Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Zoonotic transmission of tuberculosis between pastoralists and their livestock in South-East Ethiopia

Despite huge global efforts in tuberculosis (TB) control, pastoral areas remain under-investigated. During two years sputum and fine needle aspirate (FNA) specimens were collected from 260 Ethiopian pastoralists of Oromia and Somali Regional States with suspected pulmonary TB and from 32 cases with suspected TB lymphadenitis. In parallel, 207 suspected tuberculous lesions were collected from cattle, camels and goats at abattoirs. All specimens were processed and cultured for mycobacteria; samples with acid-fast stained bacilli (AFB) were further characterized by molecular methods including genus and deletion typing as well as spoligotyping. Non-tuberculous mycobacteria (NTM) were sequenced at the 16S rDNA locus. Culturing of AFB from human sputum and FNA samples gave a yield of 174 (67%) and 9 (28%) isolates, respectively. Molecular typing was performed on 173 of these isolates and 160 were confirmed as Mycobacterium tuberculosis, three as M. bovis, and the remaining 10 were typed as NTMs. Similarly, 48 AFB isolates (23%) yielded from tuberculous lesions of livestock, of which 39 were molecular typed, including 24 M. bovis and 4 NTMs from cattle, 1 M. tuberculosis and 1 NTM from camels and 9 NTMs from goats. Isolation of M. bovis from humans and M. tuberculosis from livestock suggests transmission between livestock and humans in the pastoral areas of South-East Ethiopia.     

The Characterization of Clinically Important Gram Negative Anaerobic Bacilli by Conventional Bacteriological Tests

One hundred and sixty-five reference strains and laboratory isolates of Gram negative, non-sporing, anaerobic bacilli were subjected to a series of simple laboratory tests that were initially selected for their discriminatory value. Conventional biochemical tests, tests of resistance to antibiotics, and tolerance to dyes and bile salts were included. These tests allowed a clear separation of strains into three main groups: Bacteroides fragilis, B. melaninogenicus and Fusobacterium spp. Certain tests were found useful for identifying recognized subspecies of B. fragilis and B. melaninogenicus . A scheme for the identification of unknown laboratory isolates of Gram negative anaerobic bacilli is presented.     

Comparison of Borrelia burgdorferi isolated from humans and ixodid ticks in Hokkaido, Japan.

Four spirochetal isolates (JEM1 to JEM4) were obtained from cutaneous lesions of patients manifesting erythema chronicum migrans in Hokkaido, Japan. In the protein profiles by SDS-PAGE and the reactivities with monoclonal antibodies (H5332 and H9724) by immunoblotting, all the human isolates were identical with the tick isolates from Ixodes persulcatus. These data indicate that I. persulcatus is an important vector of Lyme disease for humans in Japan.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.     

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.     

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

     

Antagonistic action of Pseudomonas aeruginosa against Staphylococci, coliform bacilli and various types of Proteus

Antagonistic activity of 2 fresh isolates and 3 collection strains of Pseudomonas aeruginosa against 177 microbial strains was determined with the method of late antagonism. Among the microbial strains there were 56 staphylococcal strains isolated from patents and carriers. 38 nontypable colon bacilli isolated from healthy persons, 59 enteropathogenic colon bacilli of various serogroups, 12 strains of Proteus and 12 colon bacilli, carriers of multiple drug resistance factors (R factors). All the cultures were sensitive to the antagonistic action of 5 or at least 3 strains of Pseudomonas used in the study. The most active antagonists were the fresh isolates of Pseudomonas as compared to the collection strains. Among the staphylococci S. aureus proved to be the most resistant to the antagonistic action of Pseudomonas as compared to S. epidermidis, the same as the strains isolated from carriers as compared to the strains isolated from patients. As for the enteric bacilli the most resistant were the strains of Proteus. Acquiring of transmissive R factors by the colon bacilli markedly increased their sensitivity to the antagonistic action of Pseudomonas.     

Isolation and characterization of Histophilus somni (Haemophilus somnus) in semen samples of rams with epididymitis

Histophilus somni (Haemophilus somnus) has been reported as the cause of epididymitis in rams. This bacterium has also been found in the preputial mucosa of rams without epididymitis lesions. H. somni is a bacterium that is difficult to characterize, since it is a pleomorphic Gram-negative bacilli of characteristics similar to Actinobacillus seminis, which is also found in ram epididymitis lesions. The objective of this work was to determine if H. somni (H. somnus) is involved in cases of sheep epididymitis. A clinical examination was performed in 160 rams, extracting semen by electro-ejaculation of 28 of them, which had epididymal lesions. The penis was exteriorized in order to avoid prepuce contamination. The semen samples were cultivated in chocolate agar in a 10% CO₂ environment. Two strains were isolated in pure culture with a colony morphology and microscopy similar to H. somni (H. somnus). These were identified using the API 20 E system, using as a control the reference strain of H. somnus (2336ATCC). One of the isolates (129H) resulted identical to the reference strain and the other (827) presented differences in the arginine decarboxylase, H₂S, catalase and inositol reactions, although these differences have been reported (in strains isolated from different geographic origins, animal species and anatomical region). To characterize the isolates, an electrophoretic analysis of total proteins was performed (PAGE–SDS) finding identical profiles between the reference strain of H. somnus and isolate 129H and similar in relation to isolate 827. The amplification of a fragment of approximately 407 bp was observed in the 129H isolate and the ATCC strain, but not in 827. In other samples, isolations were made of Brucella ovis, Corynebacterium spp., Staphylococcus and other pleomorphic Gram-negative bacilli similar to A. seminis. Therefore, it has been confirmed that H. somni is present in the reproductive tract of rams and it could be involved in the presentation of ovine epididymitis. It is important that we underline that this is the first report of H. somni isolation in Mexico from ram semen samples.     

Studies on the Tuberculin Reactivity of Tuberculin-Active Peptide

A comparative study of the tuberculin potency and reactivity to TAP and PPD-S was made in humans and guinea pigs. A comparison of reactions elicited by TAP and PPD-S was made in two groups of guinea pigs previously inoculated with killed bacilli or with living bacilli. The tuberculin reactions produced by TAP and PPD-S in human beings was studied in school children from rural districts. These children were divided randomly into three groups and each group was given a different amount of TAP and PPD-S. The results obtained are as follows: (1) While relative potency of TAP was relatively much weaker than PPD-S in guinea pigs sensitized with tubercle bacilli, in human beings the tuberculin potency of TAP was approximately 1/2 or 1/3 that of PPD-S. (2) There was a marked difference in the tuberculin reactivity to TAP and PPD-S between guinea pigs immunized with killed bacilli and guinea pigs immunized with living bacilli.     

Mycobacterium tuberculosis isolates grown under oxygen deprivation invade pulmonary epithelial cells

Mycobacterium tuberculosis has the ability to adapt to and survive under different environmental conditions, including oxygen deprivation. To better understand the pathogenesis of M. tuberculosis, we studied the invasion of human alveolar (A549) and human bronchial (BBM) epithelial cell lines by M. tuberculosis isolates cultured under oxygen deprivation. We used isolates belonging to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and the laboratory strains H37Rv and H37Ra. We determined that: (1) M. tuberculosis bacilli grown under oxygen deprivation invade epithelial cells, (2) the invasion capacity of all 17 isolates differed, and (3) oxygen deprivation influenced the invasion capacity of these isolates. All isolates invaded the A549 more effectively than the BBM cells. Three of the F15/LAM4/KZN isolates, two of which had extensively drug resistance (XDR) profiles, were at least twice as invasive (≥33%) as the most invasive Beijing isolate (15%) (P < 0.05). We conclude that for a more comprehensive understanding of the pathogenesis of M. tuberculosis, studies should include isolates that have been cultured under oxygen deprivation.     

Isolation and Identification of Psychrophilic Species of Bacillus from Milk

Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures.     

Identification of Actinomyces,Propionibacteria, Lactobacilli and Bifidobacteria by Amplified 16S rDNA Restriction Analysis

Amplified 16S rDNA restriction analysis (ARDRA) with Hae III and Hpa II was applied to 37 reference strains, 179 human clinical and four veterinary isolates of Propionibacterium, Lactobacillus andBifidobacterium and some other anaerobic, non-sporing, Gram-positive bacilli. Results were compared with those obtained by ARDRA for reference strains (26) and clinical isolates (469) of Actinomyces spp. Reference strains were clearly differentiated to species level. Clinical isolates of Propionibacterium and Lactobacillus were identified with confidence to species level. Bifidobacterium spp. were identified in ARDRA with confidence to genus, but anomalies in species level identification of some reference strains and clinical isolates may reflect unreliable identification in conventional tests. Isolates of Arcanobacterium spp., Actinobaculum schaalii, Eggerthella lenta, some Eubacterium spp., Gardnerella vaginalis, Mobiluncus spp., Atopobium vaginae, Abiotrophia defectiva, Streptococcus mutans, Streptococcus intermedius and Clostridium sp. were clearly differentiated in ARDRA. ARDRA is a simple, rapid, and highly discriminatory method for identification of anaerobic, non-sporing, Gram-positive bacilli.     

Cytochemical reactions of human leprosy bacilli and mycobacteria: ultrastructural implications

Leprosy bacilli harvested from freshly biopsied tissue from cases of lepromatous, borderline and histoid leprosy were, in conjunction with Mycobacterium lepraemurium and representative mycobacteria, examined cytochemically with and without their pyridine-extractable acid-fastness. Unlike the mycobacteria, unextracted leprosy bacilli failed to give a positive response to the periodic acid Schiff test or to take up Sudan black B, toluidine blue O, alkaline methylene blue or safranin O. Once their acid-fastness was removed with pyridine, leprosy bacilli were stained by all of the foregoing dyes except Sudan black B, under this condition they remained gram positive. While permanent loss of acid-fastness from leprosy bacilli always resulted in a loss of acid hematein-fixing material (Smith-Dietrich-Baker tests), the reverse was not true. Mild aqueous saponification, bromination, or sequential treatment with lipase and phospholipase D resulted in a loss of acid hematein-positivity but not acid-fastness. After pyridine extraction, bromination, or aqueous saponification, true mycobacteria lost neither their acid hematein-positivity nor their acid-fastness. The acid hematein-positive material and the acid-fastness of both leprosy bacilli and mycobacteria were lost after treatment with alkaline ethanol. These cytochemical findings are discussed in the light of what is known of the ultrastructure of leprosy bacilli and mycobacteria, and of the occurrence of a dl-3, 4-dihydroxyphenylalanine oxidase in leprosy bacilli but not in mycobacteria. An effort is made to explain the rather unique cytochemical properties of leprosy bacilli. Since pyridine-extractable acid-fastness (and acid hematein-positivity) serve to distinguish human leprosy bacilli from M. lepraemurium, one or the other, or both, are suggested as bases for differentiating these two organisms in animal experiments designed to show the in vivo propagation of human leprosy bacilli.     

Rib lesions in chronic pulmonary tuberculosis

The diagnosis of skeletal tuberculosis in human remains has traditionally been based upon the detection of secondary skeletal lesions which result from hemotogenous dissemination of tubercle bacilli (e.g., Pott's disease). Since such lesions develop in less than 7% of cases of human tuberculosis, the paleodemography and paleoepidemiology of this disease have been difficult to assess from skeletal remains. This study presents a new diagnostic approach to tuberculosis, focusing on the skeletal manifestations of chronic pulmonary disease (which comprises approximately 90% of human-form tuberculosis). Four hundred forty-five skeletal remains from persons dying of tuberculosis during the first half of the 20th century were examined. A total of 70/445 (16%) exhibited skeletal lesions in one or more locations as a response to infection. Of these 70, 39 (56%) were found to display a specific set of lesions restricted to the internal aspect of the ribs. These lesions take one of two forms: (1) diffuse periostitis or (2) localized abscess, and appear to correspond to areas of chronic pulmonary infection. The diffuse type of rib lesion is more commonly observed than the localized type. In our observations (and according to the natural history of tuberculosis) the occurrence of chronic pulmonary tuberculosis is usually mutually exclusive with hematogenous dissemination to secondary bone locations. Thus, the detection of rib lesions in cases of chronic pulmonary disease increases the absolute sample size of skeletal tuberculosis by a factor of two in this study.     

Occurrence of granulomatous appendicitis in rabbits

Granulomatous appendicitis was observed in all of the 45 Japanese white rabbits examined. Histologically, multiple microgranulomas were accompanied with foreign body giant cells and focal calcifications in lymph nodules of appendix. Foreign body giant cells contained hair coat and larvae of Passalurus ambiguus. In addition, the sacculus rotundus and mesenteric lymph nodes were affected with the same lesions. PAS- and Gram- positive bacilli were phagocytized in the microgranulomas and macrophages. They were also stained positively with the immunoperoxidase method for the auto-sera of rabbits. Isolation of these bacilli in pure culture has not yet been accomplished. The occurrence of granulomatous lesions due to tuberculosis, pseudotuberculosis, tularemia and actinomycosis seems unlikely in the present cases because none of these organisms nor characteristic lesions were detected. It was suggested that the Gram-positive bacilli appeared to play a role in granulomatous appendicitis.     

Functional grouping of thermophilic Bacillus strains using amplification profiles of the 16S-23S internal spacer region

Molecular and biochemical assays were used to determine the identification of thermophilic bacilli isolated from New Zealand milk powder. One hundred and forty one isolates of thermophilic bacilli were classified into six species using biochemical profiles. Geobacillus stearothermophilus represented 56% of the isolates. All isolates were also analysed by randomly amplified polymorphic DNA (RAPD) analysis, with 45 types identified. Amplification of the 16S-23S rDNA internal spacer region produced two to eight amplification products per strain. The patterns from gel electrophoresis of the internal spacer region amplicons formed two major groupings suggesting the possibility of two distinct species. Partial sequences of 16S rDNA from representatives from each group were compared with sequences in GeneBank and were found to match the 16S rDNA sequences of B. flavothermus and G. thermoleovorans. Primers were designed for these species and used to screen an arbitrary selection of 59 of the dairy isolates. This enabled the identification of 28 isolates as B. flavothermus and 31 isolates as Geobacillus species and these appear to be the predominant isolates in the New Zealand milk powder samples examined. Comparison of the fragment pattern generated by amplification of the 16S-23S rDNA internal spacer region is a simple method to differentiate thermophilic Bacillus species associated with the dairy industry.     

Liver in Leprosy: Histological and Biochemical Findings

The histological findings and their correlation with biochemical functions of the liver in 240 leprosy patients are presented. In 21% with tuberculoid leprosy and in 62% with lepromatous leprosy leprous granulomata were found in the liver. A significant prevalence of granulomatous lesions in the liver among patients with tuberculoid and borderline-tuberculoid leprosy of less than one year''s duration suggests that bacillaemia occurs early in all forms of leprosy.There was a direct correlation between bacterial index and the presence of acid-fast bacilli in the liver. Of 50 patients with negative skin smears seven had acid-fast bacilli at liver biopsy. From none of these liver homogenates were acid-fast bacilli grown on culture in Löwenstein-Jensen medium.The alterations in liver functions were more consistently seen when acid-fast bacilli were associated with the presence of leprous granulomatous lesions. The acid-fast bacilli were found to persist even after one to five years of specific antileprosy therapy and after the bacilli in the skin had cleared up. This may explain the relatively frequent recrudescence or relapse of the bacillated types of leprosy when specific antileprosy therapy is stopped soon after bacterial negativity is attained on skin smears.