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In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates.

Clavulanic Acid↗

The mycobactericidal efficacy of ortho-phthalaldehyde and the comparative resistances of Mycobacterium bovis, Mycobacterium terrae, and Mycobacterium chelonae.

OBJECTIVES: To assess the mycobactericidal efficacy of an agent relatively new to disinfection, ortho-phthalaldehyde (OPA) and to compare the resistances of three Mycobacterium species. Mycobacterium bovis (strain BCG) was compared with Mycobacterium chelonae and Mycobacterium terrae to investigate the feasibility of using either of the latter two species in tuberculocidal testing. M. chelonae (a rapid grower) and M. terrae (an intermediate grower) both grow faster and are less virulent than M. bovis (a slow grower). DESIGN: The quantitative suspension protocol specified by the Environmental Protection Agency (EPA), the Tuberculocidal Activity Test Method (EPA test), was used throughout this study. Standard suspensions of all three species were prepared in a similar manner. Two suspensions of M. bovis, created in different laboratories, were used. These were tested against two concentrations of alkaline glutaraldehyde to provide reference data. Two concentrations of OPA were evaluated against all mycobacterial test suspensions. Four replicates of each organism-disinfectant combination were performed. RESULTS: Results were assessed by analysis of variance. M. terrae was significantly more resistant to 0.05% OPA than either M. bovis or M. chelonae. At 0.21% OPA, M. terrae was slightly more susceptible than one test suspension of M. bovis, but not significantly different from the other. M. chelonae was significantly less resistant than the other species at both OPA concentrations. At their respective minimum effective concentration, OPA achieved a 6-log10 reduction of M. bovis in nearly one sixth the time required by glutaraldehyde (5.5 minutes vs. 32 minutes). CONCLUSIONS: These data, along with other recent studies, lend support to the idea that M. terrae may be a suitable test organism for use in the tuberculocidal efficacy testing of disinfectants. They also confirm the relatively rapid tuberculocidal activity of OPA.

Analysis of Variance↗

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil.

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.

Agriculture↗

Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis.

Mycobacterium bovis is the cause of tuberculosis in cattle and is a member of the Mycobacterium tuberculosis complex. In contrast to many other pathogenic bacterial species, there is little evidence for the transfer and recombination of genes between cells. The clonality of this group of organisms indicates that the population structure is dominated by reductions in diversity, caused either by population bottlenecks or selective sweeps as entire chromosomes become fixed in the population. We describe how these forces have shaped not only the phylogeny of this group but also, at a very local level, the population structure of Mycobacterium bovis in the British Isles. We also discuss the practical implications of applying this knowledge to understanding the spread of infection and the development of improved vaccines and diagnostic tests.

Animals↗

Enzymatic inactivation and reactivation of chloramphenicol by Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis and Mycobacterium bovis are inhibited by chloramphenicol. Chloramphenicol acetyltransferase (CAT) converts chloramphenicol to inactive diacetyl chloramphenicol, but a mycobacterial carboxylesterase hydrolyzes the diacetyl product to active chloramphenicol. The esterase activity was eliminated by proteinase K and heat treatment. Protein extracts of M. tuberculosis and M. bovis hydrolyzed four other ester substrates. cat was inserted into the chromosome of both M. tuberculosis and M. bovis resulting in a level of chloramphenicol resistance that could be used to select for transformants. CAT assays in the resistant strain of M. tuberculosis showed interference due to esterase activity. This interference could be eliminated with the addition of a heating step.

Biotransformation↗

The complete genome sequence of Mycobacterium bovis.

Mycobacterium bovis is the causative agent of tuberculosis in a range of animal species and man, with worldwide annual losses to agriculture of $3 billion. The human burden of tuberculosis caused by the bovine tubercle bacillus is still largely unknown. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guérin vaccine strain, the most widely used human vaccine. Here we describe the 4,345,492-bp genome sequence of M. bovis AF2122/97 and its comparison with the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. Strikingly, the genome sequence of M. bovis is >99.95% identical to that of M. tuberculosis, but deletion of genetic information has led to a reduced genome size. Comparison with M. leprae reveals a number of common gene losses, suggesting the removal of functional redundancy. Cell wall components and secreted proteins show the greatest variation, indicating their potential role in host-bacillus interactions or immune evasion. Furthermore, there are no genes unique to M. bovis, implying that differential gene expression may be the key to the host tropisms of human and bovine bacilli. The genome sequence therefore offers major insight on the evolution, host preference, and pathobiology of M. bovis.

Genome, Bacterial↗

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.

Bacteriophage Typing↗

Does resistance to pyrazinamide accurately indicate the presence of Mycobacterium bovis?

Mycobacterium bovis is best identified by screening those isolates of the Mycobacterium tuberculosis complex that have any pyrazinamide (PZA) resistance, using a confirmatory test such as spoligotyping, biochemical testing, or genomic deletion analysis. The sensitivity for detection of M. bovis is lowered to 82% when only PZA-monoresistant isolates are screened.

Adult↗

Use of sodium deoxycholate to extract cell wall components of virulent Mycobacterium bovis.

Mycobacterium bovis ATCC No. 19210 was grown on Middlebrook 7H-10 medium with pyruvate. Cells were harvested, and extracts were prepared, using 2% sodium deoxycholate (DOC) and 0.003M EDTA (in 0.1M Tris-HCl, 0.15M NaCl with 0.02% sodium azide [pH 8.4]). Phenylmethylsulfonyl fluoride was added, and the cells were extracted for 48 hours at 4 C. Cells were removed by centrifugation at 10,000 X g for 30 minutes. The supernatant was filter sterilized and separated into 2 fractions (peak A and peak B) by size-exclusion chromatography. The nonfractionated DOC extract and DOC peak A elicited delayed-type hypersensitivity responses at each of the protein concentrations tested (0.5, 1.5, and 4.5 micrograms) in M bovis-sensitized guinea pigs; responses were not detected, using DOC peak B. Significant differences were detected for each of the antigens when enzyme-linked immunosorbent assay values were compared, using sera from cattle before and 10 months after they were exposed to M bovis (P less than 0.01).

Animals↗

Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis.

Mycobacterium bovis causes tuberculosis in cattle and many other animals including humans while BCG, an attenuated form of M. bovis, has been used widely as a safe vaccine. Both strains infect host macrophages and their fate is determined by their ability to survive within these phagocytic cells. We compared interactions of these two strains with bovine alveolar macrophages in order to gain an understanding of virulence mechanisms involved in the early pathogenesis of M. bovis infection. Macrophages were infected with bacilli at varying multiplicities of infection and cultured for 1-4 days. Bacterial metabolism within macrophages was assessed by [3H]-uracil uptake and bacterial growth was assessed by culture and acid-fast staining. Induction of TNF-alpha, IL-1 beta and IL-6 cytokine mRNA transcription in macrophages was determined by reverse transcriptase-polymerase chain reaction. Infection of macrophages by virulent M. bovis resulted in enhanced bacterial metabolism, enhanced induction of macrophage cytokines and reduced viability of macrophages when compared to M. bovis BCG-infected macrophages. These differences may reflect virulence mechanisms contributing to the early pathogenesis of bovine tuberculosis.

Animals↗

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.

Animals↗

Mycobacterium bovis versus Mycobacterium tuberculosis as a cause of acute cervical lymphadenitis without pulmonary disease.

Bovine tuberculosis remains a common disease of cattle in countries such as Mexico. Children eating unpasteurized dairy products from Mexican cattle can develop Mycobacterium bovis cervical lymphadenitis. However, the bovine mycobacterium can be misdiagnosed as Mycobacterium tuberculosis based on standard laboratory testing. Accurate speciation is important for selection of the preferred antibiotic regimen for treatment of Mycobacterium bovis infection.

Acute Disease↗

Identification of a cadmium-induced gene in Mycobacterium bovis and Mycobacterium tuberculosis.

A 17-kDa protein (CadI) was induced by cadmium in Mycobacterium bovis and Mycobacterium tuberculosis. Comparison of the N-terminal sequence from M. bovis CadI with the annotated M. tuberculosis genome database identified Rv2641 as the encoding gene. Long and short promoter fragments from M. bovis cadI were fused to the lacZ reporter gene in pYUB76. Only the long fragment directed cadmium-inducible activity when electroporated into M. bovis. The cadI promoter has potential for both constitutive and inducible expression studies in M. bovis and M. tuberculosis.

Amino Acid Sequence↗

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

Animals↗

A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis.

A multiplex-polymerase chain reaction (PCR) assay based on one-step amplification and detection of two different mycobacterial genomic fragments was designed for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from a 500-bp genomic fragment which is well conserved in M. bovis and the pncA gene (based on M. tuberculosis-specific nucleotide polymorphism, a cytosine residue at position 169), specific for M. tuberculosis. The multiplex-PCR allowed detection of a single product of 500 bp in M. bovis isolates while M. tuberculosis isolates generated a single product of 185 bp, with or without an additional product of 500 bp. None of the atypical mycobacterial isolates revealed any amplification products. The method was found to be highly specific and could detect as little as 20 pg of pure DNA. This multiplex-PCR assay, based on the 500-bp fragment and the pncA gene, may be very useful for the rapid and specific differentiation of these two closely related mycobacteria and easy to use in medical and veterinary microbiological laboratories.

Amidohydrolases↗

Development of specific lymphocyte immunostimulation and tuberculin skin reactivity in swine infected with Mycobacterium bovis and Mycobacterium avium.

Swine infected with Mycobacterium bovis and Mycobacterium avium were studied for development and specificity of lymphocyte immunostimulation and tuberculin skin reactivity to purified protein derivatives (PPD) of tuberculins. Lymphocytes from infected animals had maximal blastogenic responses to PPD prepared from the homologous organisms. The time of appearance of specifically sensitized lymphocytes was studied, as was the specificity of skin reactivity. In vitro lymphocyte responses of M avium serotype 1-infected swine to M avium serotype 1 PPD were greater than were the responses of M avium serotype 2-infected swine to the same antigen. Results indicate that the specific stimulation of sensitized lymphocytes might prove useful in the diagnosis of tuberculosis in swine and in differentiating infections due to different serotypes of M avium.

Administration, Oral↗