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Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages.

The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.

Animals↗

Advance in bacterial typing methods (a review).

We give a short review about the two main groups of bacterial typing methods for epidemiological purposes: the phenotypic and genotypic methods. The advantage of the phenotypic methods is their feasibility for the less equipped laboratories, their disadvantages are the variability of the phenotypic properties, and their high labor requirement. The advantages of the genotypic typing methods are the higher discriminatory power and applicability of the same method for different species of bacteria. The disadvantage of these methods is that they are expensive and labour consuming. Beside the short description, we show the results of different authors obtained by the use of the molecular methods in the epidemiological practice and we call the attention to the insufficiency concerning the stability.

Animals↗

Somatic hybrids Solanum nigrum (+) S. tuberosum: morphological assessment and verification of hybridity.

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.

Culture Techniques↗

Fingerprinting of DNA and RNA by arbitrarily primed polymerase chain reaction: applications in cancer research.

RNA fingerprinting by RAP-PCR is a powerful tool for the temporal and spatial analysis of differential gene expression. Many biological situations exist where differential gene expression results in distinguishable phenotypes, including, for example, tissue and cell types, responses to hormones, growth factors, stress, and the heterologous expression of certain genes. There are several methods for detecting differential gene expression and cloning differentially expressed genes that do not rely on a biological assay of phenotype. Most of these methods fall into two general categories: subtractive hybridization and differential screening. RAP-PCR offers numerous advantages over these methods, including its simplicity and its ability to compare the fluctuations in gene expression between multiple samples simultaneously using minute amounts of RNA. In addition, RAP-PCR can yield information on the overall patterns of gene expression between different cell types or between different physiological conditions of the same cell type. Comparison of the RAP-PCR fingerprints from these different experimental groups permits one to draw inferences regarding the overall cellular states of gene expression and the interrelation between gene transcripts belonging to the same or different regulatory pathways. Hypotheses regarding signal transduction pathways can be obtained using this information. RAP-PCR offers applications in cancer research in the detection of tumor-specific alterations in gene expression, providing a bountiful source of tumor markers. The pleiotropic impact of oncogene activation, tumor suppressor gene inactivation, and mutator mutations, in gene regulation, can be readily assessed by RAP-PCR in model systems both in vitro and in vivo.

Base Sequence↗

Genetic diversity within Lactobacillus sakei and Lactobacillus curvatus and design of PCR primers for its detection using randomly amplified polymorphic DNA.

The genotypic and phenotypic diversity among isolates of the Lactobacillus curvatus/Lactobacillus graminis/Lactobacillus sakei group was evaluated by comparing RAPD data and results of biochemical tests, such as hydrolysis of arginine, D-lactate production, melibiose and xylose fermentation, and the presence of haem-dependent catalase. Analyses were applied to five type strains and to a collection of 165 isolates previously assigned to L. sakei or L. curvatus. Phenotypic and RAPD data were compared with each other and with previous DNA-DNA hybridization data. The phenotypic and genotypic separation between L. sakei, L. curvatus and L. graminis was clear, and new insights into the detailed structure within L. sakei and L. curvatus were obtained. Individual strains could be typed by RAPD and, after the elimination of similar or identical isolates, two sub-groups in both L. curvatus and L. sakei were defined. The presence or absence of catalase activity further distinguished the two L. curvatus sub-groups. By cloning and sequencing specific RAPD products, pairs of PCR primers were developed that can be used to specifically detect L. curvatus, L. sakei and each of the L. sakei sub-groups.

Animals↗

Molecular typing of vibrio vulnificus isolated from clinical specimens by pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis.

Primary V. vulnificus septicemia has been continuously reported in Korea. We analyzed the molecular diversity of V. vulnificus strains isolated from clinical specimens using pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. We analyzed a total of 23 V. vulnificus strains isolated from 22 patients between 1992 and 1997. RAPD analysis was performed using five random primers, and we obtained chromosomal DNA restriction patterns with NotI and SfiI by PFGE. Two isolates from one patient disclosed similarity value 1.0 by RAPD and had an indistinguishable pattern when analyzed with PFGE, which indicated that they were the same strains. The remaining 22 isolates from 22 patients could be separated into 5 different molecular types in RAPD analysis. The range of similarity values among the isolates was wide (0.29-0.81). Of 22 strains, four strains could not be typed in repeated trials by PFGE with NotI and SfiI. The PFGE patterns of 18 strains showed a remarkable polymorphism consisting 12-19 fragments (20-870 kb). These results show that V. vulnificus strains isolated from Korea are genetically diversified for the most part.

Adult↗

Expression of cartilage-specific functional matrix chondromodulin-I mRNA in rabbit growth plate chondrocytes and its responsiveness to growth stimuli in vitro.

Cartilage-specific functional matrix chondromodulin-I (ChM-I) is a 25 kDa glycoprotein purified from fetal bovine epiphyseal cartilage which stimulates the growth of rabbit chondrocytes and the colony formation of the cells in agarose. In the present study, we isolated rabbit ChM-I precursor CDNA by reverse transcription-polymerase chain reactions. Northern blot analysis revealed that the expression of ChM-I mRNA occurred in a tissue-specific manner in cartilage. Moreover, the ChM-I mRNA level was markedly changed in response to growth and differentiation stimuli in primary cultured chondrocytes. Among others, fibroblast growth factor-2, transforming growth factor-beta, and parathyroid hormone related peptide each markedly down-regulated the expression of ChM-I mRNA in chondrocytes. These results indicated that the expression ChM-I was placed under the dynamic control of local growth and differentiation factors.

Amino Acid Sequence↗

Comparison of rapid automated laser fluorescence analysis of DNA fingerprints with four other computer-assisted approaches for studying relationships between Acinetobacter baumannii isolates.

The relationships between isolates suggested by a novel DNA typing method (RAPD-ALFA) that combines randomly amplified polymorphic DNA with automated on-line laser fluorescence analysis of DNA fragments were compared with those suggested by four other computer-assisted typing strategies (biotyping, antibiogram typing, pulsed-field gel analysis of chromosomal fingerprints and arbitrarily-primed DNA amplification with three different primers) for 25 isolates of Acinetobacter baumannii obtained from 12 different hospitals in four countries over a period of 12 years. The results obtained by cluster analysis with two different software packages confirmed that the relationships suggested by RAPD-ALFA were robust and essentially similar to those suggested by the other more laborious computer-assisted typing methods. The technique of RAPD-ALFA appears to offer the possibility of routine on-line molecular identification and typing of isolates from particular hospital wards or units (e.g., intensive care units), and could, therefore, play a key role in the early recognition and prevention of outbreaks of infection.

Acinetobacter↗

Relationship of Staphylococcal isolates in a Moroccan hospital by comparing phenotypical and genotypical tests.

The relationship of Staphylococcus isolates was determined among a collection of 26 clinical strains at the Centre Hospitalier Universitaire de Rabat. These isolates originated principally from blood culture and wounds. In order to affirm the clonal origins of these isolates, six phenotype (biotype, anti-biotype, serotype, phage type), and genotype (random amplified polymorphic DNA, pulsed field gel electrophoresis) methods were used. Biotyping, anti-biotyping, phage and serotyping were generally not sufficient while many isolates remained non-phage typeable. Random amplified polymorphic DNA analysis used in epidemiological typing seemed suitable for S. epidermidis and S. haemolyticus. However, rigorous standardization will be needed to assure reliable results. Pulsed field gel electrophoresis discriminated more efficiently than random amplification polymorphic DNA analysis. This study attests to the suitability of two or more methods in combination for typing Staphylococcus isolates.

Bacterial Typing Techniques↗

Polymorphic molecular markers from anonymous nuclear DNA for genetic analysis of populations.

A simple method is presented for developing polymorphic, anonymous DNA markers suitable for population genetic studies. Anonymous DNA fragments are screened for sequence variability using a common mutation detection technique (single strand conformation polymorphism analysis; SSCP) and locus-specific PCR primers are designed for polymorphic DNA fragments. Detection of the markers by SSCP analysis coupled with sequence analysis of SSCP variants allows rapid screening while retaining information about the genealogical relationship among alleles. Variability detected for six markers was assessed in rainbow trout Oncorhynchus mykiss and was compared with variability detected by similar analysis of intron loci. Between three and 12 distinct alleles were observed at each marker locus, and average within-population heterozygosity ranged from 0.12 to 0.44. Advantages and limitations of the methodology for population genetic analysis are discussed.

Alleles↗

Rapid discrimination of Mycobacterium avium strains from AIDS patients by randomly amplified polymorphic DNA analysis.

A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform.

AIDS-Related Opportunistic Infections↗

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD, biochemical and morphological analysis.

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.

Chromatography, High Pressure Liquid↗

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains.

Streptococci from different collections and dairy materials were characterized by conventional and molecular methods. After amplification of the 16S-23S rDNA spacer region, all the strains referable to the genus Streptococcus exhibited a single polymerase chain reaction (PCR) product, allowing their differentiation from enterococci. Cleaving this PCR product with Hae III, two different restriction patterns could be observed, allowing Streptococcus salivarius DSM 20560T, Strep. thermophilus NCDO 822 and two strains of Streptococcus spp. to be gathered in one group and all the other strains in another. In order to achieve strain typing, all the cultures were investigated by random amplified polymorphic DNA (RAPD)-PCR analysis employing two selected primers. The results were treated by cluster analysis, appearing significantly consistent with both the taxonomic position and the origin of the strains. Pulsed-field gel electrophoresis (PFGE) of Sma I digests of the genomic DNA from 11 representative strains with decreasing levels of RAPD similarity allowed their diversity to be confirmed, even though RAPD-PCR proved to be less discriminating than PFGE analysis. The results are discussed with reference to the capability of the analytical procedures used to aid both identification and strain typing of streptococci, as well as the taxonomic structure of the species Strep. thermophilus.

Bacterial Typing Techniques↗

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa.

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.

Bacterial Typing Techniques↗

In vitro generation of cloned populations of Toxoplasma gondii.

Cloned populations were generated from Indian isolates of Toxoplasma gondii by transferring single tissue cysts from the brains of chronically infected mice to confluent murine macrophage (J774A.1) monolayers. The clones were then maintained continuously as tachyzoites in culture. Physical rupture of the tissue cysts and release of bradyzoites prior to seeding was found to be necessary for establishment of the parasite in culture. Although intact tissue cysts seeded over monolayers released bradyzoites spontaneously, they did not succeed in setting up an infection in the monolayers. Random amplified polymorphic DNA (RAPD)-PCR, which revealed distinct patterns for a clone and its progenitor, further confirmed the efficiency of the technique. The cloning technique was found to be simple and rapid compared to those involving limiting dilutions.

Animals↗

Monitoring of Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening of soppressata molisana, a typical Italian sausage.

AIMS: "Soppressata molisana", a fermented sausage produced in southern Italy, is commonly obtained without starter addition. However, the use of starter cultures is more and more recommended in meat fermentation processes in order to guarantee stable production performance. In this study, the survival of the Staphylococcus xylosus DSM 20266 was evaluated during the ripening of "soppressata molisana" fermented sausage. METHODS AND RESULTS: The fastest method of RAPD-PCR was employed for discrimination of the added strain from those naturally present during the ripening of the "soppressata molisana". The results obtained were confirmed by analysis of the DNA macrorestriction profile by PFGE. The electrophoretic pattern of bacterial total proteins was also studied, but clear differences between the different strains could not be detected. CONCLUSIONS: The RAPD technique was a valid tool for monitoring Staph. xylosus DSM 20266 in "sopressata molisana". SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility of monitoring the presence of Staph. xylosus strains during the ripening of fermented sausages by a reliable and repeatable technique such as RAPD.

Animals↗

Epidemiological evidence for dormant Cryptococcus neoformans infection.

To date, the time of acquisition of a Cryptococcus neoformans infectious strain has never been studied. We selected a primer, (GACA)(4), and a probe, CNRE-1, that by randomly amplified polymorphic DNA (RAPD) analysis and restriction fragment length polymorphism (RFLP), respectively, regrouped strains from control samples of C. neoformans var. grubii environmental isolates according to their geographical origins. The two typing techniques were then used to analyze 103 isolates from 29 patients diagnosed with cryptococcosis in France. Nine of the 29 patients lived in Africa a median of 110 months prior to moving to France; 17 of the patients originated from Europe. Results showed a statistically significant clustering of isolate subtypes from patients originating from Africa compared to those from Europe. We conclude that the patients had acquired the C. neoformans infectious strain long before their clinical diagnoses were made.

Cryptococcosis↗

Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction.

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.

Bacterial Toxins↗