Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis strains.

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.

Genotyping of present-day and historical Geobacillus species isolates from milk powders by high-resolution melt analysis of multiple variable-number tandem-repeat loci.

Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10(4) spores · g(-1)) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.

Correlation Analysis of Ultrasonic Stress Wave Characteristics and the Destructive Strength Measurements in Cylindrical Wooden Structure.

The utility sector has been employing ultrasonic-based nondestructive evaluation (NDE) to determine the cross-sectional groundline integrity of wooden utility poles. While it is far less invasive than other methods, its efficacy has not been thoroughly studied. This study aims to fill this technical gap by analyzing the correlation between the propagational characteristics of the ultrasonic stress wave using a novel embedded waveguide technique and the existing destructive testing methods. The proposed embedded waveguide technique excites diffusive Rayleigh mode (AW2) propagating in the shell region of the cross-sectional plane. This discovery allows a direct examination of the shell region condition through stress wave analysis. By employing the Gabor wavelet transformation and the model-based arrival region identification, this proposed technique extracts the propagation velocity and the associated spectral response of AW2. This study uses the static break assessment per ASTM 1036 Standard Test Methods And The longitudinal compression test per ASTM D143-14 "secondary method" to quantify the cross-sectional strength of the test specimen. This work performs a comprehensive correlation analysis between the extracted AW2 features and the associated destructive test. An overall correlation R2 from 0.2 to 0.5 is achieved between the AW2 features and the static break test results. An overall correlation of R2 of 0.4 is achieved for 30-35 ft poles in the longitudinal compression test.

The physico-chemical characterization of casein-modified surfaces and their influence on the adhesion of spores from a Geobacillus species.

To gain a better understanding of the factors influencing spore adhesion in dairy manufacturing plants, casein-modified glass surfaces were prepared and characterized and their effect on the adhesion kinetics of spores from a Geobacillus sp., isolated from a dairy manufacturing plant (DMP) was assessed using a flow chamber. Surfaces were produced by initially silanizing glass using (3-glycidyloxypropyl) trimethoxysilane (GPS) or (3-aminopropyl) triethoxysilane to form epoxy-functionalized (G-GPS) or amino-functionalized glass (G-NH(2)) substrata. Casein was grafted to the G-GPS directly by its primary amino groups (G-GPS-casein) or to G-NH(2) by employing glutaraldehyde as a linking agent (G-NH(2)-glutar-casein). The surfaces were characterised using streaming potential measurements, contact angle goniometry, infrared spectroscopy and scanning electron microscopy. The attachment rate of spores suspended in 0.1 M KCl at pH 6.8, was highest on the positively charged (+14 mV) G-NH(2) surface (333 spores cm(-2) s(-1)) compared to the negatively charged glass (-22 mV), G-GPS (-20 mV) or G-GPS-casein (-21 mV) surfaces (162, 17 or 6 spores cm(-2) s(-1) respectively). Whilst there was a clear decrease in attachment rate to negatively charged casein-modified surfaces compared to the positively charged amine surface, there was no clear relationship between surface hydrophobicity and spore attachment rate.

Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line.

AIM: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean-in-place regimes. METHODS AND RESULTS: Exposing sessile spores to 1% NaOH at 65°C for 30min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from -20 to -32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l(-1) KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log(10) CFU cm(-2) ) than untreated spores (3·6 log(10) CFU cm(-2) ). CONCLUSIONS: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. SIGNIFICANCE OF STUDY: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra.

AIMS: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. MATERIALS AND RESULTS: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore's negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. CONCLUSIONS: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.

Recovery of spores from thermophilic dairy bacilli and effects of their surface characteristics on attachment to different surfaces.

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of approximately 10(7) spores ml(-1); spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from -10 to -20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 x 10(4) spores cm(-2)), with fewer spores attaching to glass (3 x 10(3) spores cm(-2)). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (approximately 1 x 10(3) spores cm(-2)), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.

The effect of transient temperatures on the growth of Salmonella typhimurium LT2 in gelatin gel.

The growth of colonies of Salmonella typhimurium derived from single immobilised cells was studied while subjected to constant and sinusoidally-varying temperatures. The bacteria grew in microbiological culture media adjusted to different pH and sodium chloride (NaCl) concentration and solidified with gelatin that was contained within a cassette formed between sheets of PVC film that allowed gaseous exchange. At pH 7.0 and 0.5% (w/v) NaCl and either 12 degrees C or 20 degrees C, S. typhimurium grew at a rate similar to that in liquid medium. The decrease in growth rate at 20 degrees C at a lower pH or higher NaCl concentration was greater in the case of immobilised cells than for cells in liquid medium. The change in the numbers of viable bacteria was measured with time under sinusoidally-varying temperatures between 4 and 22 degrees C and between 12 and 22 degrees C of period in the range 12 to 480 min. The experimental growth curves were compared with predictions based on isothermal growth in liquid medium. The discrepancies between experiment and prediction were greater for gels stressed by NaCl or pH than for gels at pH 7.0 and containing 0.5% (w/v) NaCl, consistent with the isothermal observations.

Connectivity, compatibility, and transcription services.

A transcription service must be able to access the dictation system and other systems in transcribing a client's medical dictation. Hence, connectivity and compatibility are vital considerations.

Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.

Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

Suitability of live and fire-killed small-diameter ponderosa and lodgepole pine trees for manufacturing a new structural wood composite.

Finding alternative uses for raw material from small-diameter trees is a critical problem throughout the United States. In western states, a lack of markets for small-diameter ponderosa pine (Pinus ponderosa) and lodgepole pine (Pinus contorta) can contribute to problems associated with overstocking. To test the feasibility of producing structural composite lumber (SCL) beams from these two western species, we used a new technology called steam-pressed scrim lumber (SPSL) based on scrimming technology developed in Australia. Both standing green and fire-killed ponderosa and lodgepole pine logs were used in an initial test. Fire-killed logs of both species were found to be unsuitable for producing SPSL but green logs were suitable for producing SPSL. For SPSL from green material, ponderosa pine had significantly higher modulus of rupture and work-to-maximum load values than did SPSL from lodgepole pine. Modulus of elasticity was higher for lodgepole pine. The presence of blows was greater with lodgepole pine than with ponderosa. Blows had a negative effect on the mechanical properties of ponderosa pine but no significant effect on the mechanical properties of SPSL from lodgepole pine. An evaluation of non-destructive testing methods showed that X-ray could be used to determine low density areas in parent beams. The use of a sonic compression wave tester for NDE evaluation of modulus of rupture showed some promise with SPSL but requires further research.

Chromatin replication in vitro. Properties of a HeLa nuclear system.

An in vitro HeLa chromatin replication system was developed and characterized. Purified nuclei synthesized DNA from replication forks which were undergoing chain elongation at the time of cell fractionation. DNA replication in isolated nuclei was deficient in DNA maturation functions; the Okazaki fragments synthesized in vitro accumulated and were ligated to high-molecular-weight DNA with low efficiency. The chromatin protein components of nuclei incubated in the DNA replication assay conditions were investigated for displacement, degradation, and exchange. Displacement of total nuclear protein during in vitro incubation occurred to the extent of 3%, and involved only the nonhistone nuclear proteins. Degradation was not detectable, assayed both by loss of acid-soluble radioactivity and by protein electrophoretic patterns in polyacrylamide gels. No detectable protein was exchanged from chromatin to exogenous DNA.

Rapid turnover of the histone-ubiquitin conjugate, protein A24.

The specific activity of protein A24 was found to exceed that of the core histones by 2-3 fold following a brief labeling period. Accordingly, the A24 protein was found to be unstable, with a decay half-life of 90 minutes. When decay of the ubiquitin moiety was measured, it was found to turn over more extensively than the H2A moiety.