isolates. RESULTS: Among 179 isolates derived from pulmonary TB patients, distribution of spoligotypes comprised the East Africa-Indian (30.2%), T (17.9%), H (12.3%) and Beijing (9.5%) lineages. Other lineages found in smaller proportions were the Latin American-Mediterranean, MANU, S and X lineages. Nineteen isolates (10.6%) could not be grouped into any of the reported lineages or shared types. Single nucleotide polymorphism analysis of katG(463) and gyrA(95) grouped these isolates primarily into PGG1 (9/19, 47%). CONCLUSION: Only a few genetically identical clustered isolates were identified within the 9-month study period, and most isolates were genetically diverse. Furthermore, 15 spoligopatterns identified in our study have not been reported previously. To our knowledge, this is the first comprehensive study describing genotypes of M. tuberculosis clinical isolates in Sulawesi.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

Background. Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n 5 862), particularly among US-born tuberculosis patients. Methods. Representative isolates were selected using population-based genotyping data from New York City and New Jersey. Growth and cytokine/chemokine response were measured in infected human monocytes. Survival was determined in aerosol-infected guinea pigs. Results. Multiple genotyping methods and phylogenetically informative synonymous single nucleotide polymorphisms showed that all strains were related by descent. In axenic culture, all strains grew similarly. However, infection of monocytes revealed 2 growth phenotypes, slower (doubling 55 hours) and faster (25 hours). The faster growing strains elicited more tumor necrosis factor a and interleukin 1b than the slower growing strains, even after heat killing, and caused accelerated death of infected guinea pigs (9 weeks vs 24 weeks) associated with increased lung inflammation/pathology. Epidemiologically, the faster growing strains were associated with human immunodeficiency virus and more limited in spread, possibly related to their inherent ability to induce a strong protective innate immune response in immune competent hosts. Conclusions. Natural variation, with detectable phenotypic changes, among closely related clinical isolates of M. tuberculosis may alter epidemiologic patterns in human populations.