Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv.

AIM: Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical beta-lactamases activity. METHODS AND RESULTS: Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit beta-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have beta-lactamase activity by the hydrolysis of nitrocefin and other beta-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of beta-lactam antibiotics. CONCLUSION: The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having beta-lactamase or beta-lactamase-like activity, giving wide spectrum of resistance against beta-lactams. SIGNIFICANCE AND IMPACT OF THE STUDY: Systematic study of hypothetical beta-lactamases of M. tuberculosis and related species and their correlation with beta-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.

Inhibition of vancomycin-resistant enterococci by an in vitro continuous-flow competitive exclusion culture containing human stool flora.

An in vitro anaerobic continuous-flow competitive exclusion (CFCE) culture model was used to study the ability of human stool flora to inhibit the growth of vancomycin-resistant (VR) enterococci (VRE). The CFCE culture was established from a stool sample obtained from a healthy adult. When 10(3) or 10(6) cfu/mL of VR Enterococcus faecium were added to the CFCE culture, the VRE were eliminated within 6 or 9 days, respectively. When 10(7) cfu/mL of the CFCE culture was added to a continuous-flow culture that contained 10(7) cfu/mL of VRE, the density of VRE was reduced but not eliminated. These data support the hypothesis that the indigenous intestinal flora inhibit growth of VRE and suggest that CFCE cultures may be a useful means to study interactions between the indigenous flora and VRE.

Effect of oral Bacillus coagulans administration on the density of vancomycin-resistant enterococci in the stool of colonized mice.

AIMS: A mouse model of vancomycin-resistant enterococcus (VRE) stool colonization was used to study the effect of Bacillus coagulans, a biotherapeutic agent, on the density of colonization. METHODS AND RESULTS: VRE-colonized mice received orally administered B. coagulans (107 cfu) or saline daily for four days. For one VRE strain, the density of VRE at one and four days after treatment was 1.4 log10cfu x g(-1) lower in experimental vs. control mice (P=0.03), and 35% of experimental vs. 0% of control mice had no detectable VRE four days after treatment (P=0.03). For two additional strains, there was no statistically significant reduction of VRE density in the B. coagulans groups. CONCLUSION: B. coagulans therapy reduced the density of colonization for one of three VRE strains tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests a potential role for biotherapeutic agents as a means to reduce the density of VRE intestinal colonization.

Behaviour of Zebu and crossbed cows in restricted suckling groups.

The aim of this study was to investigate whether Zebu (Bos indicus) and crossbred (Bos indicusxBos taurus) cows differ in maternal related behaviour during restricted group suckling. The behaviours of 20 Zebu and 16 crossbred cows were recorded during two 30min sessions each day after milking when their calves were present for suckling, for a time period of 2 weeks per month for 6 months postpartum. The main activity in both breeds during the 0.5h observation sessions was suckling. However, the two breeds differed significantly in several behavioural aspects. The Zebu cows had longer total duration of suckling than the crossbred cows, per 30min session, 11.8 and 9.4min, respectively. The separate suckling bouts were longer in the Zebu cows (2.8min in Zebu and 2.3min in crossbred) and this breed also had more suckling bouts than the crossbred per session, 3.8 and 3.2, respectively. Zebu cows stayed in close contact with their calves for longer time and directed more agonistic actions against alien calves and cows than the crossbred cows.

Influence of restricted suckling and level of feed supplementation on postpartum reproductive performance of zebu and crossbred cattle in the semi-arid tropics.

This study was carried out in central Tanzania on a group of 45 Zebu and 37 crossbred cows which were 4 to 10 years old. At calving time, the animals were allocated to one of the 4 treatment groups. In addition to free access to grazing for all cows in the study, in group H:AR (n = 18), cows were fed a high level of concentrate supplementation (4kg/day) and calves were artificially reared; in group H:RS (n = 24), cows were fed a high level of concentrate supplementation (4kg/day) and calves were only allowed restricted suckling up until the weaning age of 6 months. In group L:AR (n = 23) cows were fed a low level of concentrate supplementation (2kg/day) and calves were artificially reared; and in group L:RS (n = 17) cows were fed a low level of concentrate supplementation (2kg/day) and calves were only allowed restricted suckling up until the weaning age of 6 months. Milk progesterone was used as a means of determining the postpartum resumption interval (PRI) and the interval from parturition to conception (PCI). The overall PRI was 47.4 +/- 0.4 days and was significantly affected by breed but not by calving season, with crossbred cows exhibiting a shorter PRI than Zebu cows. The effect of the treatments was significant, with cows in the group H:AR displaying a significantly shorter PRI than those in the other groups, while cows in group L:RS showed a significantly longer PRI than those in the other groups. The overall PCI was 149.5 +/- 3.7 days, and was not significantly affected by breed or calving season. The effect of the treatments was significant, with cows in the group H:AR having a significantly shorter PCI than cows in the other groups, while cows in group L:RS showed a significantly longer PCI than those in the other groups. Crossbred cows had higher live weights at calving (299.4 kg) than Zebu cows (272.6 kg), while all cows gained weight during the first 3 months after calving. The treatments had a significant effect on weight gain, with cows in the group H:AR gaining significantly more weight than those in the other groups. Cows which had high live weights at calving exhibited significantly shorter PRI and PCI than the lighter cows. Animals which had gained more than 5 kg during the first month after calving, or which had gained more than 8 kg during the first 3 months after calving, showed significantly shorter PRI and PCI than cows which had gained less weight. The results show that the calf rearing system and the level of feed supplementation interact with each other and can influence the postpartum anoestrous period in Zebu and Zebu crossbred cattle. Increasing the level of nutrition in restricted suckling cows tended to improve the postpartum anoestrous period, but the positive effects of supplementation could not completely compensate for the negative effects of suckling.

Clonidine in prophylaxis of migraine.

Various drugs have been used in migraine to bring down the frequency of attacks. Recently clonidine hydrochloride, an antihypertensive agent, has been reported to be effective. The mechanism of action of this drug is not clear, but it is thought to act through its effect on tyramine metabolism. A double-blind cross-over trial using clonidine and identical-looking placebo tablets was carried out in 20 patients of migraine. There was no statistically significant difference between clonidine and the placebo in reducing the frequency of attacks, as evaluated by the non-parameteric test of Wilcoxon. No side effects of clonidine were observed.