Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Putative compensatory mutations in the rpoC gene of rifampin-resistant Mycobacterium tuberculosis are associated with ongoing transmission.

Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P < 0.01). Moreover, these putative compensatory mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P < 0.001). Our study supports a role for rpoC mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.

What do we mean by normal? The mathematical concept and social dimension of normality.

1. Normality has a mathematical definition as well as a social dimension. 2. It is essential to consider the social dimension when formulating care. 3. Concepts of normality differ according to an individual's life experiences, which are shaped by age, sex, gender, race, culture and social class. 4. Concepts of normality also vary in accordance with the group defining it--for example, the developers of social policy, the recipients of that policy and the rest of the society concerned.

A confident start to a new relationship. Biological and psychological aspects of breastfeeding.

1. The biological and psychological aspects of breastfeeding (along with the common problems) need to be considered if success is to occur. 2. Most mothers can breastfeed. 3. Mothers with severe anatomical abnormalities, who are HIV positive, have active TB or whose children have inherited mono- or disaccharide intolerances should not breastfeed. 4. It is important not to induce guilt (and sour the mother/child relationship) if a mother does not choose to breastfeed.

Management of asthma in pregnancy.

1. Approximately a third of mothers find their asthma improves during pregnancy, while one third stay the same and the remainder's asthma worsens. 2. Asthma needs to be controlled; if it is not, the fetus may receive insufficient oxygen. 3. The two main types of asthma arise from different causes and have different patterns of inheritance, although in many cases the originating stimulus is the same. 4. A polluted environment contributes to the production of allergens and irritants which trigger asthma in susceptible people.

Bringing purpose to life. The relationship between unemployment and health.

It is a common assumption that a person's mental and physical health will suffer during or even under the threat of a period of unemployment. It is important that community healthcare professionals can diagnose to what extent the trauma of unemployment has affected their patients. Only then can appropriate treatment be designed and implemented.

Greater understanding enhances control. Maintaining pharmacological control of asthma.

1. Asthma is a syndrome--not a disease--which affects the bronchi in the lungs. 2. Asthma is a cause of substantial ill-health, and economic loss (due to sick leave) to both clients and society. 3. Agreed systems of management (protocols) between client and practitioner may help clients gain control over their condition.

A force for the future. The changing role of the district nurse.

Current developments in community nursing mean the district nurse's role will inevitably be enhanced. To meet the challenge posed by greater responsibility, it is vital that training is expanded to meet new requirements.

A smooth start to a new age. Helping women cope with the menopause.

The menopause is a natural event in women's lives, but its associations with loss of fertility and its side-effects often lead to it being seen in a negative light. Nurses can help women control the side-effects so they may learn to see it as a positive change.

A preventable tragedy. The nurse's role in preventing coronary heart disease.

Heart disease is the biggest killer in the UK, yet simple changes in diet and lifestyle may vastly reduce people's risk. Nurses are ideally placed to advise on the subject.

Cervical cancer: a preventable problem.

Incidence of cervical cancer appears to be increasing among younger women. With effective screening, treatment and health education, it is almost entirely preventable.