Viability and Diversity of the Microbial Cultures Available in Retail Kombucha Beverages in the USA.

Kombucha is a two-stage fermented sweetened tea beverage that uses yeast and lactic acid bacteria (LAB) to convert sugars into ethanol and lactate and acetic acid bacteria (AAB) to oxidize ethanol to acetate. Its popularity as a beverage grew from claims of health benefits derived from this vibrant microbial bioconversion. While recent studies have shed light on the diversity of cultures in Kombucha fermentation, there is limited information on the diversity, and especially viability, of cultures in retail beverages that advertise the presence of Kombucha and probiotic cultures. In this study, 12 Kombucha beverages produced by different manufacturers throughout the US were purchased and microbially characterized. Eight of the beverages contained viable Kombucha cultures, while 3 of the remaining 4 had viable Bacillus cultures as added probiotics. Amplicon profiling revealed that all contained Kombucha yeast and bacteria cells. The dominant yeasts detected were Lachancea cidri (10/12), Brettanomyces (9/12), Malassezia (6/12), and Saccharomyces (5/12). Dominant LAB included Liquorilactobacillus and Oenococcus oeni, and AAB were Komagataeibacter, Gluconobacter, and Acetobacter. One beverage had a significant amount of Zymomonas mobilis, an ethanol-producing bacterium from Agave cactus. While Kombucha beverages differ in the types and viability of cultures, all except one beverage contained detectable viable cells.

Insights from metagenome-assembled genomes on the genetic stability and safety of over-the-counter probiotic products.

The demand for and acceptance of probiotics is determined by their quality and safety. Illumina NGS sequencing and analytics were used to examine eight marketed probiotics. Up to the species level, sequenced DNA was taxonomically identified, and relative abundances were determined using Kaiju. The genomes were constructed using GTDB and validated through PATRICK and TYGS. A FastTree 2 phylogenetic tree was constructed using several type strain sequences from relevant species. Bacteriocin and ribosomally synthesized polypeptide (RiPP) genes were discovered, and a safety check was performed to test for toxins, antibiotic resistance, and genetic drift genes. Except for two products with unclaimed species, the labeling was taxonomically correct. In three product formulations, Lactobacillus acidophilus, Limosilactobacillus reuteri, Lacticaseibacillus paracasei, and Bifidobacterium animalis exhibited two to three genomic alterations, while Streptococcus equinus was found in one. TYGS and GDTB discovered E. faecium and L. paracasei in distinctly different ways. All the bacteria tested had the genetic repertoire to tolerate GIT transit, although some exhibited antibiotic resistance, and one strain had two virulence genes. Except for Bifidobacterium strains, the others revealed a variety of bacteriocins and ribosomally synthesized polypeptides (RiPP), 92% of which were unique and non-homologous to known ones. Plasmids and mobile genetic elements are present in strains of L. reuteri (NPLps01.et_L.r and NPLps02.uf_L.r), Lactobacillus delbrueckii (NPLps01.et_L.d), Streptococcus thermophilus (NPLps06.ab_S.t), and E. faecium (NPLps07.nf_E.f). Our findings support the use of metagenomics to build better and efficient production and post-production practices for probiotic quality and safety assessment.

The natural history and ecology of melanism in red wolf and coyote populations of the southeastern United States - evidence for Gloger's rule.

BACKGROUND: Gloger's rule postulates that animals should be darker colored in warm and humid regions where dense vegetation and dark environments are common. Although rare in Canis populations, melanism in wolves is more common in North America than other regions globally and is believed to follow Gloger's rule. In the temperate forests of the southeastern United States, historical records of red wolf (Canis rufus) and coyote (Canis latrans) populations document a consistent presence of melanism. Today, the melanistic phenotype is extinct in red wolves while occurring in coyotes and red wolf-coyote hybrids who occupy the red wolf's historical range. To assess if Gloger's rule could explain the occurrence and maintenance of melanistic phenotypes in Canis taxa, we investigated differences in morphology, habitat selection, and survival associated with pelage color using body measurements, GPS tracking data, and long-term capture-mark-recapture and radio-telemetry data collected on coyotes and hybrids across the southeastern United States. RESULTS: We found no correlation between morphometrics and pelage color for Canis taxa. However, we observed that melanistic coyotes and hybrids experienced greater annual survival than did their gray conspecifics. Furthermore, we observed that melanistic coyotes maintained larger home ranges and exhibited greater selection for areas with dense canopy cover and wetlands than did gray coyotes. CONCLUSIONS: In the southeastern United States, pelage color influenced habitat selection by coyotes and annual survival of coyotes and hybrids providing evidence that Gloger's rule is applicable to canids inhabiting regions with dense canopy cover and wetlands. Greater annual survival rates observed in melanistic Canis may be attributed to better concealment in areas with dense canopy cover such as coastal bottomland forests. We suggest that the larger home range sizes of melanistic coyotes may reflect the trade-off of reduced foraging efficiency in lower quality wetland habitat for improved survival. Larger home ranges and differential use of land cover by melanistic coyotes may facilitate weak assortative mating in eastern coyote populations, in which melanistic animals may have lower success of finding compatible mates in comparison to gray conspecifics. We offer that our observations provide a partial explanation for why melanism is relatively low (< 10%) but consistent within coyote populations throughout southeastern parts of their range.

Sensitivity analysis of effective transverse shear viscoelastic and diffusional properties of myelinated white matter.

Motivated by the need to interpret the results from a combined use of in vivo brain Magnetic Resonance Elastography (MRE) and Diffusion Tensor Imaging (DTI), we developed a computational framework to study the sensitivity of single-frequency MRE and DTI metrics to white matter microstructure and cell-level mechanical and diffusional properties. White matter was modeled as a triphasic unidirectional composite, consisting of parallel cylindrical inclusions (axons) surrounded by sheaths (myelin), and embedded in a matrix (glial cells plus extracellular matrix). Only 2D mechanics and diffusion in the transverse plane (perpendicular to the axon direction) was considered, and homogenized (effective) properties were derived for a periodic domain containing a single axon. The numerical solutions of the MRE problem were performed with ABAQUS and by employing a sophisticated boundary-conforming grid generation scheme. Based on the linear viscoelastic response to harmonic shear excitation and steady-state diffusion in the transverse plane, a systematic sensitivity analysis of MRE metrics (effective transverse shear storage and loss moduli) and DTI metric (effective radial diffusivity) was performed for a wide range of microstructural and intrinsic (phase-based) physical properties. The microstructural properties considered were fiber volume fraction, and the myelin sheath/axon diameter ratio. The MRE and DTI metrics are very sensitive to the fiber volume fraction, and the intrinsic viscoelastic moduli of the glial phase. The MRE metrics are nonlinear functions of the fiber volume fraction, but the effective diffusion coefficient varies linearly with it. Finally, the transverse metrics of both MRE and DTI are insensitive to the axon diameter in steady state. Our results are consistent with the limited anisotropic MRE and co-registered DTI measurements, mainly in the corpus callosum, available in the literature. We conclude that isotropic MRE and DTI constitutive models are good approximations for myelinated white matter in the transverse plane. The unidirectional composite model presented here is used for the first time to model harmonic shear stress under MRE-relevant frequency on the cell level. This model can be extended to 3D in order to inform the solution of the inverse problem in MRE, establish the biological basis of MRE metrics, and integrate MRE/DTI with other modalities towards increasing the specificity of neuroimaging.

Development of an endolysin enzyme and its cell wall-binding domain protein and their applications for biocontrol and rapid detection of Clostridium perfringens in food.

Clostridium perfringens is a well-known pathogen that causes food-borne illnesses. Although bacteriophages can be effective natural food preservatives, phage endolysin and cell wall-binding domain (CBD) provide useful materials for lysis of C. perfringens and rapid detection. The genome of phage CPAS-15 consists of 51.8-kb double-stranded circular DNA with 78 open reading frames, including an endolysin gene. The apparent absence of a virulence factor or toxin gene suggests its safety in food applications. C. perfringens endolysin (LysCPAS15) inhibits host cells by up to a 3-log reduction in 2 h, and enhanced green fluorescent protein (EGFP)-fused CBD protein (EGFP-LysCPAS15_CBD1) detects C. perfringens within 5 min. Both exhibit broader host range spectra and higher stabilities than a bacteriophage. Tests in milk show the same host lysis and specific detection activities, with no hindrance effect from food matrices, indicating that endolysin and its CBD can provide food extended protection from C. perfringens contamination.

Use of Lactococcus lactis as a production system for peptides and enzymes encoded by a Lantibiotic gene cluster from Bifidobacterium longum.

Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic, but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production, genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters, its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack of DNA binding motifs did not support this, and one current analysis suggested a high likelihood of it interacting with LanD. Therefore, gene lanR1 together with lanADMIT were codon optimized and synthesized. Those genes were then cloned into an efficient dual-plasmid nisin-controlled expression system in L. lactis. The addition of the lanR1 gene exhibited toxicity in E. coli, specifically causing a shorter cell size as observed by SEM. No toxicity was observed in L. lactis. While this production system did not result in the production of a bioactive lantibiotic by L. lactis, it did successfully produce all the peptides and enzymes encoded by the original lantibiotic gene cluster from B. longum, as confirmed by LC-MS. This will now facilitate efforts into determining the proper conditions required for these enzymes to produce a bioactive lantibiotic.

Psychotherapy Termination Practices with Older Adults: Impact of Patient and Therapist Characteristics.

OBJECTIVES: The aims of this study were to survey clinicians' opinions regarding psychotherapy practices in mutual termination with a specified population (depressed older adult outpatients) and to examine the patient and therapist characteristics that may influence such practices. METHODS: We surveyed psychologists' (N = 96) psychotherapy termination practices, using a hypothetical depressed older adult as a referent, to assess consensus on the appropriateness of various guidelines to termination and to examine whether these differ as a function of patient and therapist characteristics. RESULTS: Several practices were generally agreed to be "extremely appropriate" when terminating psychotherapy with older adults, including collaborating to determine the end date of treatment and discussing patient growth. Data also indicate that patient factors, such as personality pathology, and therapist factors, such as having an Integrative theoretical orientation were associated with differential endorsement of termination practices. Identification as a geropsychologist or working regularly with older adults were associated with a more cautious approach to termination. CONCLUSIONS: There is substantial consensus regarding many approaches to termination, but modifications might be appropriate depending on patient characteristics. CLINICAL IMPLICATIONS: Clinicians agree on a set of fundamental termination practices when working with older adults, but modify these based on orientation and diagnosis.

Thermus and the Pink Discoloration Defect in Cheese.

A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch's postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.

Organic contaminants in Great Lakes tributaries: Prevalence and potential aquatic toxicity.

Organic compounds used in agriculture, industry, and households make their way into surface waters through runoff, leaking septic-conveyance systems, regulated and unregulated discharges, and combined sewer overflows, among other sources. Concentrations of these organic waste compounds (OWCs) in some Great Lakes tributaries indicate a high potential for adverse impacts on aquatic organisms. During 2010-13, 709 water samples were collected at 57 tributaries, together representing approximately 41% of the total inflow to the lakes. Samples were collected during runoff and low-flow conditions and analyzed for 69 OWCs, including herbicides, insecticides, polycyclic aromatic hydrocarbons, plasticizers, antioxidants, detergent metabolites, fire retardants, non-prescription human drugs, flavors/fragrances, and dyes. Urban-related land cover characteristics were the most important explanatory variables of concentrations of many OWCs. Compared to samples from nonurban watersheds (<15% urban land cover) samples from urban watersheds (>15% urban land cover) had nearly four times the number of detected compounds and four times the total sample concentration, on average. Concentration differences between runoff and low-flow conditions were not observed, but seasonal differences were observed in atrazine, metolachlor, DEET, and HHCB concentrations. Water quality benchmarks for individual OWCs were exceeded at 20 sites, and at 7 sites benchmarks were exceeded by a factor of 10 or more. The compounds with the most frequent water quality benchmark exceedances were the PAHs benzo[a]pyrene, pyrene, fluoranthene, and anthracene, the detergent metabolite 4-nonylphenol, and the herbicide atrazine. Computed estradiol equivalency quotients (EEQs) using only nonsteroidal endocrine-active compounds indicated medium to high risk of estrogenic effects (intersex or vitellogenin induction) at 10 sites. EEQs at 3 sites were comparable to values reported in effluent. This multifaceted study is the largest, most comprehensive assessment of the occurrence and potential effects of OWCs in the Great Lakes Basin to date.

Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese.

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses.

BACKGROUND: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. RESULTS: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. CONCLUSIONS: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.

Temporal and spatial differences in microbial composition during the manufacture of a continental-type cheese.

We sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine-salted continental-type cheese in cheeses produced early and late in the production day. Differences in the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas, and Bifidobacterium, not routinely associated with a continental-type cheese produced from pasteurized milk, were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high-throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.

A review of advances in collaborative pharmacy practice to improve adherence to standards of care in diabetes management.

The prevalence of diabetes in the United States is increasing and so is the need to provide diabetes care. Given the time commitment and complexity of diabetes management, an interdisciplinary approach is recommended. Pharmacists are integral members of the diabetes care team because of their accessibility and expertise in medication management. Pharmacists are receiving specialized training and becoming more involved in direct patient care through collaborative practice opportunities such as medication therapy management and collaborative drug therapy management. These collaborative practice models increase patient access to care and allow pharmacists to optimize drug therapy and provide important education to promote diabetes self-management. Studies show pharmacists practicing in a variety of outpatient environments can reduce HbA1c, LDL and BP as well as improve adherence to recommended American Diabetes Association guidelines (yearly monofilament exams, dilated eye exams, microalbumin screening, etc). Pharmacists working as part of the health care team can ensure optimal diabetes management.

Complications in pediatric scoliosis surgery.

BACKGROUND: Scoliosis surgery in childhood is associated with a range of postoperative complications that may require admission to the pediatric intensive care unit (PICU) or high-dependency unit (HDU). AIM: The aim of this study was to identify preoperative factors associated with PICU and HDU admissions after corrective surgery and devise a scoring system that could be used by clinicians to predict the level of dependency required postoperatively. METHODS: A retrospective case note review was carried out in 90 patients who underwent corrective scoliosis surgery at Sheffield Children's Hospital (SCH) between January 2008 and October 2010. Predictors of PICU and HDU requirement postoperatively were identified and a simple scoring system created using multiple logistic regression and receiver operator characteristic (ROC). RESULTS: There was a statistically significant difference in the preoperative parameters (pulmonary function, Cobb angle, and number of vertebrae fused) of those patients who required PICU or HDU care compared with those who did not. The area under the receiver operator characteristic curve for the final scoring system was 0.95 for PICU admission and 0.87 for HDU admission at the optimal cut-off point, demonstrating good diagnostic accuracy. CONCLUSIONS: The authors have identified a significant relationship between preoperative variables and the levels of dependency required postoperatively and have proposed a scoring system which can be used to aid decision-making involving bed planning for patients after corrective scoliosis surgery. However, this work is based on the clinical course of a single set of patients who had surgery in a single tertiary center and has not been tested on patients from other centers.

Diabetes and renal disease: who does what?

Care of patients with diabetes and chronic kidney disease (CKD) in the UK is divided between primary care, diabetologists and nephrology. In a retrospective analysis, we examined the distribution of care provision for patients with diabetes and CKD. Nephrology services see a minority of diabetic patients with CKD, but they see the majority of those with an estimated glomerular filtration rate (eGFR) of <30 ml/min. Of those followed in nephrology, 70% showed no evidence of progressive renal dysfunction. The nephrology cohort were significantly younger that those seen by primary care physicians or diabetologists. Half of the patients with diabetes and CKD seen in either the primary care and diabetology cohorts, with no nephrology input, had a rate of fall of eGFR of >5 ml/min/yr. This suggests that older age might deter referral to nephrology, which is based predominantly on CKD stage. This results in a significant proportion of patients with stable renal function being seen by nephrology, and in the under-referral of a large cohort of patients with progressive CKD.

Aortic annular sizing for transcatheter aortic valve replacement using cross-sectional 3-dimensional transesophageal echocardiography.

OBJECTIVES: This study compared cross-sectional three-dimensional (3D) transesophageal echocardiography (TEE) to two-dimensional (2D) TEE as methods for predicting aortic regurgitation after transcatheter aortic valve replacement (TAVR). BACKGROUND: Data have shown that TAVR sizing using cross-sectional contrast computed tomography (CT) parameters is superior to 2D-TEE for the prediction of paravalvular aortic regurgitation (AR). Three-dimensional TEE can offer cross-sectional assessment of the aortic annulus but its role for TAVR sizing has been poorly elucidated. METHODS: All patients had severe symptomatic aortic stenosis and were treated with balloon-expandable TAVR in a single center. Patients studied had both 2D-TEE and 3D imaging (contrast CT and/or 3D-TEE) of the aortic annulus at baseline. Receiver-operating characteristic curves were generated for each measurement parameter using post-TAVR paravalvular AR moderate or greater as the state variable. RESULTS: For the 256 patients studied, paravalvular AR moderate or greater occurred in 26 of 256 (10.2%) of patients. Prospectively recorded 2D-TEE measurements had a low discriminatory value (area under the curve = 0.52, 95% confidence interval: 0.40 to 0.63, p = 0.75). Average cross-sectional diameter by CT offered a high degree of discrimination (area under the curve = 0.82, 95% confidence interval: 0.73 to 0.90, p < 0.0001) and mean cross-sectional diameter by 3D-TEE was of intermediate value (area under the curve = 0.68, 95% confidence interval: 0.54 to 0.81, p = 0.036). CONCLUSIONS: Cross-sectional 3D echocardiographic sizing of the aortic annulus dimension offers discrimination of post-TAVR paravalvular AR that is significantly superior to that of 2D-TEE. Cross-sectional data should be sought from 3D-TEE if good CT data are unavailable for TAVR sizing.

Nucleic acid-based approaches to investigate microbial-related cheese quality defects.

The microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.

Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria.

Bifidobacteria are widely used as probiotics and have attracted increasing research interest worldwide. However, molecular techniques are still very scarce mainly due to the low efficiencies and strain-specific electroporation protocols that have been developed. Bacterial conjugation enables the transfer of genetic material among a relatively wide range of organisms and with virtually no size limitation. A conjugation protocol was developed based on the RP4 conjugative machinery in the Escherichia coli strain WM3064(pBB109). Using this machinery, the newly constructed transmissible E. coli-Bifidobacterium shuttle vector, pDOJHR-WD2, was successfully and consistently transferred into several strains representing four Bifidobacterium species at efficiencies which correlated with the E. coli to bifidobacteria ratios. Higher ratios were found to significantly improve transfer frequency per recipient, with almost 100 % transfer frequency occurring when the ratio was 10(5) : 1. The incompatible resident plasmid, pDOJH10S, in Bifidobacterium longum DJO10A was able to coexist, albeit at lower copy numbers, with the incoming vector pDOJHR-WD2 even though they possess the same ori. In some cases the copy number of this resident plasmid was too low to observe via gel electrophoresis, but it could be detected by Southern hybridization. Plasmid curing resulted in a strain, DJO10A-W3, that had lost both plasmids and this showed a one-log increase in conjugation efficiency due to the lack of plasmid incompatibility. In conclusion, this novel conjugative gene transfer protocol can be used for the introduction of genetic material (without size restriction) into Bifdobacterium species and is particularly useful for strains that are recalcitrant to electroporation.

Contribution of the Actinobacteria to the growing diversity of lantibiotics.

Currently, 76 lantibiotics have been described; the vast majority being produced by members of the Firmicute phylum of bacteria. There is a growing number being identified from the Actinobacteria phylum and some of these exhibit novel modifications leading to an increased functional diversity among lantibiotics. In this review, we discuss the currently characterized lantibiotics highlighting the expanding diversity provided by those from the Actinobacteria. This increased diversity has the potential to expand lantibiotic applications as antimicrobials in foods and pharmaceuticals. In addition, a phylogenetic classification system based on the full prepropeptide sequences showed remarkable consistency with current classification systems and may provide a more rapid and convenient means for classifying lantibiotics.