Bioinformatics analyses of <Emphasis Type="Italic">Shigella</Emphasis> CRISPR structure and spacer classification

Clustered regularly interspaced short palindromic repeats (CRISPR) are inheritable genetic elements of a variety of archaea and bacteria and indicative of the bacterial ecological adaptation, conferring acquired immunity against invading foreign nucleic acids. Shigella is an important pathogen for anthroponosis. This study aimed to analyze the features of Shigella CRISPR structure and classify the spacers through bioinformatics approach. Among 107 Shigella, 434 CRISPR structure loci were identified with two to seven loci in different strains. CRISPR-Q1, CRISPR-Q4 and CRISPR-Q5 were widely distributed in Shigella strains. Comparison of the first and last repeats of CRISPR1, CRISPR2 and CRISPR3 revealed several base variants and different stem-loop structures. A total of 259 cas genes were found among these 107 Shigella strains. The cas gene deletions were discovered in 88 strains. However, there is one strain that does not contain cas gene. Intact clusters of cas genes were found in 19 strains. From comprehensive analysis of sequence signature and BLAST and CRISPRTarget score, the 708 spacers were classified into three subtypes: Type I, Type II and Type III. Of them, Type I spacer referred to those linked with one gene segment, Type II spacer linked with two or more different gene segments, and Type III spacer undefined. This study examined the diversity of CRISPR/cas system in Shigella strains, demonstrated the main features of CRISPR structure and spacer classification, which provided critical information for elucidation of the mechanisms of spacer formation and exploration of the role the spacers play in the function of the CRISPR/cas system.     

Occurrence and characterisation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">diarizonae</Emphasis> serovar 61: k: 1, 5, (7) in sheep in the federal state of Thuringia,Germany

Background

The occurrence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) and other Salmonella organisms in sheep in the German federal state of Thuringia was examined for the first time. Pooled faecal samples from 90 flocks located in this state were monitored.

Results

Only SASd was detected in 74 (82.2%) out of the 90 sheep herds, other Salmonella serovars were not identified. A positive correlation was found between the flock size and the detection probability of SASd. Despite the agent’s high prevalence, clinical symptoms of a disease exclusively due to SASd have not been observed. The SASd strains were characterised by macrorestriction analysis, antimicrobial testing and the biochemical profile. All strains were sensitive to 13 out of 14 antimicrobial substances and resistant to only sulfamethoxazole. The high number of macrorestriction groups of SASd strains indicated a low clonality of the serovar.

Conclusions

Data from sheep derived foods and public health data in Germany strongly suggest that the significance of SASd for public health is considerably lower than that of serovars belonging to Salmonella enterica subspecies enterica. For this reason and because of the low disease-causing potential of SASd in sheep, it is worthwile to consider a reduction in ongoing activities from combating to monitoring serovar 61: k: 1, 5, (7) in the sheep population.

     

Metabolomic analysis of the mechanism of action of yerba mate aqueous extract on <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Introduction

Salmonella enterica serovar Typhimurium is a Gram-negative enteropathogen that infects millions of people worldwide each year; the emergence of drug-resistant strains has heightened the need for novel treatments. Aqueous extracts of yerba mate (Ilex paraguariensis) effectively inhibit drug-resistant S. Typhimurium in vitro. Some chemical constituents that contribute to the extract’s antibacterial activity have been identified, but the mechanism of action of the extract is still unknown.

Objectives

This study sought to gain insight into the antibacterial mechanism of yerba mate extract against S. Typhimurium.

Methods

Assays for catalase activity and membrane permeability were used to select time points for an LC-MS metabolomics analysis of S. Typhimurium intracellular components.

Results

Yerba mate extract induced changes in central carbon metabolism in S. Typhimurium, reduced catalase activity by means other than direct inhibition, and did not change membrane integrity despite a significant increase in the production of a cell wall precursor. Additional significant differences were observed in the global metabolic regulators alpha-ketoglutarate and acetylphosphate, the energy-related molecule NAD⁺, and in an unexpected match to the antibacterial compound yohimbine.

Conclusion

This work provides the first evaluation of the mechanism of action of yerba mate extract on S. Typhimurium, revealing a major impact on central carbon metabolism, catalase activity, and possible metabolic links to interference in energy production and membrane integrity. The putative identification of the antibacterial compound yohimbine and the many unidentified compounds provides additional avenues for future investigations of yerba mate compounds capable of traversing or binding to S. Typhimurium’s membrane.

     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Evaluation of lytic bacteriophages for control of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

Background

The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium.

Methods

The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.

Results

P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).

Conclusion

The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.

     

<Emphasis Type="Italic">In vivo</Emphasis> Proinflammatory Cytokine Production by CD-1 Mice in Response to Equipotential Doses of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG and <Emphasis Type="Italic">Salmonella enterica</Emphasis> Lipopolysaccharides

The capacities of relatively nontoxic lipopolysaccharide (LPS) from Rhodobacter capsulatus PG and highly potent LPS from Salmonella enterica serovar Typhimurium to evoke proinflammatory cytokine production have been compared in vivo. Intravenous administration of S. enterica LPS at a relatively low dose (1 mg/kg body weight) led to upregulation of TNF-α, IL-6, and IFN-γ production by non-sensitized CD-1 mice. LPS from R. capsulatus PG used at a four-times higher dose than that from S. enterica elicited production of almost the same amount of systemic TNF-α; therefore, the doses of 4 mg/kg LPS from R. capsulatus PG and 1 mg/kg LPS from S. enterica were considered to be approximately equipotential doses with respect to the LPS-dependent TNF-α production by CD-1 mice. Rhodobacter capsulatus PG LPS was a weaker inducer of the production of TNF-α, IL-6, and IFN-γ, as compared to the equipotential dose of S. enterica LPS. Administration of R. capsulatus PG LPS before S. enterica LPS decreased production of IFN-γ, but not of TNF-α and IL-6, induced by S. enterica LPS. Rhodobacter capsulatus PG LPS also suppressed IFN-γ production induced by S. enterica LPS when R. capsulatus PG LPS had been injected as little as 10 min after S. enterica LPS, but to a much lesser extent. Rhodobacter capsulatus PG LPS did not affect TNF-α and IL-6 production induced by the equipotential dose of S. enterica LPS. In order to draw conclusion on the endotoxic activity of particular LPSs, species-specific structure or arrangement of the animal or human immune systems should be considered.     

Design of a CRISPR-Cas system to increase resistance of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> to bacteriophage SPP1

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes.     

On the global CRISPR array behavior in class I systems

Background

Much effort is underway to build and upgrade databases and tools related to occurrence, diversity, and characterization of CRISPR-Cas systems. As microbial communities and their genome complements are unearthed, much emphasis has been placed on details of individual strains and model systems within the CRISPR-Cas classification, and that collection of information as a whole affords the opportunity to analyze CRISPR-Cas systems from a quantitative perspective to gain insight into distribution of CRISPR array sizes across the different classes, types and subtypes. CRISPR diversity, nomenclature, occurrence, and biological functions have generated a plethora of data that created a need to understand the size and distribution of these various systems to appreciate their features and complexity.

Results

By utilizing a statistical framework and visual analytic techniques, we have been able to test several hypotheses about CRISPR loci in bacterial class I systems. Quantitatively, though CRISPR loci can expand to hundreds of spacers, the mean and median sizes are 40 and 25, respectively, reflecting rather modest acquisition and/or retention overall. Histograms uncovered that CRISPR array size displayed a parametric distribution, which was confirmed by a goodness-of fit test. Mapping the frequency of CRISPR loci on a standardized chromosome plot revealed that CRISPRs have a higher probability of occurring at clustered locations along the positive or negative strand. Lastly, when multiple arrays occur in a particular system, the size of a particular CRISPR array varies with its distance from the cas operon, reflecting acquisition and expansion biases.

Conclusions

This study establishes that bacterial Class I CRISPR array size tends to follow a geometric distribution; these CRISPRs are not randomly distributed along the chromosome; and the CRISPR array closest to the cas genes is typically larger than loci in trans. Overall, we provide an analytical framework to understand the features and behavior of CRISPR-Cas systems through a quantitative lens.

Reviewers

This article was reviewed by Eugene Koonin (NIH-NCBI) and Uri Gophna (Tel Aviv University).

     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

Isolation and identification of <Emphasis Type="Italic">Salmonella</Emphasis> spp. from red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) and badgers (<Emphasis Type="Italic">Meles meles</Emphasis>) in northern Italy

Background

Salmonella spp. have been isolated from a wide range of wild animals. Opportunistic wild carnivores such as red foxes (Vulpes vulpes) and badgers (Meles meles) may act as environmental indicators or as potential sources of salmonellosis in humans. The present study characterizes Salmonella spp. isolated from the intestinal contents of hunted or dead red foxes (n?=?509) and badgers (n?=?17) in northern Italy.

Findings

Thirty-one strains of Salmonella belonging to 3 Salmonella enterica subspecies were isolated. Fourteen different serovars of S. enterica subsp. enterica were identified, among which were serovars often associated with human illness.

Conclusions

Wild opportunistic predators can influence the probability of infection of both domestic animals and humans through active shedding of the pathogen to the environment. The epidemiological role of wild carnivores in the spread of salmonellosis needs to be further studied.

     

In Vitro Characterization of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains with Inhibitory Activity on Enteropathogens for Use as Potential Animal Probiotics

The present study evaluates the probiotic properties of three Lactobacillus plantarum strains MJM60319, MJM60298, and MJM60399 possessing antimicrobial activity against animal enteric pathogens. The three strains did not show bioamine production, mucinolytic and hemolytic activity and were susceptible to common antibiotics. The L. plantarum strains survived well in the simulated orogastrointestinal transit condition and showed adherence to Caco-2 cells in vitro. The L. plantarum strains showed strong antimicrobial activity against enterotoxigenic Escherichia coli, Shiga toxin-producing E. coli, Salmonella enterica subsp. enterica serovar Typhimurium, Choleraesuis and Gallinarum compared to the commercial probiotic strain Lactobacillus rhamnosus GG. The mechanism of antimicrobial activity of the L. plantarum strains appeared to be by the production of lactic acid. Furthermore, the L. plantarum strains tolerated freeze-drying and maintained higher viability in the presence of cryoprotectants than without cryoprotectants. Finally, the three L. plantarum strains tolerated NaCl up to 8% and maintained >60% growth. These characteristics of the three L. plantarum strains indicate that they could be applied as animal probiotic after appropriate in vivo studies.     

<Emphasis Type="Italic">Sordaria fimicola</Emphasis>-like ascomycete isolated from <Emphasis Type="Italic">Pinus coulteri</Emphasis> needles in Slovakia

This is the first report of Sordaria fimicola-like ascomycete which was encountered during a diversity study of injured tissues of coulter pine in Slovakia. The fungus was identified as Sordaria fimicola by morphological analyses. Sequence analysis of internal transcribed spacer region (ITS) showed that the fungus is highly related to the ITS sequences of several S. fimicola isolates documenting wide ecological valence and geographical distribution of S. fimicola-like ascomycetes.     

Nucleotide diversity and phylogenetic relationships among <Emphasis Type="Italic">Gladiolus</Emphasis> cultivars and related taxa of family Iridaceae

The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA–trnH and trnL–trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA–trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL–trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.     

Combining de novo and reference-guided assembly with scaffold_builder

Genome sequencing has become routine, however genome assembly still remains a challenge despite the computational advances in the last decade. In particular, the abundance of repeat elements in genomes makes it difficult to assemble them into a single complete sequence. Identical repeats shorter than the average read length can generally be assembled without issue. However, longer repeats such as ribosomal RNA operons cannot be accurately assembled using existing tools. The application Scaffold_builder was designed to generate scaffolds – super contigs of sequences joined by N-bases – based on the similarity to a closely related reference sequence. This is independent of mate-pair information and can be used complementarily for genome assembly, e.g. when mate-pairs are not available or have already been exploited. Scaffold_builder was evaluated using simulated pyrosequencing reads of the bacterial genomes Escherichia coli 042, Lactobacillus salivarius UCC118 and Salmonella enterica subsp. enterica serovar Typhi str. P-stx-12. Moreover, we sequenced two genomes from Salmonella enterica serovar Typhimurium LT2 G455 and Salmonella enterica serovar Typhimurium SDT1291 and show that Scaffold_builder decreases the number of contig sequences by 53% while more than doubling their average length. Scaffold_builder is written in Python and is available at http://edwards.sdsu.edu/scaffold_builder. A web-based implementation is additionally provided to allow users to submit a reference genome and a set of contigs to be scaffolded.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

Diversity of CRISPR-Cas-mediated mechanisms of adaptive immunity in prokaryotes and their application in biotechnology

CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

Analysis of the ITS1/ITS2 nuclear spacers and the secondary structure of <Emphasis Type="Italic">5.8S</Emphasis> rRNA gene in endemic species <Emphasis Type="Italic">Bellevalia sarmatica</Emphasis> (Pall. ex Georgi) Woronow and related species of the subfamily scilloideae

Sequence variability of the ITS spacers and 5.8S rRNA gene was examined in 11 accessions of the subfamily Scilloideae, including seven accessions of rare and endangered species Bellevalia sarmatica from Volgograd region. The intraspecific polymorphism level of the examined ITS1–5.8S–ITS2 sequence of B. sarmatica accessions constituted 1.3%. The phylogenetic position of B. sarmatica within the genus Bellevalia was determined. It was demonstrated that B. sarmatica belonged to the section Nutantes, and the most closely related species were B. webbiana and B. dubia. Nucleotide substitutions in the 5.8S rRNA gene sequence of the analyzed Scilloideae accessions were identified and studied. The predicted secondary structure of 5.8S rRNA gene was constructed. It was demonstrated that in the examined accessions, mutations in the 5.8S rRNA gene were mainly localized in the third hairpin region and had no effect on the secondary structure of the 5.8S rRNA molecule.