Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli.

The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli.     

A rapid method for viability and drug sensitivity of Mycobacterium leprae cultured in macrophages and using fluorescein diacetate

The ability of viable M. leprae to hydrolyze Fluorescein diacetate and retain fluorescein inside the bacteria was used to identify viable M. leprae inside the cultured in vitro macrophages. The subjective microscopic count of the FDA test was demonstrated as useful routine test by confirming the results obtained therein with a quantitative and non subjective measurement of fluorescence in spectrofluorimeter. Using this method loss of viability of M. leprae in presence of dapsone and rifampicin was demonstrated. Such an assay, was well correlated with another in vitro assay, the Fc receptor test and also the in vivo mouse foot test. The drug resistance of clinical isolates of M. leprae demonstrated by mouse foot pad was also correlated with FDA test system. Thus we have reported a reliable, consistent and rapid in vitro test system for determining viability and drug sensitivity of M. leprae.     

The mouse foot-pad technique for cultivation of Mycobacterium leprae

Although multiplication of Mycobacterium leprae in the foot pads of immune-competent mice is limited, and no leprosy-like lesions are produced in these animals, the mouse foot-pad system represents the first truly useful and reproducible animal model of M. leprae infection. Its employment has enabled research into basic questions with respect to the microbiology of M. leprae, and the epidemiology, treatment and control of leprosy. The mouse foot-pad technique is labour-intensive and time-consuming, and is expensive in terms of the costs of animal purchase and maintenance. In addition, the technique appears to be rather imprecise and insensitive, compared with the techniques employed in working with cultivable micro-organisms. For these reasons, and also as a by-product of the success of multi-drug therapy, the technique has been abandoned in many research centres. Nevertheless, until a more simple and sensitive technique for demonstrating the viability of M. leprae is developed, the mouse foot-pad system remains an essential tool for leprosy research. In this review, we discuss the mouse foot-pad technique in detail, analyse its precision, point out its shortcomings, describe its most important applications, and prescribe a method by which to assess the ability of an alternative technique to serve in place of this established technique.     

RT-PCR检测16S rRNA基因片段对麻风菌活性的评价

目的:探讨麻风病患者皮损组织中麻风菌16S rRNA基因片段判断麻风菌活性的应用价值。方法:根据BI值和联合化疗(MDT)的疗期对27例麻风患者分组,以RT-PCR法检测皮损组织中麻风菌16S rRNA的特异性片段。结果:(1)无论BI高低,未经治疗的患者16S rRNA均为阳性。(2)MDT治疗、BI≥3～6者的11例患者中:有9例16SrRNA为阳性,MDT疗期小于和大于6个月的两组中均各有1例患者16S rRNA阴性。(3)MDT治疗、BI≤2的6例患者:有1例MDT小于6个月病人其16S rRNA阳性,其余5例患者16SrRNA均为阴性。结论:随MDT治疗的进行,麻风菌16S rRNA阳性患者比例减少;诊断时BI值越低的患者中,经治疗后皮损中麻风菌16S rRNA阴转的比例增大,提示麻风菌16S rRNA与患者皮损中麻风菌活性具有相关性。     

Searching for secreted proteins of Mycobacterium leprae.

In mycobacteria secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in Mycobacterium tuberculosis culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied for Mycobacterium leprae since secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of M. tuberculosis were compared with sequences of M. leprae. Genes for homologues of the 85a, 85b, 85c, mpt32 (apa), mpt51, erp, mtc28, mtb12, Rv3354 and Rv0526 genes were identified. All of these and six genes of the mcel operon contain signal sequences for secretion in M. leprae as well. In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organization of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis, the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.     

The activity of rifabutin against Mycobacterium leprae.

Minimal effective doses of rifabutin and rifampicin were determined in Mycobacterium leprae isolated from skin biopsies of newly diagnosed, previously untreated lepromatous leprosy patients. Rifabutin was more potent than rifampicin. Our previous report that rifabutin was fully active against rifampicin-resistant M. leprae could not be confirmed. Examination of two strains of rifampicin-resistant M. leprae from elsewhere, and a repeat experiment on our original strain of rifampicin-resistant bacilli, showed full cross-resistance between rifampicin and rifabutin. A clinical trial in three newly diagnosed, previously untreated lepromatous patients showed that rifabutin has rapid bactericidal activity.     

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).     

Study on the micromorphology of Mycobacterium leprae

Summary The micromorphology of Mycobacterium leprae is described. After fixation with osmium tetroxide supplemented with calcium ions, the cell wall was seen to be composed of three layers; the cytoplasmic membrane exhibited the architecture of an elementary membrane. The mesosomes were best visualized after fixation with glutaraldehyde; they were sometimes in contact with the nuclear equivalent. Only one sort of phosphate body was found. The nucleoid was best visualized after fixation with osmium tetroxide.     

Phagocytosis of Mycobacterium leprae by cardiac muscle cells--a preliminary report

Fetal cardiac muscle cells were shown to ingest M. leprae easily within 20 minutes of exposure in vitro. This phagocytosis is considered nonspecific and facilitated by the lipid coat of the mycobacteria. The presence of M. leprae free in the cytoplasm of the muscle cells did not seriously affect the morphology or rhythmic contractions of the cells. The significance of the presence of M. leprae in somatic cells needs further study.