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Pollutants in an environment can have long-term implications for the species living there, resulting in local adaptations with implications for their genetic structure. Heavy metal pollutants infiltrate soils and groundwater, bioaccumulate in food webs, and negatively impact biota. In this study, we investigated the degree to which the genetic structure and variability of the slender green-winged grasshopper (Aiolopus thalassinus (Fabricius) (Orthoptera: Acrididae)) were impacted by heavy metal pollution and distance. We used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method to examine the genetic variability of populations in 3 heavy metal-polluted and 3 unpolluted locations across varying geographical distances in Egypt. The heavy metal concentrations of cadmium, copper, lead, and zinc were measured from the grasshopper tissue and soils. Sixty-nine unique and polymorphic bands were produced by 4 primers. Cluster and principal component analyses separated the populations inside and outside Cairo into 2 main branches, which were further divided into smaller branches corresponding to their geographical regions. We found no differences in the Shannon genetic diversity index between populations or with increasing heavy metal concentrations in either the soil or the grasshopper tissue. Our results showed a greater genetic variation among populations than between populations within the same location, indicating populations within locations were less differentiated than those between locations. The moderate correlation between genetic similarity and spatial distance suggests geographical isolation influenced grasshopper population differentiation. Based on the RAPD analysis, environmental pollutants and geographical distances impact the A. thalassinus population structure, potentially restricting gene flow between sites even at small spatial scales. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as an important global threat in recent years. The objective of the present study was to characterize the molecular characteristics, antibiotic resistance pattern and the distribution of virulence factors in CRKP isolated from different clinical specimens. A total of 60 clinical CRKP isolates were collected from clinical samples. Based on Clinical Laboratory Standards Institute guidelines, antimicrobial susceptibility testing was assessed by the disk diffusion method. Carbapenem and aminoglycoside resistance determinants in addition to virulence genes were inspected by PCR. Molecular characteristics of CRKP isolates were analyzed by random amplified polymorphic DNA (RAPD) PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR. All isolates were resistant to imipenem, meropenem, cefoxitin, levofloxacin, cefotaxime, ceftazidime and ciprofloxacin. Resistance to tetracycline, gentamicin and kanamycin were detected in 53%, 75% and 72% of isolates, respectively. The most common carbapenem resistance genes were OXA-48 (28.5%) and NDM (22%). The most common aminoglycosides resistance genes were aac6´Ib (57%) and aac(3)-IVa (28%). The most prevalent virulence genes were mrkD (82%), entB (62%) and ybts (58%). ERIC and RAPD analyses revealed 55 and 53 different patterns of CRKP isolates, respectively. We conclude that CRKP infections have been associated with different genotypes and that the carbapenemase type (OXA-48) and AME gene (aac6´-Ib) were widely distributed in CRKP isolates in our hospital. Continued compliance with existing phenotypes and genotypes, and strict enforcement of infection control guidelines, are recommended approaches for the prevention and dissemination of these strains. [ABSTRACT FROM AUTHOR]

Aims Brewing yeasts are classified into two species- Saccharomyces pastorianus and Saccharomyces cerevisiae. Most of the brewing yeast strains are natural interspecies hybrids typically polyploids and their identification is thus often difficult giving heterogenous results according to the method used. We performed genetic characterization of a set of the brewing yeast strains coming from several yeast culture collections by combination of various DNA-based techniques. The aim of this study was to select a method for species-specific identification of yeast and discrimination of yeast strains according to their technological classification. Methods and Results A group of 40 yeast strains were characterized using PCR-RFLP analysis of ITS-5·8S, NTS, HIS4 and COX2 genes, multiplex PCR, RAPD-PCR of genomic DNA, mtDNA-RFLP and electrophoretic karyotyping. Reliable differentiation of yeast to the species level was achieved by PCR-RFLP of HIS4 gene. Numerical analysis of the obtained RAPD-fingerprints and karyotype revealed species-specific clustering corresponding with the technological classification of the strains. Taxonomic position and partial hybrid nature of strains were verified by multiplex PCR. Differentiation among species using the PCR-RFLP of ITS-5·8S and NTS region was shown to be unreliable. Karyotyping and RFLP of mitochondrial DNA evinced small inaccuracies in strain categorization. Conclusions PCR-RFLP of HIS4 gene and RAPD-PCR of genomic DNA are reliable and suitable methods for fast identification of yeast strains. RAPD-PCR with primer 21 is a fast and reliable method applicable for differentiation of brewing yeasts with only 35% similarity of fingerprint profile between the two main technological groups (ale and lager) of brewing strains. Significance and Impact of the Study It was proved that PCR-RFLP method of HIS4 gene enables precise discrimination among three technologically important Saccharomyces species. Differentiation of brewing yeast to the strain level can be achieved using the RAPD-PCR technique. [ABSTRACT FROM AUTHOR]

Viruses are highly abundant in soils with their numbers exceeding those of cooccurring bacterial cells by 10- to over 1000-fold. Water and organic matter content influence the magnitude of the viral-to-bacterial ratio in soils; thus, ecosystem type and land use shape interactions between viral and host microbial communities in soils. Less understood are the shorter term interactions between viral and host communities that ultimately maintain the large viral standing stock within soils. This study examined short-term dynamics of viral and bacterial communities in soils to determine whether the growth of soil bacterial communities results in the production of soil viruses, and if viral community responses occur within specific populations. In microcosms amended with different carbon sources, increases in viral abundance (VA) accompanied increases in bacterial abundance (BA) and bacterial respiration rate (BRR). The timing and intensity of increases in BA, VA and BRR were different across C sources suggesting differences in the predominant mode of viral replication within growth-stimulated bacterial populations. Moreover, compositional changes occurred in soil bacterial and viral communities indicating that new viral production arose from a subset of host populations. To our knowledge, these are the first observations of soil viral populations responding to short-term changes in soil bacterial communities [ABSTRACT FROM AUTHOR]

The diversity of 55 Salmonella-specific bacteriophages isolated from 191 fecal samples of poultry and swine from farms located in diverse geographic areas of Spain was determined using lysis profiling, DNA restriction and random amplification of polymorphic DNA (RAPD-PCR). Among them, lysis profiling and RAPD-PCR exhibited 100% typeability and DNA restriction 96%, with discriminatory power of 0.978 (± 0.016), 0.938 (± 0.028) and 0.982 (± 0.013), respectively. The highest concordance (0.974) was that between RAPD-PCR and lysis profiling. None of the bacteriophages isolated from poultry and swine shared any DNA restriction or RAPD-PCR patterns and only two lysis profiles were common to bacteriophages isolated from poultry and swine. The major part of the lysis and RAPD-PCR profiles from the bacteriophages isolated from poultry included only one or two bacteriophages, while those obtained from swine contained more than two bacteriophages. Overall, our results provide evidence of the remarkable diversity exhibited by bacteriophages of Salmonella in farm animals. Moreover, they also show that RAPD-PCR may also be suitable for the pre-screening of the diversity of Salmonella bacteriophages for further use in biocontrol and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Aims The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli. Methods and Results Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L. innocua and E. coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable ( VBNC) state after the pulsed light treatment. A high percentage of L. innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E. coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species ( ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH- DA and RAPD- PCR, respectively. Conclusions A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample−1. Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes. Significance and Impact of the Study The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment. [ABSTRACT FROM AUTHOR]

Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies. [ABSTRACT FROM AUTHOR]

The recent boom in phage therapy and phage biocontrol requires the design of suitable cocktails of genetically different bacteriophages. The current methods for typing phages need significant quantities of purified DNA, may require a priori genetic information and are cost and time consuming. We have evaluated the randomly amplified polymorphic DNA (RAPD)-PCR technique to produce unique and reproducible band patterns from 26 different bacteriophages infecting Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus lactis, Escherichia coli, Streptococcus thermophilus, Bacillus subtilis and Lactobacillus casei bacterial strains. Initially, purified DNA and phage suspensions of seven selected phages were used as a template. The conditions that were found to be optimal 8 μM of 10-mer primers, 3 μM magnesium oxalacetate and 5% dimethyl sulfoxide. The RAPD genomic fingerprints using a phage titer suspension higher than 109 PFU mL−1 were highly reproducible. Clustering by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm correlated largely with genetically different phages infecting the same bacterial species, although closely related phages with a similar DNA restriction pattern were indistinguishable. The results support the use of RAPD-PCR for quick typing of phage isolates and preliminary assessment of their genetic diversity bypassing tedious DNA purification protocols and previous knowledge of their sequence. [ABSTRACT FROM AUTHOR]

Aims: Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages. Methods and Results: Different points during the production of a traditional fermented sausage type ( fuet) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers’ hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source. Conclusions: The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin. Significance and Impact of the Study: This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment. [ABSTRACT FROM AUTHOR]

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non- Saccharomyces yeasts ( Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced. [ABSTRACT FROM AUTHOR]

Aim:  The aim of this study was to develop a specific and sensitive identification method for two Aureobasidium pullulans biocontrol strains, CF10 and CF40, based on a sequence-characterized amplified region (SCAR) derived from RAPD – and multiplex-RAPD PCR analysis. Methods and Results:  The random amplified polymorphic DNA (RAPD) and multiplex RAPD-PCR techniques were used for a preliminary screening of A. pullulans genetic variability among 200 isolates. This approach allowed the selection of ten fragments present solely in strains CF10 and CF40. The RAPD fragments were cloned, sequenced and used to design two SCAR primers. Two primer pairs obtained from SCH3RAPD fragment of CF 40 and 6RAPD of CF10 were highly specific and sensitive. Conclusions:  In this study, we developed strain-specific multiplex-PCR based on sequence-characterized amplified region (SCAR) markers to simultaneously detect both strains in a single PCR. Significance and Impact of the Study:  This new multiplex-PCR provides a valuable tool for specific and sensitive identification of CF10 and CF40, and could be used in studies on the efficacy and persistence of introduced strains of A. pullulans for fire blight control. [ABSTRACT FROM AUTHOR]

A multiphasic approach was used to investigate the yeast ecology in Italian fermented sausages. Culture-dependent and -independent methods were applied to identify the yeast species during the maturation process and to characterize the numerically dominant species. Plating analysis and subsequent molecular identification of the isolates highlighted the dominance of Debaryomyces hansenii, but at least other three yeast species – Candida zeylanoides, Pichia triangularis and Metschnikowia pulcherrima – contributed to the fermentation as well. Direct denaturing gradient gel electrophoresis analysis confirmed that D. hansenii was the main yeast species present and its activity was also demonstrated. No other yeasts species were detected on the direct denaturing gradient gel electrophoresis gels, whereas DNA of Penicillium farinosum, Penicillium viridicatum and Mucor racemosus were present. Molecular characterization by RAPD-PCR analysis of the D. hansenii isolates demonstrated a shift in its population from the beginning to the end of the maturation of the sausages. Strains present during the early stages of the fermentation were grouped in clusters that differed from those isolated in the final phases of the maturation, underlining the genetic differences between these two populations of D. hansenii. However, all the isolates were able to grow in the presence of 3.5% sodium chloride and at 10°C, evidence that these parameters did not select the species present at the end of the maturation period. [ABSTRACT FROM AUTHOR]

Aims: To monitor and select genetically characterized strains of Beauveria brongniartii to be used as microbiological control agents against Melolontha melolontha in different climatic conditions of the Valley of Aosta (northwest Italy). Methods and Results: Molecular random amplified polymorphic DNA markers allowed monitoring of five B. brongniartii strains (C2, F, K2, N3 and W2) in field trials. Ten sites were chosen at Jovençan, Saint-Pierre and Quart areas, where a mixture of the five strains colonizing rye kernels was applied to the soil of each M. melolontha infested site. Growth, persistence and virulence on M. melolontha larvae of five fungal strains were evaluated in two subsequent 24-month studies. Beauveria brongniartii grew best at the Jovençan sites. Not only did strain F persist better than the other strains in most soil samples but it was also the most virulent strain. Strain F was isolated the most frequently from infected M. melolontha larvae recovered from the test sites. A general decrease in the larvae rate was detected in the test field soil. Conclusions: Strain F of B. brongniartii was better than other strains in growth, persistence and virulence against M. melolontha larvae in the test site soil. Significance and Impact of the Study: Results obtained from preliminary field trials support the use of strain F as a biological control agent against M. melolontha in the Valley of Aosta even if further targeted studies are still necessary. [ABSTRACT FROM AUTHOR]

The objectives of the study were to develop a specific procedure for quantification and identification of Bacillus thuringiensis kurstaki HD1, which is used as a biopesticide, and to quantify its presence in different kinds of cabbage for human consumption. We found that B. thuringiensis kurstaki HD1 can be distinguished from other B. thuringiensis strains by its unique random amplification of polymophic DNA-PCR pattern with the OPA9 primer and the presence of the flagellin genes, as detected by the primers FLAB1 and FLAB2. We detected from one to 100 Bacillus cereus-like bacteria in 10 batches of five different cabbage products for consumption. As many as 73 out of 134 isolates (53.7%) were identical with B. thuringiensis kurstaki HD1. The results show that B. thuringiensis kurstaki HD1 from biopesticides can be found in vegetables for human consumption. [ABSTRACT FROM AUTHOR]

Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga. A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galvão & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses support monophyly for A. albitarsis s.s., A. albitarsis B, and A. deaneorum. Anopheles marajoara, as identified by RAPD banding patterns, was either polyphyletic or paraphyletic in all phylogenetic analyses. The phylogenetic pattern and within-species genetic distances observed in A. marajoara suggest the existence of a previously unidentified species (species E) in northern Brazil and Venezuela. Diagnostic RAPD bands were unable to distinguish between A. marajoara and species E, probably because of the low number of correlated bands used to identify species and weaknesses of the RAPD technique, in particular, violations of the untested assumption of homology of comigrating bands. A. marajoara (even without species E) is paraphyletic with respect to A. deaneorum: if A. deaneorum is a separate species from A. marajoara, then A. marajoara may consist of two or more species in Amazonian Brazil. Based on mtDNA COI sequences, there are at least four phylogenetic species within the Albitarsis Complex: A. albitarsis s.s., A. albitarsis B, A. marajoara, and species E; the species status of A. deaneorum is ambiguous. [ABSTRACT FROM AUTHOR]

Abstract: Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel’s ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92–99%) between them. [Copyright &y& Elsevier]

b. martin, m. garriga, m. hugas and t. aymerich. 2005.To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects.Species-specific PCR and partial sequencing of 16S rRNA andsodAgenes were used to identify enterococcal population.Enterococcus faeciumwas the most frequently isolated species followed byE. faecalis,E. hiraeandE. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All theE. faecalisstrains carried virulence genes (efaAfs,esp,aggandgelE) and allE. faeciumisolates carriedefaAfmgene.Enterococcus faecalisshowed higher antibiotic resistance than the other species. Only oneE. faeciumstrain showedvanAgenotype (high-level resistance to glycopeptides) andE. gallinarumandE. casseliflavus/flavescensisolates showedvanC1andvanC2/C3genotypes (low-level resistance only to vancomycin) respectively.E. faecalishas been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products.The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed. [ABSTRACT FROM AUTHOR]

• Background and Aims The saltbush Atriplex halimus is a chenopodiaceous plant well adapted to dry saline habitats and widely distributed in the Mediterranean Basin. A study was carried out to analyse the genetic diversity of A. halimus at the level of the Mediterranean Basin.• Methods To assess the intra- and interpopulational variation of A. halimus a total of 51 populations and six plants per populations was analysed with the RAPD-PCR technique. For the study of the phylogeny of the populations, 21 samples of A. halimus and seven samples of other species of Atriplex were analysed by the sequencing of the ITS (internal transcribed spacer) region of the ribosomal DNA.• Key Results The AMOVA analysis of the RAPD results showed that populations were divided into two discrete genetic groups, as the variation among groups accounted for 54·36 % of the total variance of the collection. At the same time, the intrapopulational diversity was high, as 301 out of 306 plants analysed constituted an individual RAPD haplotype. The sequencing of the ITS region also showed a significant separation of the two genetic groups, with a genetic distance of 0·023 nucleotide substitutions per site. Using A. breweri, A. canescens, A. glauca and A. prostrata as outgroups in the phylogenetic analysis, A. breweri and A. canescens are the species closest to A. halimus from this group, while A. prostrata is the most distant.• Conclusions The present work indicates that two genetic groups of A. halimus can be distinguished after analysing the genetic diversity of 51 populations from ten countries in the Mediterranean Basin. [ABSTRACT FROM AUTHOR]

Abstract: Saccharomyces cerevisiae strains isolated from two different wineries in central Italy were subjected to enological and molecular characterization to investigate the influence of the winery environment. One of the selected wineries is a modern, working winery, whereas the second one was abandoned since 1914 and was located in an artificial cavern. The results obtained by our analysis of the fermentation traits underline the selectivity of the winery environment (winery effect), since strains isolated from the industrial winery showed higher values for characters typically subjected to selective pressure, such as maximum capability to produce ethanol, fermentation rate and SO2 resistance. Pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD)-PCR and SAU-PCR were carried out to assesss genetic differences between the two populations studied. Only RAPD-PCR could distinguish between the two populations based on their provenience, whereas PFGE and SAU-PCR gave profiles shared between strains isolated from the industrial and former winery. Moreover, analysis of the karyotypes suggested the presence of chromosomal-length polymorphism; differences in the size and number of chromosomes between the two groups of isolates, as well as within each group, were observed. [Copyright &y& Elsevier]

s. barnini, c. dodi and m. campa. 2004. To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Randomly amplified polymorphic DNA (RAPD)-PCR was used to verify the species composition of commercial dairy starters and to detect possible shifts in strain composition of these cultures. After RAPD-PCR analysis, not all the strains isolated in the years 2001 and 2002 fell within the same dendrogram cluster of the strains isolated in the year 2000 and used as reference strains. Changes in composition of the microbial population and/or voluntary immission of new biotypes with respect to the original strain formulation had occurred in the starters. The microbial composition of modern dairy starters represents a key point because the complex relationships among microorganisms can easily be altered. Little variations in the microbial composition could have unexpected effects on cheese quality. [Copyright &y& Elsevier]

l.h. travassos, m.n. pinheiro, f.s. coelho, j.l.m. sampaio, v.l.c. merquior and e.a. marques. 2004. To investigate phenotypic aspects including biotyping, drug susceptibility and production of extracellular enzymes and genetic diversity of Stenotrophomonas maltophilia clinical strains obtained from seven hospitals in Rio de Janeiro, Brazil. Thirty-nine S. maltophilia strains were investigated by biotying, susceptibility testing, extracellular enzymes detection and by randomly amplified polymorphic DNA (RAPD)-PCR. Biotyping distinguished 13 biotypes among 39, and one of them was prevalent. The majority of the strains produced DNase, gelatinase and haemolysin. Protease, lipases and phospholipase C activities were observed in highly variable amounts. None of the strains was elastase producer. The percentage of full susceptibility, by agar dilution, was 100, 94·8, 81·6 and 26·3% for trimethoprim/sulphametoxazole, ticarcillin/clavulanate, ciprofloxacin and ceftazidime, respectively. Thirty-three RAPD-PCR profiles were obtained suggesting multiple sources of acquisition. The results pointed out the necessity of monitoring S. maltophilia especially in critical hospital wards, to assure effective control measures. Despite of the genetic diversity among the strains, in two situations it was observed indistinguishable RAPD-PCR profiles among strains isolated from different patients who had been hospitalized in the same hospital ward, suggesting the possibility of nosocomial transmission that until now has been rarely related. [ABSTRACT FROM AUTHOR]

c. andrighetto, g. marcazzan and a. lombardi. 2004. This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time. [ABSTRACT FROM AUTHOR]

Abstract Aims: The goal of this study was to develop a reproducible method for molecular typing strains of Oenococcus oeni , and also to apply it in the study of population dynamics of these strains during malolactic fermentation of wine. Methods and Results: A new method of multiplex randomly amplified polymorphic DNA (RAPD)-PCR has been developed, based on the combination of one random 10-mer and one specific 23-mer oligonucleotide in a single PCR. This method generates unique and discriminant DNA profiles for strains of O. oeni . The strains of this species were also clearly distinguished from other species of lactic acid bacteria. The method was applied to study the dynamics of O. oeni strains during malolactic fermentation, in three vintages in the same cellar. Conclusions: A fast and reliable method for typing strains of O. oeni has been designed and optimized. It improves the reproducibility and rapidity of conventional RAPD-PCR, and it has been validated monitoring the population dynamics during malolactic fermentation. Significance and Impact of the Study: This method will be a good tool to study the population dynamics of bacteria during malolactic fermentation and to evaluate the performance of new malolactic starter cultures and their dominance over the native microbiota. [ABSTRACT FROM AUTHOR]

Abstract Aims: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. Methods and Results: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis . RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. Conclusion: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. Significance and Impact of the Study: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread. [ABSTRACT FROM AUTHOR]

The aim of the study was to characterise, quantitatively and qualitatively, the yeast microbiota found during spontaneous fermentation of grape musts obtained from the red grape varieties 'Rondo' and 'Regent'. For the research, grapes originating from two vineyards (Srebrna Góra and Spotkaniówka) located in southern Poland were used. Musts of the studied grape varieties provided a favourable environment for the growth of microorganisms. Musts obtained from fruits from the Spotkaniówka vineyard were characterised by a higher overall number of yeasts. A maximum number of cells were recorded from the fourth to the ninth day of the process, in all of the analysed musts. The growing season of 2013 proved to be the least favourable for the growth of microorganisms on grapes, which resulted in their smaller number in the batches during spontaneous fermentation. During the process, succession of different groups of yeasts was observed. It was initiated by Hanseniaspora uvarum and Candida railenensis strains, which, with the progress of spontaneous fermentation, were replaced by Saccharomyces cerevisiae strains. Metschnikowia pulcherrima and Pichia membranifaciens yeasts were identified at both the beginning and the end of the process. During fermentation, Zygoascus meyerae, Kluyveromyces lactis and Nakazawaea ishiwadae strains were also identified. [ABSTRACT FROM AUTHOR]