A Trial of Improving Antituberculous Regimens inMice Model of Tuberculosis

Hussein A. Abdul Hussein
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Keywords : A Trial of Improving,Antituberculous,Regimens inMice,Model of Tuberculosis
Medical Journal of Babylon  6:1 , 2014 doi:1812-156X-6-1
Published :15 June 2014

Abstract

Dormancy is a mycobacterial property of persistence in a viable nonreplicating form. It stands for a real community health threat because of its contribution to drug resistance and recurrence of tuberculosis. In order to arrange for a trial of solving this problem, the basic mechanisms by which mycobacterium become dormant were considered in order to partially reverse these factors. Factors that induce partial reactivation from dormancy include: presence of oxygen free radicals and Lalanine nutrient supply. For evaluating antituberculous regimens that include the test agents added to the conventional rifampicin plus INH, both in vitro (MIC) and in vivo (mice) models were used. Two fold serial dilution of modified Lwenstein-Jensen medium have showed equipotent inhibitory effect of metranidazole, L-alanine and metronidazole plus ginsenoside when each added to rifampicin plus INH but those were significantly more potent than rifampicin plus INH only regimen; P< 0.05 at intial concentrations of 4 microgram/ ml for each except ginsenoside 8 microgram/ ml. In vivo assessment include dividing 36 mice into 6 groups 5 of them were induced with tuberculosis by inoculation of subcutaneous aspect of left abdominal side with 5000 CFU of M. tuberculosis and monitored for temperature, nodule diameter and intramacrophage mycobacteria count in histopathological analysis along 4 weeks of treatment course. Regimen that included metronidazole plus ginsenoside added to rifampicin plus INH had the more potent antituberculous activity in comparism with other regimens in regard to prevention of increase in body temperature and decreasing nodule diameter from 7 mm to 2 mm whereas nodular intramacrophage bacilli were just 4 bacilli in comparison with untreated mean 8 3. Other regimens came second in potency and were significant in comparism with rifampicin plus INH only regimen. From the overall results, there was a significant augmentation of antituberculous potency upon adding some dormancy reversal metronidazole and L-alanine that could be a promising future regimen for eradicating dormant persistant tuberculosis.

Introduction

many microorganisms exert a considerable threat for community due to their variable virulence factors. One of the most obstacling virulence factors is their abstinence from taking antimicrobials owing to their cell signal directed metabolic inactivitation, a phenomenon called dormant state [1]. The most common pathogenic microorganisms that reveal dormancy are Mycobacteria, Salmonellae, Brucellae and some viral infections. Dormancy is a major contributor of 1- drug resistance by these microorganisms[2] 2- mandation of prolonged antimicrobial course 3- reactivation of the same disease after cessation of acute phase [3] 4- necessitation of multiple drugs regimens to avoid resistance. Variable molecular mechanisms can mediate microbial dormancy , these include 1- presence of toxic environmental factors like immune cytokines secreted by innate cells; macrophages, dentritic cells, NK and phagocytes examples of these cytokines are IL1, IL12, CC, Igs, INF lysosomal proteases and phospholipases in addition to those secreted by T and B lyphocytes and free radicals[4] 2- Host tissue PH 3- Lack of vital requirements for microbial survival [5] like hypoxia [6] or low CO2 tension whether as a disease condition like fibrosed, granulomatous or caseous tuberclous center or tissue specific relative low CO2 tension that explains a diminished incidence of development of tuberculosis in lower aerated organs like kidneys and higher incidence in right upper lung. Another important vital factor for mycobacteria growth and replication are the nutrients and energy sources including amino acids, cofactors, sugar and carboxylic acids [5]. A trial of partial reactivation of mycobacteria by provision of oxygen free radicals could theoretically overcome drug abstinence behavior and hence improves outcome of antituberculous regimens [7], however free radicals could be scavenged by different electron scavengers and reducing agent as antistress factors that can regulate phases of partial reactivation of these microorganisms. Animal model of M. tuberculosis is very important for surveillance and assessment of new strategies of treating this community threatening disease. From the most commonly used animals are mice which are considered to be a reliable model of monitoring experimental tuberculosis [8,9].

Materials and methods

The used materials
Test agents
1- Metronidazole vial (500 mg;
Kimadia. Jordan)
2- L- alanine powder (8 mg;
Chemika-Fulka; Germany)
3- Gensinoside tablet (200 mg;
Gensing; Canada)
Antituberculous agents
1- Rifampicin cap (300 mg; Ajanta;
India)
2- INH tablet (100 mg; Ajant; India)
Staining with Ziehl-Neelsen stain.
Culture media
1- Brain-Heart nutrient medium as
transport medium
2- Lowenstein-Jensen medium
Racks for MIC of 2 fold dilution of
blank solvents with modified
Lowenstein-Jensen medium [10].
Instrument
1- light microscope 2- Sensitive
thermotransducer 3- physiograph
4- Dissecting set 5- incubator.
In vitro methods of assessing
antimycobacterial potency.
Mycobacterium tuberculosis was
requested from the Institute of
Respiratory Diseases where it had
been identified and tested for
rifampicin and INH susceptibility. It
had been transported in a brain-heart
medium to be cultured and incubated
in Lowenstein-Jensen medium for
cultivation and preparing of a unique
subcutaneous inoculum of 5000 CFU
to be injected subcutaneously into the
mice.
Mice Model of Tuberculosis
Thirty-six mice aging 2-3 months
with 25-30 grams average body
weights of both sexes were given a
standard oxoid diet and water ad
libitum. They were bred in standard
breeding cages under a specially care
and monitoring. They were divided
into 6 groups:
Healthy group; N = 6 were injected
0.1 ml of transport media (lacking M.
tuberculosis) subcutaneously at the
left side of the abdomen as a healthy
control group. The same procedure
done for the following groups except
for the content of injection and
treatment.
Tuberculosis induced groups include:
Induced untreated; N = 6 injected
with 0.1 ml of normal saline solution
contains (5000 CFU) Mycobacterium
tuberculosis subcutaneously at left
lower abdominal aspect. These mice
were given 0.5 ml distilled water
orally daily for 4 weeks as a
controlled untreated
Control treated group N = 6 were
given 1mg rifampicin + 1mg INH in
0.5 ml distilled water orally daily for
4 weeks as a controlled treated group
Test regimen 1 group N = 6 were
given 1mg rifampicin + 1mg INH +
2mg metronidazole in 0.5 ml distilled
water orally for 4 weeks.
Test regimen 2 group N = 6 were
given 1mg rifampicin + 1mg INH +
2mg metronidazole in addition to
2mg gensinoside in 0.5 ml distilled
water orally for 4 weeks.
Test regimen 3 group N = 6 were
given 1mg rifampicin + 1mg INH in
addition to 2mg L-alanine powder in 0.5 ml distilled water orally for 4
weeks.
For all groups, sublingual
temperature was weekly measured
with a sensitive thermotransducer in
addition to repeated measuring of the
lesion diameter weekly for 4 weeks
after which period the autopsy
histopathologic examination of the
induced tuberculous nodule was done
with assessing the severity of the
lesion after treatment.


Results

Combining metronidazole 4 micrograms/ml plus 4microgram/ml gensinoside with 4 microgram/ml rifampicin and 4 microgram/ml INH had the same mycobacterium inhibitory potency to the regimens that include adding either 4 microgram/ ml metronidazole or 8 microgram/ml L-alanine to rifampicin plus INH in the 2 fold diluted modified Lowenstein-Jensen medium at the 3rd dilution (0.5 microgram/ml for each) in comparison with rifampicin plus INH only treatment :MIC at 1st dilution (2 microgram rifampicin and 2 microgram INH) There were no significant changes between different regimens, however, it was obvious that antituberulous drugs prevent progressive rise in temperature noticed with the untreated group, measured as a cumulative correlation coefficient R at P<0.05. The most potent improving activity in the nodule size was obtained by combining both metronidazole and ginsenoside with rifampicin plus INH: r = 0.988 at P< 0.05. This combination caused a reduction of nodular size from 7 mm to 2 mm at the end of the treatment course whereas a clear progressive increase in size if untreated.Adding metronidazole or L-alanine to rifampicin plus INH caused significant decrease in intramacrophage bacilli in comparison to rifampicin plus INH only; P< 0.05 after the end of treatment course.

Discussions

Dormancy is a dangerous bacterial response that despite avoidance of serious acute proliferation, microorganisms like mycobacteria remain viable but inactive for years or even decades which endangers human life upon reactivation by any reason for example immune compromisation, malnutrition, immune deficiency and iatrogenic immunosuppression [11]. However, dormancy process makes reactivated M.tuberculosis even more virulent due to abstinence from antimicrobial intake by these bacteria giving them a further chance of mutation and escape from antibacterial activity [12]. This property necessitates a prolonged treatment course with multiple antimicrobial regimens that means more toxicity and less patient compliance. On the other hand, M. tuberculosis usually resides in a thick inflammatory caseous or granulomatous center that cause diminished drug distribution kinetics for tuberculous center which will further predispose for mycobacterial resistance [13]. A close monitoring of M. tuberculosis pathogenesis gives a clue regarding the mechanisms by which dormancy could occur. Different environmental factors such as immune system elements including phagocytes, lymphocytes and their secretions in addition to mycobacterial surrounding PH, O2 tension, free radicals and nutrients are the triggering factors for [3,4] enhancing signal directed dormant state. A partial reversing of one or more of these factors will theoretically overcome antimicrobial abstinence by these microorganisms. In this current study, a trial of provision of oxygen free radicals with metronidazole was assessed; on the other hand, addition of M. tuberculosis nutrients factors such as L-alanine amino acid was also monitored. These groups were controlled by standard treatment with INH and rifampicin with and without gensinoside supplement group as an electron scavenger factor [14] in form of alternating administration with oxygen free radicals inducer metronidazole. Tuberculosis model included an induction of subcutaneous isolated, identified and cultivated M. tuberculosis to the left lateral aspect of abdomen of the mice with constitutional clinical signs and histopathological follow up of the lesion. In vitro assessment of inhibitory potency for the test regimens with MIC; table 1. revealed that combining metronidazole 4 micrograms/ml plus 4microgram/ml gensinoside with 4microgram/ml rifampicin and 4 microgram/ml INH had the same mycobacterium inhibitory potency to the regimens that include adding either 4 microgram/ ml metronidazole or 8 microgram/ml L-alanine to rifampicin plus INH in the 2 fold diluted modified Lowenstein-Jensen medium at the 3rd dilution (0.5 microgram/ml for each) in comparison with rifampicin plus INH only treatment :MIC at 1st dilution (2 microgram rifampicin and 2 microgram INH). That means there was no influence by the gensinoside as a direct antimycobacterial agent although gensinoside intensified the anti-tuberculous activity in the mice model. So, in vivo activity of gensinoside may include modulation of mice immune response against tuberculosis. One study regarding assessment of MIC values of rifampicin and INH revealed mycobacterial inhibitoy concentrations that approximate findings of this study: at 1 micrograms/ml for each [15]. In regard to L-alanine, antimycobacterial potentiation is attributed according to in vivo findings that alanine induces resuscitation of the dormant M. tuberculosis owing to activation of bacterial cell wall enzymes including alanine dehydrogenase. This process could render the bacteria more susceptible to INH inhibitory effects [16, 17]. Concerning the constitutional evaluation of the sublingual temperature in figure 1, all antituberculous regimens showed the same antipyretic effect with nonsignificant variations in between groupd although it was expected that group contains gensinoside will cause obvious and more potent reduction of mice temperature due to its scavenger activity [14], however, this similarity may be explained by the rapid and potent cidal activity of the applied regimens as compared with the control at P<0.05. The cumulative tuberculous nodule reducing effect was also evaluated; figure 2. It was highly significant upon combining metronidazole and ginsenoside with rifampicin and INH regimen which had reduced nodule diameter from 7 mm +/- 2 mm at ist week to 2 mm +/- 1mm at the end of treatment that was even more potent than other regimens, R = 0.988, P< 0.05. Similar findings were obtained upon estimating the mean intramacrophage tubercle bacilli as an indicator for the killing activity of both macrophages and drugs; figure 3 so that adding metronidazole alone or with ginsenoside to rifampicin plus INH or adding L-alanine to rifampicin plus INH had approximately the same activity, however their potency was significantly more than rifampicin plus INH only regimen at P< 0.05. Many studies have assessed the intramacrophage mycobacteria as a reliable parameter to assess antituberculous activity for test drugs like clarithromycin and gatifloxacin [18]. Metronidazole will exert oxygen free radicals toxic to maycobacterial bacilli, an effect which is under trial for arranging for eradication of tuberculosis [19]. Whereas potentiation of antimycobacterial activity by adding alanine could be attributed to ability of alanine to enhance a partial reactivation of mycobacterial cell wall synthesis a step of triggering reactivation of the dormant bacteria by inducing the enzyme alanine dehydrogenase [20].

Conclusions

There was a promising tuberculosis eradicating activity obtained from adding factors that can partially reverse the dormant state like metronidazole and L-alanine.

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