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.

Adrenomedullary responses to acute and chronic ethanol administration to rats.

The variations in levels of adrenal dopamine (DA), noradrenaline (NA) and adrenaline (A) after acute and chronic ethanol administration have been studied in rats. A relatively moderate dose of ethanol (2 g/kg) induced significant increases in DA levels, while NA and A concentrations did not change, or decreased depending on the interval of time elapsed after ethanol injection. These findings, together with those obtained in rats pretreated with alpha-methyl-p-tyrosine methyl ester (AMT), indicate an increased turnover of adrenal catecholamines (CA) after acute ethanol treatment. Chronic ethanol intake leads to significant increases in DA levels in the adrenal glands of rats subjected to ethanol feeding for 12 and 16 days; no changes were observed in NA or A concentrations in these groups of animals. After 30 days of ethanol ingestion, the levels of the three CA are within the control range, a fact that could suggest some adaptation of the sympatho-adrenal system to ethanol. After 16 days of treatment, tolerance to acute effects of ethanol on adrenomedullary system was not clear.

Effect of naloxone administration upon responses of adrenal hormones to withdrawal from ethanol.

Rats maintained on an ethanol-liquid diet developed physical dependence after 16 days. Activation of adrenocortical function and overactivity of the sympathoadrenal system were observed during withdrawal from ethanol. The opiate antagonist naloxone prevented the adrenomedullary response, and attenuated, though not significantly, the increases in serum corticosterone induced by ethanol deprivation. These findings suggest that endogenous opioid pathways may be involved in the ethanol-withdrawal syndrome.

Effects of adrenaline on the acquisition and maintenance of ethanol preference in a taste conditioning paradigm.

The effects of subcutaneous adrenaline administration on preference for ethanol (2.5% solution) have been investigated, using a two-bottle choice situation. Administration of the amine (50 micrograms/kg) immediately after the conditioning session significantly attenuated ethanol preference. Adrenaline treatment (10, 50 or 100 micrograms/kg) prior to the first retention test induced a significant reduction in ethanol preference. When the amine was injected prior to conditioning only the dose of 100 micrograms/kg reduced later ethanol preference. Our results indicate that systemically administered adrenaline impairs the acquisition of preference to a weak ethanol solution. It is suggested that this effect of the amine may be linked to interference with consolidation of memory and retrieval processes.

Adrenocortical response to acute and chronic ethanol administration in rats.

Acute ethanol administration (2 g/kg IP) induced a significant rise in serum corticosterone levels which seemed to be related to blood ethanol concentration. Chronic ethanol administration, in the form of a liquid diet for 16 or 30 days, did not alter the levels of serum corticosterone. Chronic treatment of rats with a liquid diet containing ethanol resulted in the development of tolerance.

The effects of acute and chronic administration of morphine on the turnover of brain and adrenal catecholamines in rats.

Brain and adrenal catecholamine turnover in adult female rats treated with morphine was investigated. A different time course response of brain and adrenal catecholamines to alpha-methyl-p-tyrosine methyl-ester (AMT) administration in normal rats was observed; the catecholamine turnover rate in adrenal glands appeared to be much slower than in the brain. Acute morphine increased the turnover of brain dopamine and noradrenaline as well as of adrenal catecholamines, whereas chronic morphine treatment induced a decrease in the turnover of brain noradrenaline. Withdrawal induced by nalorphine produced an increase in the utilization of brain noradrenaline and adrenal catecholamines; this effect could be related to the withdrawl stress situation induced by the opiate antagonist. Although the mechanism of morphine action may implicate other neurotransmitters besides catecholamines, our results contribute to evidence that brain and adrenal catecholamines could be involved in the mechanism of morphine tolerance and/or dependence.

Chronic administration of morphine in cats: effects on adrenal and urinary catecholamines.

Adrenaline and noradrenaline levels in the adrenal glands and the excretion of both bioamines in urine of adult cats were investigated after chronic administration of morphine and nalorphine-induced withdrawal. After 7 days of daily consecutive morphine treatment, a significant increase in the adrenal noradrenaline content and a drop in adrenaline content were observed. After 2 weeks of daily injection of morphine, no significant changes were observed in the adrenal catecholamine level. One month of treatment with the opioid caused a significant increase in the adrenal content of both adrenaline and noradrenaline. Urinary excretion of catecholamines was significantly increased during the 4 weeks of treatment. In animals subjected to spontaneous or induced withdrawal with nalorphine, the adrenal content of catecholamines was altered and the ratio adrenaline/noradrenaline in the adrenal gland was shifted towards noradrenaline. A first injection of morphine produced an excitant manic response characterized by hyperexcitement and aggressive behaviour; animals chronically treated with the drug showed a progressively diminished response to this effect of the drug. It is concluded that physical dependence on morphine is reached by cats chronically treated with morphine and that this effect of the drug influences adrenomedulllary function in a different fashion depending on the stage of morphine treatment.

Adrenal, plasma and urinary corticosteroids during single or repeated administration of morphine in cats.

Corticosteroids and ascorbic acid in the adrenal glands of adult cats have been investigated after single or repeated administration of morphine. Also plasma levels and urinary excretion of corticosteroids were determined. A significant increase in the content of corticosteroids in the glands and plasma was found after initial injection of morphine. After 7 days of consecutive morphine treatment a fall of corticosteroids in the glands was observed; after 2 weeks of daily injections the content of adrenal corticosteroids was significantly lower than in the control animals but no change was found in the plasma. Administration of the drug during 1 month led to a highly significant decrease of corticosteroids in the glands as well as in plasma. No significant change in adrenal ascorbic acids was found whether the adrenal corticosteroids were higher or lower than in the control cats. Urinary corticosteroids were high during the first week of morphine treatment but thereafter the excretion declined progressively and was lower than the control level after 13 days of treatment. The significant decrease of corticosteroids observed after repeated administration of morphine and the rise in adrenal corticosteroids found after the injection of nalorphine to the morphinized animals suggest that some kind of morphine dependence had been developed in the cats after repeated administration of the drug.

Brain catecholamines and adrenocortical activation by morphine in the cat.

Following the administration of 250 mg/kg of alpha-methyl-p-tyrosine (H 44/68) to adult cats, an increase in plasma cortisol was found before any decrease in the contents of noradrenaline in the diencephalon could be observed; this suggests that the induced release of ACTH by H 44/68 administration may be independent of the synthesis impairment of catecholamines. The results observed in cats treated with H 44/68 plus morphine, suggest that the opioid causes an increase in the turnover of noradrenaline. Although different mechanisms for the action of morphine on the pituitary-adrenal system and on brain catecholamines cannot be ruled out, the increased secretion of ACTH induced by morphine could be related to the increased turnover of noradrenaline observed in this study.

Modifications in adrenal hormones response to ethanol by prior ethanol dependence.

Ethanol was administered to rats by means of a liquid diet for 16 days; after an ethanol-free interval of four weeks, animals received a test (IP) dose of ethanol (2 g/kg), and the adrenocortical and adrenomedullary responses were evaluated. Chronically ethanol-exposed animals showed tolerance to the stimulatory effect of ethanol in the pituitary-adrenal axis. Likewise, previously dependent rats showed tolerance to the increase in the activity of the adrenomedullary function induced by acute administration of the drug. Our results indicate that chronic ethanol ingestion can induce persistent changes after complete alcohol abstinence.

Pituitary-adrenal responses to sub-chronic treatment with phenobarbital and/or phenytoin (diphenylhydantoin) in rats.

The response of the pituitary-adrenal axis of the male rat to sub-chronic dose treatment with phenobarbital and/or phenytoin under basal and stress conditions was investigated. Plasma corticosterone levels were measured in rats sacrificed either in the morning or in the afternoon, subjected or not to 2 hours of immobilization stress. Phenobarbital did not seem to significantly affect the pituitary-adrenal activity under basal conditions or in the response to stress. Phenytoin induced a disruption of the corticosterone diurnal variation present in the rat under basal conditions and seemed to partially inhibit the pituitary-adrenal response to stress when applied in the morning. The combined treatment with phenobarbital and phenytoin affected the afternoon rise in corticosterone levels present under basal conditions, as well as stress response at the same time of the day. The reported results agree with the hypothesis about the existence of mechanisms controlling ACTH release under basal conditions, dissociable from those controlling ACTH release in response to stress situations, and that phenytoin could influence some or others differently, depending on the animal's endocrine situation.

Effects of morphine upon the pituitary-adrenal system and adrenal catecholamines: a comparative study in cats and rats.

The aim of the present study was to investigate the effects of acute and chronic administration of morphine upon the pituitary-adrenal activity and adrenal catecholamines in rats and cats, two animal species with very different behavioural patterns of response to the opiate. Acute administration of the drug induced in both animal species an activation of the pituitary-adrenal system. Chronic administration of morphine to cats and rats induced a depression in the pituitary-adrenal function. No significant changes in the adrenal levels of catecholamines were observed in rats treated chronically with the drug. However, in the cat, the effects of morphine on adrenomedullary function seemed to depend on the stage of morphine treatment. The behavioural patterns of response in both animal species during chronic administration of the opiate, as well as the effects of induced withdrawal with nalorphine (an antagonist of morphine), indicated that dependence on morphine had developed, not only in the rats, but also in the cats. Acute morphine administration had a sedative effect, while in the cats the opiate produced a species-specific manic response characterized by hyperexcitement and aggressive behavior.

Genetic diversity of Mycobacterium tuberculosis in Madang, Papua New Guinea.

SETTING: Madang and surroundings, Papua New Guinea (PNG). OBJECTIVE: To characterise the genetic diversity and drug susceptibility of Mycobacterium tuberculosis isolates collected in Madang and surroundings. DESIGN: M. tuberculosis was isolated from sputum samples from active pulmonary tuberculosis cases. Drug resistance profiles were obtained by drug susceptibility testing. M. tuberculosis lineages were identified by single nucleotide polymorphisms and sub-typing was performed by spoligotyping. Spoligotyping and 24 locus mycobacterial interspersed repetitive units-variable number of tandem repeats were combined to identify clustered isolates. RESULTS: The 173 M. tuberculosis isolates collected belonged predominantly to the Euro-American lineage (Lineage 4) and the East-Asian lineage (Lineage 2). Multidrug-resistant M. tuberculosis were observed in 5.2% of isolates. Lineage 2 M. tuberculosis, which includes the 'Beijing' genotype, was significantly associated with any drug resistance (OR 5.2, 95%CI 1.8-15.1). Cluster analyses showed 44% molecularly clustered isolates, suggesting transmission of M. tuberculosis in the community, including transmission of primary drug-resistant M. tuberculosis. CONCLUSION: These data provide the first insight into the molecular characteristics of M. tuberculosis in the Madang area of PNG, and indicate substantial drug resistance with evidence of ongoing transmission.

Strain diversity, epistasis and the evolution of drug resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis harbours little DNA sequence diversity compared with other bacteria. However, there is mounting evidence that strain-to-strain variation in this organism has been underestimated. We review our current understanding of the genetic diversity among M. tuberculosis clinical strains and discuss the relevance of this diversity for the ongoing global epidemics of drug-resistant tuberculosis. Based on findings in other bacteria, we propose that epistatic interactions between pre-existing differences in strain genetic background, acquired drug-resistance-conferring mutations and compensatory changes could play a role in the emergence and spread of drug-resistant M. tuberculosis.

Tuberculosis transmission patterns among Spanish-born and foreign-born populations in the city of Barcelona.

During a 2-year period (2003-2004), tuberculosis (TB) transmission in Barcelona and the factors related to transmission among the Spanish- and foreign-born populations were studied by molecular epidemiology. Data were obtained from TB cases and Conventional Contact Tracing registries and genotyping was performed using restriction fragment length polymorphism (RFLP)-IS6110 and MIRU12 as a secondary typing method. Of the 892 TB cases reported, 583 (65.3%) corresponded to Spanish-born and 309 (34.6%) to foreign-born. Six hundred and eighty-seven cases (77%) were confirmed by culture. RFLP typing of 463/687 (67.4%) isolates was performed, revealing 280 (60.5%) unique and 183 (39.5%) shared patterns, which were grouped into 65 clusters. Spanish-born individuals were significantly more clustered than foreign-born individuals (44.6% vs. 28.8%; p 0.016). Clustering in foreign-born individuals was associated with HIV (p 0.051, odds ratio = 3.1, 95% confidence interval 1-10.9) and alcohol abuse (p 0.022), whereas, in the Spanish-born individuals, clustering was associated with age in the range 21-50 years, (p 0.024). Of the total clusters, 36/65 (55.3%) included only Spanish-born patients, whereas 22/65 (33.8%) included individuals from both populations. In mixed clusters, the index case was Spanish-born in 53% and foreign-born in 47%. Among the foreign-born, 2.8% were ill on arrival, 30% developed TB within the first year and 50.3% developed TB within the first 2 years; 58.3% were from South America. In conclusion, half of the foreign-born TB patients developed the disease during the first 2 years after arrival, which, in most cases, was the result of endogenous reactivation. Recent TB transmission among Spanish-born and foreign-born populations, as well as bidirectional transmission between communities, contributed significantly to the burden of TB in Barcelona, suggesting the need to improve Public Health interventions in both populations.

Infectiousness, reproductive fitness and evolution of drug-resistant Mycobacterium tuberculosis.

Mathematical models predict that the future of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) epidemic will depend to a large extent on the transmission efficiency or relative fitness of drug-resistant Mycobacterium tuberculosis compared to drug-susceptible strains. Molecular epidemiological studies comparing the spread of drug-resistant to that of drug-susceptible strains have yielded conflicting results: MDR strains can be up to 10 times more or 10 times less transmissible than pan-susceptible strains. Experimental work performed with model organisms has highlighted a level of complexity in the biology of bacterial drug resistance that is generally not considered during standard epidemiological studies of TB transmission. Recent experimental studies in M. tuberculosis indicate that drug resistance in this organism could be equally complex. For example, the relative fitness of drug-resistant strains of M. tuberculosis can be influenced by the specific drug resistance-conferring mutation and strain genetic background. Furthermore, compensatory evolution, which has been shown to mitigate the fitness defects associated with drug resistance in other bacteria, could be an important factor in the emergence and spread of drug-resistant M. tuberculosis. However, much more work is needed to understand the detailed molecular mechanisms and evolutionary forces that drive drug resistance in this pathogen. Such increased knowledge will allow for better epidemiological predictions and assist in the development of new tools and strategies to fight drug-resistant TB.