Re-growth of Mycobacterium tuberculosis populations exposed to antibiotic combinations is due to the presence of isoniazid and not bacterial growth rate.

Modulation of growth rate in Mycobacterium tuberculosis is key to its survival in the host; particularly with regard to its adaptation during chronic infection when the growth rate is very slow. The resulting physiological changes will influence the way this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of frontline antimycobacterials, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), using continuous cultures. Time-course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (23.1. mean generation time (MGT)) or slow growth rate (69.3h MGT), were analysed with the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat, and profiled kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a higher kill rate associated with INH (compared to RIF or PZA) and the appearance of re-growth. In line with this observation, re-growth was not observed with RIF-exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate re-growth. We consider here that faster, early bactericidal activity is not what is required for successful sterilisation of M. tuberculosis, but instead slower elimination of bacilli followed by reduced recovery of the bacterial population.

The effect of growth rate on pyrazinamide activity in Mycobacterium tuberculosis - insights for early bactericidal activity?

BACKGROUND: Pyrazinamide (PZA) plays an essential part in the shortened six-month tuberculosis (TB) treatment course due to its activity against slow-growing and non-replicating organisms. We tested whether PZA preferentially targets slow growing cells of Mycobacterium tuberculosis that could be representative of bacteria that remain after the initial kill with isoniazid (INH), by observing the response of either slow growing or fast growing bacilli to differing concentrations of PZA. METHODS: M. tuberculosis H37Rv was grown in continuous culture at either a constant fast growth rate (Mean Generation Time (MGT) of 23.1 h) or slow growth rate (69.3 h MGT) at a controlled dissolved oxygen tension of 10 % and a controlled acidity at pH 6.3 ± 0.1. Cultures were exposed to step-wise increases in the concentration of PZA (25 to 500 µgml(-1)) every two MGTs, and bacterial survival was measured. PZA-induced global gene expression was explored for each increase in PZA-concentration, using DNA microarray. RESULTS: At a constant pH 6.3, actively dividing mycobacteria were susceptible to PZA, with similar responses to increasing concentrations of PZA at both growth rates. Three distinct phases of drug response could be distingished for both slow growing (69.3 h MGT) and fast growing (23.1 h MGT) bacilli. A bacteriostatic phase at a low concentration of PZA was followed by a recovery period in which the culture adapted to the presence of PZA and bacteria were actively dividing in steady-state. In contrast, there was a rapid loss of viability at bactericidal concentrations. There was a notable delay in the onset of the recovery period in quickly dividing cells compared with those dividing more slowly. Fast growers and slow growers adapted to PZA-exposure via very similar mechanisms; through reduced gene expression of tRNA, 50S, and 30S ribosomal proteins. CONCLUSIONS: PZA had an equivalent level of activity against fast growing and slow growing M. tuberculosis. At both growth rates drug-tolerance to sub-lethal concentrations may have been due to reduced expression of tRNA, 50S, and 30S ribosomal proteins. The findings from this study show that PZA has utility against more than one phenotypic sub-population of bacilli and could be re-assessed for its early bactericidal activity, in combination with other drugs, during TB treatment.

A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315.

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50-90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.

Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix.

A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP) M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease.

Application of continuous culture for measuring the effect of environmental stress on mutation frequency in Mycobacterium tuberculosis.

The ability of all pathogens to survive within the host is key to their success in establishing disease. Environmental conditions that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying the response of Mycobacterium tuberculosis (M. tuberculosis) exposed to these relevant host conditions in vitro will further increase our understanding of how these environments have an impact on the molecular mechanisms M. tuberculosis adopts to combat the effects of external influences such as antimycobacterials. The methods presented here are used to investigate the effect of environmental factors on the development of drug-resistant M. tuberculosis. Cultures grown under controlled conditions in continuous culture are sampled and the frequency with which resistant mutants develop are determined. These studies provide data that aid our understanding of the complex interaction between the host environment and invading bacterium that allow resistant strains to develop and continue to cause disease.

An integrated machine learning approach for predicting DosR-regulated genes in Mycobacterium tuberculosis.

BACKGROUND: DosR is an important regulator of the response to stress such as limited oxygen availability in Mycobacterium tuberculosis. Time course gene expression data enable us to dissect this response on the gene regulatory level. The mRNA expression profile of a regulator, however, is not necessarily a direct reflection of its activity. Knowing the transcription factor activity (TFA) can be exploited to predict novel target genes regulated by the same transcription factor. Various approaches have been proposed to reconstruct TFAs from gene expression data. Most of them capture only a first-order approximation to the complex transcriptional processes by assuming linear gene responses and linear dynamics in TFA, or ignore the temporal information in data from such systems. RESULTS: In this paper, we approach the problem of inferring dynamic hidden TFAs using Gaussian processes (GP). We are able to model dynamic TFAs and to account for both linear and nonlinear gene responses. To test the validity of the proposed approach, we reconstruct the hidden TFA of p53, a tumour suppressor activated by DNA damage, using published time course gene expression data. Our reconstructed TFA is closer to the experimentally determined profile of p53 concentration than that from the original study. We then apply the model to time course gene expression data obtained from chemostat cultures of M. tuberculosis under reduced oxygen availability. After estimation of the TFA of DosR based on a number of known target genes using the GP model, we predict novel DosR-regulated genes: the parameters of the model are interpreted as relevance parameters indicating an existing functional relationship between TFA and gene expression. We further improve the prediction by integrating promoter sequence information in a logistic regression model. Apart from the documented DosR-regulated genes, our prediction yields ten novel genes under direct control of DosR. CONCLUSIONS: Chemostat cultures are an ideal experimental system for controlling noise and variability when monitoring the response of bacterial organisms such as M. tuberculosis to finely controlled changes in culture conditions and available metabolites. Nonlinear hidden TFA dynamics of regulators can be reconstructed remarkably well with Gaussian processes from such data. Moreover, estimated parameters of the GP can be used to assess whether a gene is controlled by the reconstructed TFA or not. It is straightforward to combine these parameters with further information, such as the presence of binding motifs, to increase prediction accuracy.

Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH.

OBJECTIVES: The aim of this study was to gain an insight into the molecular mechanisms of the evolution of rifampicin resistance in response to controlled changes in the environment. METHODS: We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2. RESULTS: The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10(-8) and 8.9 x 10(-8); however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12. CONCLUSIONS: The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive.

Continuous culture of mycobacteria.

Batch cultures have predominately been used for the study of physiology and gene expression in mycobacteria. This chapter describes the assembly of chemostats and the methodology that is being used for growing Mycobacterium tuberculosis in continuous culture, which provides the greatest control over experimental conditions. It is difficult to determine the underlying genetic changes that enable M. tuberculosis to adapt to the host environment, but in vitro experiments aid the interpretation of gene expression profiles of the bacillus in vivo. Selecting relevant host conditions for study presents a major challenge. Oxygen availability has been identified as an important environmental stimulus and is a simple parameter to adjust and monitor. Described here are continuous culture methods to determine the response of M. tuberculosis to low oxygen environments.

A Bayesian Change point model for differential gene expression patterns of the DosR regulon of Mycobacterium tuberculosis.

BACKGROUND: Low oxygen availability has been shown previously to stimulate M. tuberculosis to establish non-replicative persistence in vitro. The two component sensor/regulator dosRS is a major mediator in the transcriptional response of M. tuberculosis to hypoxia and controls a regulon of approximately 50 genes that are induced under this condition. The aim of this study was to determine whether the induction of the entire DosR regulon is triggered as a synchronous event or if induction can unfold as a cascade of events as the differential expression of subsets of genes is stimulated by different oxygen availabilities. RESULTS: A novel aspect of our work is the use of chemostat cultures of M. tuberculosis which allowed us to control environmental conditions very tightly. We exposed M. tuberculosis to a sudden drop in oxygen availability in chemostat culture and studied the transcriptional response of the organism during the transition from a high oxygen level (10% dissolved oxygen tension or DOT) to a low oxygen level (0.2% DOT) using DNA microarrays. We developed a Bayesian change point analysis method that enabled us to detect subtle shifts in the timing of gene induction. It results in probabilities of a change in gene expression at certain time points. A computational analysis of potential binding sites upstream of the DosR-controlled genes shows how the transcriptional responses of these genes are influenced by the affinity of these binding sites to DosR. Our study also indicates that a subgroup of DosR-controlled genes is regulated indirectly. CONCLUSION: The majority of the dosR-dependent genes were up-regulated at 0.2% DOT, which confirms previous findings that these genes are triggered by hypoxic environments. However, our change point analysis also highlights genes which were up-regulated earlier at levels of about 8% DOT indicating that they respond to small fluctuations in oxygen availability. Our analysis shows that there are pairs of divergent genes where one gene in the pair is up-regulated before the other, presumably for a flexible response to a constantly changing environment in the host.

Comparative transcriptomics reveals key gene expression differences between the human and bovine pathogens of the Mycobacterium tuberculosis complex.

Members of the Mycobacterium tuberculosis complex show distinct host preferences, yet the molecular basis for this tropism is unknown. Comparison of the M. tuberculosis and Mycobacterium bovis genome sequences revealed no unique genes in the bovine pathogen per se, indicating that differences in gene expression may play a significant role in host predilection. To define the key gene expression differences between M. tuberculosis and M. bovis we have performed transcriptome analyses of cultures grown under steady-state conditions in a chemostat. This revealed that the human and bovine pathogens show differential expression of genes encoding a range of functions, including cell wall and secreted proteins, transcriptional regulators, PE/PPE proteins, lipid metabolism and toxin-antitoxin pairs. Furthermore, we probed the gene expression response of M. tuberculosis and M. bovis to an acid-shock perturbation which triggered a notably different expression response in the two strains. Through these approaches we have defined a core gene set that shows differential expression between the human and bovine tubercle bacilli, and the biological implications are discussed.

Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester.

The low level of available iron in vivo is a major obstacle for microbial pathogens and is a stimulus for the expression of virulence genes. In this study, Mycobacterium tuberculosis H37Rv was grown aerobically in the presence of limited iron availability in chemostat culture to determine the physiological response of the organism to iron-limitation. A previously unidentified wax ester accumulated under iron-limited growth, and changes in the abundance of triacylglycerol and menaquinone were also observed between iron-replete and iron-limited chemostat cultures. DNA microarray analysis revealed differential expression of genes involved in glycerolipid metabolism and isoprenoid quinone biosynthesis, providing some insight into the underlying genetic changes that correlate with cell-wall lipid profiles of M. tuberculosis growing in an iron-limited environment.

An assay to compare the infectivity of Mycobacterium tuberculosis isolates based on aerosol infection of guinea pigs and assessment of bacteriology.

The aim of this study was to establish an assay to compare Mycobacterium tuberculosis strains, and cells grown under different growth conditions, in terms of their ability to cause a lung infection and disseminate to the spleen. M. tuberculosis strains H37Rv, Erdman, South Indian (TMC120, SI) and H37Rv cells grown aerobically or under low oxygen/iron limitation in a chemostat were assayed for infectivity. Groups of 8 animals were challenged with 3 different doses of each strain. Lung and spleen bacteriology was assessed at 16 days post-infection for all strains. Bacteriology and lung pathology at day 56 was studied for H37Rv, Erdman and SI. Strains H37Rv and Erdman had a statistically significantly higher pathogenic potential than SI and this was confirmed by analysis of lung pathology performed at 8 weeks post-infection, although the Erdman strain caused more extensive caseation without calcification and little encapsulation. The model could discriminate between cells grown under different growth conditions; low-oxygen/iron-limited cells had a significantly higher infectivity than those grown aerobically. This study presents a quick and reliable method for comparing with statistical confidence, the pathogenic potential of M. tuberculosis strains and the impact of in vitro growth conditions on the infectivity of M. tuberculosis in vivo.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.