Culture-dependent and independent techniques to monitor yeast species during cold soak carried out at different temperatures in winemaking.

Transformation of grape must into wine is a process that may vary according to the consumers' requirements. Application of cold soak prior to alcoholic fermentation is a common practice in cellars in order to enhance flavor complexity and extraction of phenolic compounds. However, the effect of this step on wine yeast microbiota is not well-known. The current study simultaneously analyzed the effect of different cold soak temperatures on the microbiological population throughout the process and the use of culture-dependent and independent techniques to study this yeast ecology. The temperatures assayed were those normally applied in wineries: 2.5, 8 and 12°C. PCR-DGGE allowed detection of the most representative species such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae. As could be expected, highest diversity indices were obtained at the beginning of each process, and survival of H. uvarum or S. bacillaris depended on the temperature. Our results are in agreement with those obtained with culture independent methods, but qPCR showed higher precision and a different behavior was observed for each yeast species and at each temperature assayed. Comparison of both culture-independent techniques can provide a general overview of the whole process, although DGGE does not reveal the diversity expected due to the reported problems with the sensitivity of this technique.

Dielectrophoresis-based purification of antibiotic-treated bacterial subpopulations.

Persistence of bacteria during antibiotic therapy is a widespread phenomenon, of particular importance in refractory mycobacterial infections such as leprosy and tuberculosis. Persistence is characterized by the phenotypic tolerance of a subpopulation of bacterial cells to antibiotics. Characterization of these "persister" cells is often difficult due to the transient, non-heritable nature of the phenotype and due to the presence of contaminating material from non-persisting cells, which usually comprise the larger fraction. In this study, we use 3D carbon-electrode arrays for dielectrophoresis-based separation of intact cells from damaged cells, revealed by differential staining with propidium iodide, and we use this procedure to purify intact cells from cultures of Mycobacterium smegmatis treated with isoniazid, a frontline anti-tuberculosis drug. The method presented in this study could be used for rapid label-free enrichment of intact persister cells from antibiotic-treated cultures while preserving the metastable persister phenotype. This approach would facilitate the downstream analysis of low-frequency subpopulations of cells using conventional omics techniques, such as transcriptomic and proteomic analysis.

Microbial ecology of Gorgonzola rinds and occurrence of different biotypes of Listeria monocytogenes.

In this study we investigated the microbiota of Gorgonzola rinds and maturing shelf swabs collected in 5 different maturing cellars in the Northwest part of Italy, in association with the detection and characterization of Listeria monocytogenes. Culture-dependent and -independent methods were performed in order to profile the main microbial populations present on the rinds and in the maturing shelves and species-specific PCR and Pulsed Field Gel Electrophoresis (PFGE) were used to identify and type L. monocytogenes isolates. The microflora was predominated by lactic acid bacteria and coagulase negative cocci, while enterococci and yeasts were very variable between the samples. Arthrobacter sp., Carnobacterium sp., Staphylococcus sp. and Brevibacterium linens, as bacteria, and Debaryomyces hansenii, as yeast, were detected by Denaturing Gradient Gel Electrophoresis (DGGE). Cluster analysis of the DGGE profiles clearly highlighted a cellar-specific microflora. L. monocytogenes was isolated in 11.1% of the rinds and 29.4% of the swabs and the molecular characterization of the isolates suggests a route of contamination from the maturing shelves to the rinds. No correlation was found between DGGE profiles and presence or absence of L. monocytogenes.

Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing gradient gel electrophoresis.

Doenjang is a traditional Korean fermented soybean paste that provides a major source of protein. The microbial diversity of 10 samples of doenjang (5 commercially manufactured products and 5 homemade products) was investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). In the first step, the nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers. Subsequently, these products were used as a template in a nested PCR to obtain fragments suitable for DGGE. The bacterial DGGE profile targeting the V3 region of the 16S rRNA gene indicated that lactic acid bacteria such as Leuconostoc mesenteroide, Tetragenococcus halophilus, and Enterococcus faecium were the predominant species. However, bands corresponding to Bacillus species, known to be the main organisms in doenjang, were not detected under the conditions described above. When selective PCR was conducted using a primer specific for Bacillus species, Bacillus subtilis and B. licheniformis were detected in several doenjang samples. In analysis of fungi, Mucor plumbeus, Aspergillus oryzae, and Debaryomyces hansenii were the most common species in the doenjang samples. On the basis of DGGE, a few differences in community structure were found for different samples. Also, cluster analysis of the DGGE profile revealed that the microbial diversity did not differ clearly between commercially manufactured and homemade products. The nested PCR-DGGE technique was used for the first time in this study to asses a microbial community in doenjang and proved to be effective in profiling microbial diversity.

Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias / Utility of molecular biology in the microbiological diagnosis of mycobacterial infections

Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial. En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar. El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado. La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB. Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas. En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available (AU)

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

Cutaneous vasculitis as a presenting feature of multiple myeloma: a report of 2 cases.

We report two male patients who presented with symmetrical, painful purpura that evolved into bullae and necrotic ulcers, predominantly on the extremities, over two months in spite of conventional therapy including oral steroids. Examination showed livedoid and purpuric patches with necrotic centers in starburst pattern over the extremities and buttocks. The first case also had similar lesions over the ears. The clinical presentation and the histopathological examination suggested a diagnosis of necrotizing leukocytoclastic vasculitis (LCV). Blood testing ruled out connective tissue disease, hepatitis B or C infection or streptococcal infection as underlying cause of vasculitis. Serum antinuclear factor, antineutrophilic cytoplasmic antibody and anticardiolipin anticoagulant were negative in both cases. Cryoglobulins were positive in case 2. An incidental finding was raised serum proteins and globulins in case 2. Further investigations revealed M band on electrophoresis and features of multiple myeloma on bone marrow biopsy in both cases. These cases emphasize the importance of simple investigations like serum proteins in the evaluation of LCV.

Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages.

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (> 100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.

Co-infection of Mycobacterium tuberculosis and Mycobacterium leprae in human archaeological samples: a possible explanation for the historical decline of leprosy.

Both leprosy and tuberculosis were prevalent in Europe during the first millennium but thereafter leprosy declined. It is not known why this occurred, but one suggestion is that cross-immunity protected tuberculosis patients from leprosy. To investigate any relationship between the two diseases, selected archaeological samples, dating from the Roman period to the thirteenth century, were examined for both Mycobacterium leprae and Mycobacterium tuberculosis DNA, using PCR. The work was carried out and verified in geographically separate and independent laboratories. Several specimens with palaeopathological signs of leprosy were found to contain DNA from both pathogens, indicating that these diseases coexisted in the past. We suggest that the immunological changes found in multi-bacillary leprosy, in association with the socio-economic impact on those suffering from the disease, led to increased mortality from tuberculosis and therefore to the historical decline in leprosy.

[Comparative characterization of the antigenic composition of M. leprae and M. lufu]. / Sravnitel'naia kharakteristika antigennogo sostava M. leprae i M.lufu.

The antigenic structure of M. leprae and M. lufu was comparatively studied for the first time. M. lufu was found to have M. leprae-specific protein with a molecular weight of 36 kDa. M. leprae and M. lufu were similar in their fractional composition of proteins and an antibody response to determinants with equal molecular weights in patients with different forms of leprosy and its varying severity. The findings may improve a diagnostic system in leprosy by using M. lufu antigens as an alternative.

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products.

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.

Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis.

Rat hepatic peroxisomes (PO) were separated from other cell organelles by free flow electrophoresis (FFE) in combination with immunocomplexing PO prior to FFE with an antibody directed against the cytoplasmic aspect of the peroxisomal membrane protein PMP 70. This novel approach is based on a method termed antigen-specific electrophoretic cell separation (ASECS) which was originally introduced for the isolation of human T and B lymphocyte subpopulations by Hansen and Hannig (J. Immunol. Methods 1982, 51, 197-208). We adapted this technique to PO isolation from a crude peroxisomal fraction, streamlining it by the following modifications: (i) The sandwich-technique recommended to further lower a negative surface charge was renounced. (ii) Instead, the pH of the electrophoresis buffer was raised from 7.2 to 8.0, thus minimizing the electrophoretic mobility of the particles immunocomplexed due to the fact that the isoelectric point (pI) of IgG molecules is close to pH 8.0. PO isolated by this modification, referred to as immune free flow electrophoresis (IFFE), are as pure, intact, and structurally well-preserved as are highly purified PO obtained by density gradient centrifugation. The technique is currently applied for the isolation of peroxisomal subpopulations that are difficult to obtain by means of density gradient centrifugation.

On the beta-glucuronidase binding protein (BGBP) of microorganisms. Its purification, the antiserum preparation against that and its localization in leproma and the other infectious lesions shown by immunohistologic method.

Our previous studies suggested that M. leprae (ML) grow in peripheral nerves and lepra cells because ML metabolize hyaluronic acid (HA), and use its component for their growth by the aid of host enzyme combined to the bacilli derived beta-glucuronidase binding protein (BGBP). In this study, therefore, we examined the method to purify BGBP from a mycobacterium HI-75 originally separated from a leproma and cultured by modified Ogawa's medium containing split products of HA (glucuronic acid and N-acetylglucosamine). The distribution of BGBP in leproma and the other lesions consisting of hepatitis B virus infected liver and M. avium-intracellulare infected lung tissue were also immunohistologically examined. As the result, the best method to get BGBP was preparatory electrophoresis in the final step of the purification and not the molecular sieving. The BGBP was actually proven in leproma and the other infected tissues as described, indicating the abilities of these microorganisms to utilize the metabolic machinery of the host with the similar ways to that of ML.

Quantitation of the soluble E-receptor of human T lymphocytes by rocket electrophoresis in the serum of patients with lepromatous and tuberculoid leprosy.

Human T lymphocytes carry a membrane receptor for sheep erythrocytes (E) which is responsible for the well-known phenomenon of E-rosette formation. This receptor has been related to CD2 molecules; it is present in a soluble form (Rs) in normal serum and may play an immunoregulatory role. In this study we quantitated soluble E-receptor in serum samples of 43 normal controls, 32 patients with tuberculoid leprosy and 53 with lepromatous leprosy, using rocket electrophoresis and an anti E receptor serum (anti-Rs) obtained from an adult sheep immunized with autologous E treated with Rs. In the 3 groups studied, the rocket means were respectively 5.0, 7.5 and 10.9 mm (p less than 0.001). We found abnormally high levels of Rs in the serum of various diseases associated with a depression of cell-mediated immunity. The increase of Rs levels in the serum may be one of the mechanisms responsible for the depression of cellular immunity in leprosy.

Quantitation of the soluble E-receptor of human T lymphocytes by rocket electrophoresis in the serum of patients with lepromatous and tuberculoid leprosy

Los linfocitos T humanos poseen, a nivel de membrana, receptores para eritrocitos de carnero (E) que les permite la unión espontánea con estas células, llevando al fenómeno de formación de rosetas. Este receptor fue relacionado con la molécula CD2. Se produjeron varios anticuerpos monoclonales contra la estructura CD2; la mayoría de ellos inhibe alguna de las funciones de los linfocitos T (blasogénesis, formación de rosetas y reacción en cultivo mixto de linfocitos). Es posible obtener este receptor de membrana en forma soluble (Rs), en el sobrenadante de células T incubadas por una hora a 4-C y en el suero de individuos normales. En nuestro laboratorio fueron estandarizados varios métodos para detección y ccuantificacióne de Rs, a partir de un antisuero (anti-Rs) obtenido al inmunizar un carnero con eritrocitos autólogos tratados con Rs (ERs). Este antisuero inhibe la formación de rosetas, es citotóxico para células T, aglutina complejos ERs e identifica linfocitos T por inmunofluorescencia. El método más práctico para cuantificacióne de Rs es el suero de pacientes con patologías asociadas a depresión de la respuesta inmune mediada por células (anemia, lepra, neoplasias, leucemias, linfomas y aplasia medular). Numerosos trabajos han demostrado que los pacientes portadores de lepra presentan alteraciones inmunológicas. Se sabe que los pacientes con lepra lepromatosa presentan deficiencia primaria específica de células T contra antígenos del M. leprae y deficiencia secundaria, inespecífica, de la respuesta inmune celular. En este trabajo, cuantificamos Rs por inmunoelectrdifusión en el suero de 43 individuos normales, 32 pacientes con lepra tuberculoide y 53 con lepra lepromatosa. El promedio de los picos obtenidos fue de 5,0, 7,5 y 10,9 mm., respectivamente. Este aumento fue estadísticamente significativo (Kruskal-Wallis p<0,001), entre los 2 grupos de pacientes y comparados con el grupo control. El aumento de Rs en el suero de los pacientes con lepra puede ser uno de los mecanismos responsables de la depresión de la respuesta inmune celular encontrada en esta enfermedad

Quantitation of the soluble E-receptor of human T lymphocytes by rocket electrophoresis in the serum of patients with lepromatous and tuberculoid leprosy

Los linfocitos T humanos poseen, a nivel de membrana, receptores para eritrocitos de carnero (E) que les permite la unión espontánea con estas células, llevando al fenómeno de formación de rosetas. Este receptor fue relacionado con la molécula CD2. Se produjeron varios anticuerpos monoclonales contra la estructura CD2; la mayoría de ellos inhibe alguna de las funciones de los linfocitos T (blasogénesis, formación de rosetas y reacción en cultivo mixto de linfocitos). Es posible obtener este receptor de membrana en forma soluble (Rs), en el sobrenadante de células T incubadas por una hora a 4-C y en el suero de individuos normales. En nuestro laboratorio fueron estandarizados varios métodos para detección y ccuantificacióne de Rs, a partir de un antisuero (anti-Rs) obtenido al inmunizar un carnero con eritrocitos autólogos tratados con Rs (ERs). Este antisuero inhibe la formación de rosetas, es citotóxico para células T, aglutina complejos ERs e identifica linfocitos T por inmunofluorescencia. El método más práctico para cuantificacióne de Rs es el suero de pacientes con patologías asociadas a depresión de la respuesta inmune mediada por células (anemia, lepra, neoplasias, leucemias, linfomas y aplasia medular). Numerosos trabajos han demostrado que los pacientes portadores de lepra presentan alteraciones inmunológicas. Se sabe que los pacientes con lepra lepromatosa presentan deficiencia primaria específica de células T contra antígenos del M. leprae y deficiencia secundaria, inespecífica, de la respuesta inmune celular. En este trabajo, cuantificamos Rs por inmunoelectrdifusión en el suero de 43 individuos normales, 32 pacientes con lepra tuberculoide y 53 con lepra lepromatosa. El promedio de los picos obtenidos fue de 5,0, 7,5 y 10,9 mm., respectivamente. Este aumento fue estadísticamente significativo (Kruskal-Wallis p<0,001), entre los 2 grupos de pacientes y comparados con el grupo control. El aumento de Rs en el suero de los pacientes con lepra puede ser uno de los mecanismos responsables de la depresión de la respuesta inmune celular encontrada en esta enfermedad (AU)

Anomalous isoenzymes of lactate dehydrogenase in sera of patients with leprosy.

In an earlier study (Saoji et al, 1980) we looked for anomalous (additional) isoenzymes in homogenates of 17 active and 25 of regressing lepromatous leprosy, three of erythema nodosum leprosum, eight of BT, 19 of TT and one of indeterminate leprosy. Anomalous bands were found in 17 cases. They correlated with large numbers of viable organisms and were thought to originate from Mycobacterium leprae. This communication describes the serum LDH isoenzymes of the same 78 cases. Six of 17 cases with anomalous LDH isoenzymes in tissues showed anomalous bands even in serum samples. The bands were much fainter but had similar mobility in terms of Ef values. Therefore, LDH isoenzymes originating from Mycobacterium leprae were discernible in sera of cases, though not on the same scale as in the tissues.

Lack of interaction of circulating T cells with phytohemagglutinin in bacillary positive untreated lepromatous leprosy patients--identification of subpopulation of lymphocytes by shifts in electrophoretic mobility.

Incubation of human peripheral blood lymphocytes from normal healthy subjects with phytohamagglutinin (PHA), causes the reduction of the surface charge of a subpopulation of T cells by 1363 +/- 242 e.s.u./cm2. The affected subpopulation was predominantly the high charge-bearing cells identifiable with early (10 min) rosette-forming cells with sheep erythrocytes. Purified lymphocytes obtained from untreated bacillary-positive, lepromatous leprosy patients contained high charge-bearing T lymphocyte subpopulation. However, incubation with PHA did not result in the shift of electrophoretic mobility of these cells, suggesting the absence of interacting sites for the mitogen on the surface of these cells. The absence of mitogen-interacting sites is not an inherent trait of leprosy patients; the surface charge of lymphocytes from Dapsone-treated bacillary-negative subjects was reduced upon incubation with PHA. A close correlation was found between the number of cells whose charge alters on incubation with PHA and the transformation index obtained with this mitogen.