Super-adhesive polymer-silica nanocomposite layers.

Atomized spray plasma deposition (ASPD) using a precursor mixture of 2-hydroxyethyl methacrylate and methacryloyl-functionalized 15 nm silica nanoparticles leads to the formation of poly(2-hydroxyethyl methacrylate)-silica nanocomposite layers. The direct application of these coatings to overlapping glass-glass joints gives rise to excellent in situ adhesion reaching 84 MPa shear bond strength and 6 GPa shear modulus prior to the onset of adherent (bulk glass) failure. This significant enhancement in interfacial adhesion arises due to the silica nanoparticle surface methacryloyl groups enhancing cross-linking throughout the nanocomposite layer.

Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada.

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.

SMILE: Simple, Mental Health, Initiative in Learning and Education.

CONTEXT: SMILE is a Simple, Mental health, Initiative in Learning and Education. SMILE was a pilot project introduced into an undergraduate clinical nursing program, Southern Cross University, Australia 2010. The program aimed to improve the knowledge and skills of third-year nursing students participating in their first clinical placement in mental healthcare. METHODS: Complementary to the clinical nursing program and the university curriculum, SMILE provided further training and support for student learning in mental healthcare. The SMILE project was a structured 15-day education program that covered the following topics: suicide prevention; psychosis; drugs and alcohol education; mental state exam; families and carers in mental health; and the Mental Health Act. The education sessions were one hour in duration. The educational material and resources were created from current research, literature and health service policy. A problem-based learning approach was used to support this education project. The dynamic factor related to SMILE was that it was based in the field. SMILE enabled the students to bridge a theory-practice gap and expand upon their current knowledge base as well as participate in ward activity. Twenty students attending their first clinical placement in mental healthcare participated in SMILE and were asked to complete a pre- and post- evaluation questionnaire before starting and upon completion of the 15-day project. RESULTS: The students participating in SMILE reported a greater understanding of mental healthcare issues and expressed a developing knowledge base and improved practical skill level. CONCLUSION: SMILE was a positive initiative that provided valuable feedback and opportunity to improve on clinical education in mental healthcare.

Mimicking a Stenocara beetle's back for microcondensation using plasmachemical patterned superhydrophobic-superhydrophilic surfaces.

A simple two-step plasmachemical methodology is outlined for the fabrication of microcondensor surfaces. This comprises the creation of a superhydrophobic background followed by pulsed plasma deposition of a hydrophilic polymer array. Microcondensation efficiency has been explored in terms of the chemical nature of the hydrophilic pixels and their dimensions. These results are compared to the hydrophilic-hydrophobic pattern present on the Stenocara beetle's back, which is used by the insect to collect water in the desert. Potential applications include fog harvesting, microfluidics, and biomolecule immobilization.

Substrate-independent approach for polymer brush growth by surface atom transfer radical polymerization.

A simple method for growing polymer brushes by atom transfer radical polymerization (ATRP) off solid surfaces has been devised. This entails pulsed plasmachemical deposition of a halogen-containing initiator layer, followed by either organic or aqueous phase controlled surface polymerization. The wide-scale applicability of this approach is exemplified by functionalizing flat substrates, microbeads, and nonwoven textiles.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.

AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity.

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.

Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at. 1999) as Streptococcus macedonicus (Tsakalidou et al. 1998).

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Nucleotide sequence and characterization of the cell envelope proteinase plasmid in Lactococcus lactis subsp. cremoris HP.

AIMS: The major cell envelope proteinase (lactocepin; EC 3.4.21.96) produced by Lactococcus lactis cheese starter bacteria is required for starter growth and acid production in milk. The aim of this study was to characterize a lactocepin plasmid from a L. lactis subsp. cremoris cheese starter strain. METHODS AND RESULTS: A restriction map of the lactocepin plasmid pHP003 from strain HP was constructed, fragments were cloned in Escherichia coli vectors, and the complete DNA sequence (13,433 bp) was determined. Among 120 industrial L. lactis starter strains screened, five contained the same specificity-type lactocepin as pHP003. The lactocepin gene in these strains was invariably linked with a partially-deleted abiB gene. CONCLUSION: The lactocepin specificity type of strain HP, conferred by a known configuration of key residues, is relatively uncommon. The gene is invariably linked with a partially deleted abiB gene on each lactocepin plasmid. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first complete sequence reported for a lactocepin plasmid, and provides the basis for better understanding, or manipulation, of lactocepin production.

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

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

Novel sucrose transposons from plant strains of Lactococcus lactis.

Lactococcus lactis strains isolated from vegetable products transferred the ability to ferment sucrose in conjugation experiments with the recipient strain L. lactis MG1614. Nisin production and sucrose fermentation were transferred together from two strains, but transfer also occurred from several other strains which did not produce nisin. Pulsed-field gel electrophoresis analysis showed that all transconjugants had acquired large chromosomal insertions at two main sites. Nisin sucrose transconjugants had gained inserts of 70 kb, while those that fermented sucrose without nisin production contained inserts of between 50 and 110 kb. Transconjugants from one donor had acquired a separate insertion of 55 kb which correlated with enhanced bacteriophage resistance, but contained neither nisin nor sucrose fermentation genes.

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction.

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.

Streptococcus waius sp. nov., a thermophilic Streptococcus from a biofilm.

Thermophilic streptococci were isolated from biofilms on stainless steel samples exposed to pasteurized skimmed milk and from dairy products from a dairy manufacturing plant. The phenotypic characters of these isolates were distinct from those of other thermophilic streptococci of dairy origin (Streptococcus thermophilus and Streptococcus bovis). Genotypic data [restriction endonuclease analysis, ribotyping, random amplified polymorphic DNA (RAPD) profiles, DNA-DNA hybridization and G + C contents] support the classification of these isolates as a new species. The sequence of the 16S rRNA was compared with that of 29 species of streptococci and shown to be significantly different. The sequence of the 16S-23S rRNA intergenic spacer region also differed from published sequences of closely related species. A fluorescent in situ hybridization probe prepared to a specific part of the 16S rRNA gene sequence was able to distinguish the unknown isolates from reference isolates of S. thermophilus and S. bovis. It is proposed that these thermophilic streptococcal isolates from a dairy environment be classified in the genus Streptococcus as a new species, Streptococcus waius (from waiu, the New Zealand Maori word for milk). The type strain is 3/1T (= NZRCC 20100T).

Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence.

The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp. lactis and ssp. cremoris differ by 9-10 bp (depending on strain), within the first 200 bp of the sequence. These differences were used to develop two methods of genetically differentiating lactis and cremoris strains. Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene. A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences. The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence. Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris. This method was used to examine lactococci isolated from raw milk. Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.

Characterization of lactococci isolated from minimally processed fresh fruit and vegetables.

Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus lactis subsp. lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the 16S rRNA gene. Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic DNA. Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products during the period they are intended to be eaten. Although these plant strains showed many similarities to strains of L. lactis used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental origin. Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose. Most of the bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin activity, or that produced a different bacteriocin with a narrow spectrum of activity. One strain produced nisin but was unable to ferment sucrose, properties that have been generally regarded as linked. These strains may have uses as biopreservatives for minimally processed plant products.

Improved microscopic identification of Clavibacter michiganensis subsp. sepedonicus cells by combining in situ hybridization with immunofluorescence.

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine-labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein-labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.

Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain.

The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISS1-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISS1-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1.

Detection of dairy Leuconostoc strains using the polymerase chain reaction.

This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe.

Molecular biology of lactococcal bacteriophage c2.

The 22163 bp genome of the lytic prolate-headed lactococcal phage c2 was fully sequenced. Mapping of restriction sites and RNA transcripts demonstrated the presence of early and late genes. Early and late promoters were identified. The early region contained 21 ORFs, with predicted protein products of 4.4-32.9 kDA, all reading right to left. Significant similarity was found between a putative protein encoded by an early region ORF and the erf (essential recombination function) gene product of Salmonella phage P22. The late genes for which a function has been identified, all of which read from left to right, included a possible holin gene, and genes encoding three major and six minor phage structural proteins. Analysis of the cohesive termini revealed complementary, non-symmetrical, 9-base single-stranded 3' extended DNAs. The exploitation of phage sequence data and analysis of phage genomes to find ways of inhibiting phage replication is discussed.