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Methodological challenges associated with structural equation modeling (SEM) and structured means modeling (SMM) in research on school violence and related topics in the social and behavioral sciences are examined. Problems associated with multiyear implementations of large-scale surveys are discussed. Complex sample designs, part of any large-scale survey, introduce multiple methodological challenges into SEM analysis. Problems with missing data and data imputation procedures are explored, along with commonly occurring departures from normality. Hypothesis testing of models and associated fit indices are explicated. Issues surrounding the use of Lagrange Multiplier tests of constraint validity in SMM are examined. Finally, basic problems associated with developing viable models of complex social phenomena with SEM are considered. [ABSTRACT FROM AUTHOR]

Violent extremism in the United States has surged over the past 25 years, with attacks on and threats to major governmental and other institutions, infrastructure (e.g., electric grid), and specific segments of the population, including immigrant and BIPOC (Black, Indigenous, and other People of Color) communities. Violent extremism can take multiple and diverse forms, such as bombings in public spaces or specific sites (e.g., house of worship, governmental office), mass and more targeted types of shootings, bombings, fire setting, and vehicle-based ramming attacks. This article provides an overview of key issues surrounding terrorism and violent extremism, especially as they can impact youth, schools, and families. Characteristics of violent extremism, radicalization processes, subsequent types of harm, and prevention approaches are discussed. Issues impacting students and schools and the work of school psychologists are also considered. The article closes with broader recommendations for moving forward., Competing Interests: Declaration of competing interest The co-authors declare that they have no conflict of interest., (Copyright © 2024 Society for the Study of School Psychology. Published by Elsevier Ltd. All rights reserved.)

Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG) 5 repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms., (© 2024. The Author(s).)

Bacteriophages and their endolysins are potential biocontrol agents for the anaerobic spoilage organism Clostridium tyrobutyricum, which causes cheese late blowing defect. This study sequenced and compared the genomes of eight bacteriophages from Spanish dairy farms that were active against C. tyrobutyricum, to identify novel species and phage proteins. Phages vB&#95;CtyS-FA67 and vB&#95;CtyS-FA70 shared >94% intergenomic similarity to each other but neither phage had significant similarity to ΦCTP1, the unique C. tyrobutyricum phage sequenced to date. Taxonomic analysis indicated that both phages belong to the class Caudoviricetes and are related to dsDNA viruses with long non-contractile tails. vB&#95;CtyS-FA67 had no other close relatives and encoded a novel endolysin, LysFA67, predicted to belong to the glycoside hydrolase GH24 family. LysFA67 lysed 93% of C. tyrobutyricum cells after 4 min in turbidity reduction assays, retaining lytic activity at pHs 4.2-8.1 and at 30-45 °C. The endolysin remained stable after 30 d storage at 4, 12 and 25 °C, while its activity decreased at -20 °C. LysFA67 lysed several clostridia species, while common dairy bacteria were not affected. Lactococcus lactis INIA 437, used as a cheese starter, was engineered to deliver LysFA67 and red fluorescent LysFA67-mCherry to dairy products. We demonstrated that these engineered strains were able to maintain lytic activity and fluorescence without affecting their technological properties in milk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sulfate- and sulfite-reducing bacteria (SRB) are a group of strict anaerobes found within the human gut. Bilophila wadsworthia , a sulfite-reducing bacterium which produces hydrogen sulfide (H 2 S) from taurine and isethionate respiration, is a common member of the healthy commensal human gut microbiota but has been implicated in several disease states including inflammatory bowel disease and colorectal cancer. Bacteroides thetaiotaomicron , one of the most prominent gut bacteria, has sulfatases which release sulfate, serving as a potential substrate for sulfate-reducing bacteria. Here, we showed that when B. thetaiotaomicron and B. wadsworthia were in co-culture, there was a significant increase in B. thetaiotaomicron 's growth and in H 2 S production by B. wadsworthia . Differential gene expression analysis revealed increased expression of B. wadsworthia 's dsrMKJOP complex in co-culture, which delivers electrons for sulfite reduction to H 2 S. This was accompanied by a decreased expression of genes associated with taurine, sulfolactate, and thiosulfate respiration, indicating that B. thetaiotaomicron may provide an alternative source of sulfite to B. wadsworthia . We hypothesized adenosine 5'-phosphosulfate (APS) to be this intermediate. Indeed, B. wadsworthia was able to grow using APS or sulfite as electron acceptors. Endometabolomic and transcriptomic analyses revealed decreased production of indole by B. thetaiotaomicron in co-culture with B. wadsworthia due to enhanced tryptophan utilization by B. wadsworthia . The results of this microbe-microbe interaction could have significant pro-inflammatory effects in the human gut environment.

Background: Gamma-aminobutyric acid (GABA) is a non-protein amino acid with neuroinhibitory, antidiabetic, and antihypertensive properties and is used as a drug for treating anxiety and depression. Some strains of lactobacilli are known to produce GABA and strengthen the gut barrier function which play an important role in ameliorating the effects caused by the pathogen on the gut barrier. The probiotic bacteria are also known to modulate the human fecal microbiota, however, the role of GABA-producing strains on the gut epithelium permeability and gut microbiota is not known., Results: In this study, we report the production of high levels of GABA by potential probiotic bacterium Limosilactobacillus fermentum L18 for the first time. The kinetics of the production of GABA by L18 showed that the maximum production of GABA in the culture supernatant (CS) occurred at 24 h, whereas in fermented milk it took 48 h of fermentation. The effect of L18 on the restoration of lipopolysaccharide (LPS)-disrupted intestinal cell membrane permeability in Caco-2 monolayers showed that it significantly restored the transepithelial electrical resistance (TEER) values, by significantly increasing the levels of junction proteins, occludin and E-cadherin in L18 and LPS-treated Caco-2 cells as compared to only LPS-treated cells. The effect of GABA-secreting L18 on the metataxonome of human stool samples from healthy individuals was investigated by a batch fermentor that mimics the conditions of the human colon. Although, no differences were observed in the α and β diversities of the L18-treated and untreated samples at 24 h, the relative abundances of bacterial families Lactobacillaceae and Bifidobacteriaceae increased in the L18-treated group, but both decreased in the untreated groups. On the other hand, the relative abundance of Enterobacteriaceae decreased in the L18 samples but it increased in the untreated samples., Conclusion: These results indicate that Li. fermentum L18 is a promising GABA-secreting strain that strengthens the gut epithelial barrier by increasing junction protein concentrations and positively modulating the gut microbiota. It has the potential to be used as a psychobiotic or for the production of functional foods for the management of anxiety-related illnesses., (© 2023. The Author(s).)

Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Glucosinolates are plant natural products which on degradation by myrosinases give rise to the beneficial bioactive isothiocyanates. Recently, a myrosinase activity was detected in a Citrobacter strain isolated from soil. This enzyme was purified enabling its amino acid sequence and gene sequence (cmyr) to be determined. In order to study this myrosinase it was necessary to establish an expression system that would enable future work such as a structural determination of the protein to be carried out. The myrosinase gene was amplified, cloned and expressed in Escherichia coli with a 6XHis-tag. The heterologous expression of cmyr enabled relatively large amounts of myrosinase to be produced (3.4 mg cmyr/100 ml culture). Myrosinase activity was determined by mixing substrate and enzyme and determining glucose release. Optimum pH and temperature were determined to be pH 6.0 and 25 °C for the Ni-NTA purified protein. The kinetic parameters of the purified myrosinase were determined using sinigrin as a substrate. K m and V max were estimated as 0.18 mM and 0.033 mmol/min/mg respectively for sinigrin under optimum conditions and compared to other kinetic data for myrosinases. The substrate specificity of myrosinase was determined having the highest affinity for sinigrin followed by glucoiberin, progoitrin, glucoerucin, glucoraphanin and glucotropaeolin., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

History A 37-year-old man from the United States presented with a 1-year history of neck pain and stiffness that had been unsuccessfully treated with manipulative therapy by a chiropractor at another institution. Past medical history was remarkable only for marijuana and air duster abuse. He denied use of any prescription medications. Physical examination was notable for markedly reduced range of motion of the cervical spine. Laboratory work-up revealed an elevated alkaline phosphatase level (302 U/L [5.0 μkat/L]; normal range, 40-100 U/L [0.7-1.67 μkat/L]), but all other laboratory findings, including complete blood count, renal function, liver function, vitamin A level, serum protein electrophoresis, and hepatitis C antibodies were within normal limits. Cervical spine radiography was performed, followed by MRI. Subsequently, a full skeletal survey was ordered. Included are representative radiographs of the pelvis, left forearm, and distal right leg with ankle.

History A 37-year-old man from the United States presented with a 1-year history of neck pain and stiffness that had been unsuccessfully treated with manipulative therapy by a chiropractor at another institution. Past medical history was remarkable only for marijuana and air duster abuse. He denied use of any prescription medications. Physical examination was notable for markedly reduced range of motion of the cervical spine. Laboratory work-up revealed an elevated alkaline phosphatase level (302 U/L [5.0 μkat/L]; normal range, 40-100 U/L [0.7-1.67 μkat/L]), but all other laboratory findings, including complete blood count, renal function, liver function, vitamin A level, serum protein electrophoresis, and hepatitis C antibodies were within normal limits. Cervical spine radiography was performed (Fig 1), followed by MRI (Fig 2). Subsequently, a full skeletal survey was ordered. Included are representative radiographs of the pelvis (Fig 3), left forearm (Fig 4), and distal right leg with ankle (Fig 5).

Hausa koko is an indigenous porridge processed from millet in Ghana. The process involves fermentation stages, giving the characteristic organoleptic properties of the product that is produced largely at a small-scale household level and sold as a street food. Like many other indigenous foods, quality control is problematic and depends on the skills of the processor. In order to improve the quality of the product and standardize the process for large-scale production, we need a deeper understanding of the microbial processes. The aim of this study is to investigate the microbial community involved in the production of this traditional millet porridge and the metabolites produced during processing. High-throughput amplicon sequencing was used to identify the bacterial (16S rRNA V4 hypervariable region) and fungal [Intergenic Transcribed Spacer (ITS)] communities associated with the fermentation, while nuclear magnetic resonance (NMR) was used for metabolite profiling. The bacterial community diversity was reduced during the fermentation processes with an increase and predominance of lactobacilli. Other dominant bacteria in the fermentation included Pediococcus , Weissella , Lactococcus , Streptococcus , Leuconostoc , and Acetobacter. The species Limosilactobacillus fermentum and Ligilactobacillus salivarius accounted for some of the diversities within and between fermentation time points and processors. The fungal community was dominated by the genus Saccharomyces . Other genera such as Pichia , Candida , Kluyveromyces , Nakaseomyces , Torulaspora , and Cyberlindnera were also classified. The species Saccharomyces cerevisiae , Stachybotrys sansevieriae , Malassezia restricta , Cyberlindnera fabianii , and Kluyveromyces marxianus accounted for some of the diversities within some fermentation time points. The species S. sansevieria and M. restricta may have been reported for the first time in cereal fermentation. This is the most diverse microbial community reported in Hausa koko . In this study, we could identify and quantify 33 key different metabolites produced by the interactions of the microbial communities with the millet, composed of organic compounds, sugars, amino acids and intermediary compounds, and other key fermentation compounds. An increase in the concentration of organic acids in parallel with the reduction of sugars occurred during the fermentation process while an initial increase of amino acids followed by a decrease in later fermentation steps was observed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Atter, Diaz, Tano-Debrah, Kunadu, Mayer, Colquhoun, Nielsen, Baker, Narbad and Amoa-Awua.)

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.

The gut microbiota has been identified as a target of toxic metals and a potentially crucial mediator of the bioavailability and toxicity of these metals. In this study, we show that aluminum (Al) exposure, even at low dose, affected the growth of representative strains from the human intestine via pure culture experiments. In vitro, Lactobacillus plantarum CCFM639 could bind Al on its cell surface as shown by electron microscopy and energy dispersive X-ray analysis. The potential of L. plantarum CCFM639 to reverse changes in human intestine microbiota induced by low-dose dietary Al exposure was investigated using an in vitro colonic fermentation model. Batch fermenters were inoculated with fresh stool samples from healthy adult donors and supplemented with 86 mg/L Al and/or 10 9  CFU of L. plantarum CCFM639. Al exposure significantly increased the relative abundances of Bacteroidetes (Prevotella), Proteobacteria (Escherichia), Actinobacteria (Collinsella), Euryarchaeota (Methanobrevibacter), and Verrucomicrobiaceae and decreased Firmicutes (Streptococcus, Roseburia, Ruminococcus, Dialister, Coprobacillus). Some changes were reversed by the inclusion of L. plantarum CCFM639. Alterations in gut microbiota induced by Al and L. plantarum CCFM639 inevitably led to changes in metabolite levels. The short-chain fatty acid (SCFAs) contents were reduced after Al exposure, but L. plantarum CCFM639 could elevate their levels. SCFAs had positive correlations with beneficial bacteria, such as Dialister, Streptococcus, Roseburia, and negative correlations with Erwinia, Escherichia, and Serratia. Therefore, dietary Al exposure altered the composition and structure of the human gut microbiota, and this was partially mitigated by L. plantarum CCFM639. This probiotic supplementation is potentially a promising and safe approach to alleviate the harmful effects of dietary Al exposure.

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Intranasal antihistamines and steroids should be delivered in a volume and with a technique that allow for optimal drug retention within the entire nasal cavity, maximize local absorption by the nasal mucosa, and, subsequently, increase the potential for the most desirable local availability and therapeutic effect. Objective: This in vitro evaluation simulated nasal medication deposition and evaluated the extent of runoff. MP-AzeFlu, a novel intranasal formulation of azelastine hydrochloride (AZE) plus fluticasone propionate (FP), was compared with sequential sprays of available commercial products with the individual medication components. Methods: A model of a normal adult human nasal cavity was used to visualize deposition of nasal spray products. A single spray of MP-AzeFlu (0.137 mL [137 μg of AZE/50 μg of FP]) or single sequential sprays of AZE nasal spray (0.137 mL [137 μg]) followed by brand name or generic FP nasal spray (0.100 mL [50 μg]) were manually actuated into the model. The interior was coated with a water-sensitive dye that changes to magenta when exposed to aqueous-based formulations. A slight vacuum was applied during spray delivery to simulate sniffing. The results were photographed by using anterior and lateral views. Results: Three replicates of MP-AzeFlu showed no dripping from the front of the nostril or backflow from the nasal cavity. However, three replicates of AZE nasal spray, followed by a brand name or generic FP nasal spray, showed significant dripping from the front of the nostril and backflow from the nasal cavity. Conclusion: A single spray of MP-AzeFlu resulted in no runoff compared with sequential dosing of the two other therapeutic products. Product runoff is likely due to the volume exceeding the capacity of the nasal cavity model. Furthermore, the common clinical dosing regimen of two sprays per nostril of each of the individual components would promote even greater increased undesirable flooding and leakage.

Background: Management of bone loss in recurrent traumatic anterior shoulder instability remains a topic of debate and controversy in the orthopedic community. The purpose of this study was to survey members of 4 North American orthopedic surgeon associations to assess management trends for bone loss in recurrent anterior shoulder instability., Methods: An online survey was distributed to all members of the American Shoulder and Elbow Surgeons, American Orthopaedic Society for Sports Medicine, and Canadian Orthopaedic Association and to fellow members of the Arthroscopy Association of North America. The survey comprised 3 sections assessing the demographic characteristics of survey respondents, the influence of prognostic factors on surgical decision making, and the operative management of 12 clinical case scenarios of varying bone loss that may be encountered in clinical practice., Results: A total of 150 survey responses were returned. The age of the patient and quantity of bone loss were consistently considered important prognostic criteria. However, little consensus was reached for critical thresholds of bone loss and how this affected the timing (ie, primary or revision surgery) and type of bony augmentation procedure to be performed once a critical threshold was reached, especially in the context of critical humeral and bipolar bone loss., Conclusions: Consistent trends were found for the management of recurrent anterior shoulder instability in cases in which no bone loss existed and when isolated critical glenoid bone loss was present. However, inconsistencies were observed when isolated critical humeral bone loss and bipolar bone loss were present., (© 2020 The Authors.)

Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

A novel Gram-positive, catalase negative, rod-shaped strain, FI11369 T , was isolated from gari , a traditional West African fermented food derived from cassava. Based on 16S rRNA gene sequence similarity, the closest type strains were Lactobacillus xiangfangensis LMG 26013 T (99.4 % similarity), Lactobacillus plajomi NBRC 107333 T (99.1 %), Lactobacillus paraplantarum DSM 10667 T (99.1 %), Lactobacillus pentosus DSM 20314 T (99.0 %), Lactobacillus plantarum subsp. plantarum ATCC 14917 T (99.0 %), Lactobacillus modestisalitolerans NBRC 107235 T (98.9 %), Lactobacillus plantarum subsp. argentoratensis DSM 16365 T (98.9 %) and Lactobacillus daowaiensis NCIMB 15183 T (98.8 %). The genome of strain FI11369 T was sequenced and the average nucleotide identity (ANI) was compared with its closest relatives. ANI analysis showed that the closest relative, L. xiangfangensis DSM 27103 T , had only a 82.4 % similarity. The main fatty acids of FI11369 T were saturated C 16 : 0 (18.2 %), unsaturated C 18 : 1   ω9 c (43.8 %) and cyclopropane C 19 : 0 cyclo ( ω 10 c and/or ω 6; 22.5 %). Based on the genotypic and phenotypic data obtained in this study, a novel Lactobacillus species, Lactobacillus garii sp. nov., with the type strain FI11369 T (=NCIMB 15148=DSM 108249), is proposed.

Staphylococcus epidermidis is a commensal species that has been increasingly identified as a nosocomial agent. Despite the interest, little is known about the ability of S. epidermidis isolates to adapt to different ecological niches through comparisons at genotype or phenotype levels. One niche where S. epidermidis has been reported is the human gut. Here, we present three S. epidermidis strains isolated from feces and show that they are not phylogenetically distinct from S. epidermidis isolated from other human body sites. Both gut and skin strains harbored multiple genes associated with biofilm formation and showed similar levels of biofilm formation on abiotic surfaces. High-throughput physiological tests using the BIOLOG technology showed no major metabolic differences between isolates from stool, skin, or cheese, while an isolate from bovine mastitis showed more phenotypic variation. Gut and skin isolates showed the ability to metabolize glycine-conjugated bile acids and to grow in the presence of bile, but the gut isolates exhibited faster anaerobic growth compared to isolates of skin origin., (Copyright © 2020 Garcia-Gutierrez, Walsh, Sayavedra, Diaz-Calvo, Thapa, Ruas-Madiedo, Mayer, Cotter and Narbad.)

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785&#95;242 (242), was less abundant in the Δ eps&#95;cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785&#95;242 gene ( 242 ) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsonii IMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell., (Copyright © 2020 Mayer et al.)

Nisin P is a natural nisin variant, the genetic determinants for which were previously identified in the genomes of two Streptococcus species, albeit with no confirmed evidence of production. Here we describe Streptococcus agalactiae DPC7040, a human faecal isolate, which exhibits antimicrobial activity against a panel of gut and food isolates by virtue of producing nisin P. Nisin P was purified, and its predicted structure was confirmed by nanoLC-MS/MS, with both the fully modified peptide and a variant without rings B and E being identified. Additionally, we compared its spectrum of inhibition and minimum inhibitory concentration (MIC) with that of nisin A and its antimicrobial effect in a faecal fermentation in comparison with nisin A and H. We found that its antimicrobial activity was less potent than nisin A and H, and we propose a link between this reduced activity and the peptide structure.

Fermented foods play a major role in the diet of people in Africa, where a wide variety of raw materials are fermented. Understanding the microbial populations of these products would help in the design of specific starter cultures to produce standardized and safer foods. In this study, the bacterial diversity of African fermented foods produced from several raw materials (cereals, milk, cassava, honey, palm sap, and locust beans) under different conditions (household, small commercial producers or laboratory) in 8 African countries was analysed by 16S rRNA gene amplicon sequencing during the Workshop "Analysis of the Microbiomes of Naturally Fermented Foods Training Course". Results show that lactobacilli were less abundant in fermentations performed under laboratory conditions compared to artisanal or commercial fermentations. Excluding the samples produced under laboratory conditions, lactobacilli is one of the dominant groups in all the remaining samples. Genera within the order Lactobacillales dominated dairy, cereal and cassava fermentations. Genera within the order Lactobacillales, and genera Zymomonas and Bacillus were predominant in alcoholic beverages, whereas Bacillus and Lactobacillus were the dominant genera in the locust bean sample. The genus Zymomonas was reported for the first time in dairy, cereal, cassava and locust bean fermentations.

Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacteria, Archaea, Eukarya and viruses coexist in the human gut, and this coexistence is functionally balanced by symbiotic or antagonistic relationships. Antagonism is often characterized by the production of antimicrobials against other organisms occupying the same environmental niche. Indeed, close co-evolution in the gut has led to the development of specialized antimicrobials, which is attracting increased attention as these may serve as novel alternatives to antibiotics and thereby help to address the global problem of antimicrobial resistance. The gastrointestinal (GI) tract is especially suitable for finding novel antimicrobials due to the vast array of microbes that inhabit it, and a considerable number of antimicrobial producers of both wide and narrow spectrum have been described. In this review, we summarize some of the antimicrobial compounds that are produced by bacteria isolated from the gut environment, with a special focus on bacteriocins. We also evaluate the potential therapeutic application of these compounds to maintain homeostasis in the gut and the biocontrol of pathogenic bacteria.

A previously reported gene cluster encoding four nisin-like peptides, three with the same sequence (NsoA1-3) and the unique NsoA4, produced antimicrobial activity in the presence of trypsin after heterologous expression in Lactococcus lactis. Protein extracts were separated by SDS gel electrophoresis or immunoprecipitation using an antibody to the NsoA2 leader. Tryptic peptides observed by LC-MS/MS covered the complete sequence of preNsoA1-3 and part of the leader sequence of preNsoA4 and confirmed the expression and the predicted sequences of the preNsoA peptides. Further, the data revealed that the preNsoA1-3 peptides were partly modified with dehydrations and formation of lanthionine rings. A certain amount of fully modified preNsoA1-3 was observed. Details of modifications of the core peptide and the C-terminal tryptic peptide TATCGCHITGK covering rings D and E indicated that 22% of these preNsoA1-3 peptides were completely modified. A lower amount of ring formation is estimated for rings A-C. Intact masses of immunoprecipitation-derived peptides determined by LC-MS accurately matched the expected preNsoA precursor peptides. The most abundant peptides detected were preNsoA2-3-8H 2 O followed by preNsoA1-8H 2 O and other states of dehydration. The results confirm incomplete processing of preNsoA peptides in the heterologous system, with the formation of a certain amount of fully modified peptides.

Ochrobactrum haematophilum FI11154 was isolated from kunu-zaki, a Nigerian traditional fermented millet-based food. Here, we present the first complete genome sequence of this species. The genome consists of five replicons and contains genes related to iron uptake and phosphatase activities., (Copyright © 2018 Diaz et al.)

Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall-binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy-related Clostridium species by fluorescence microscopy. GFP-CTP1L and GFP-CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP-EAD did not label any Clostridium strain tested. Unexpectedly, GFP-CTP1L and GFP-CBD were also able to bind to clostridial spores. In addition, GFP-CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site-directed mutants of GFP-CTP1L and GFP-CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP-CTP1L and GFP-CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aim: Docetaxel, the first-line chemotherapy for metastatic castration-resistant prostate cancer (mCRPC), provides certain survival benefits, but is associated with significant toxicity. A novel therapeutic approach for mCRPC is combining docetaxel with a chemosensitizing agent. We hypothesized that metformin, a potential chemosensitizer, would improve docetaxel efficacy in CRPC cells., Materials and Methods: MTS assays were used to determine the effect of metformin-docetaxel treatment on PC3 and DU145 cell viability. Wound-healing and ATP concentration assays were used to evaluate cell migration and intracellular ATP levels following metformin-docetaxel treatment. Western blotting was used for mechanistic evaluation., Results: Metformin-docetaxel treatment significantly reduced PC3 cell viability. Metformin-docetaxel treatment did not significantly affect cell migration or intracellular ATP levels. Western blotting revealed metformin-docetaxel treatment did not significantly change AMPK or P-AMPK expression patterns., Conclusion: Metformin may be an effective chemosensitizer for certain types of CRPC cells, but further investigation is needed., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

1. Campylobacter jejuni is the most common bacterial cause of human food-borne gastroenteritis in the world. A major source of human infection is the consumption of contaminated meat, particularly poultry. New control measures to reduce or eliminate this pathogen from the animal gastrointestinal tract are urgently required, and the use of probiotics as competitive exclusion agents is a promising biocontrol measure to reduce C. jejuni in the food chain. 2. In this study, we assessed the potential of Lactobacillus johnsonii FI9785, which has shown efficacy against Clostridium perfringens, to combat C. jejuni. The effect of prophylactic administration of L. johnsonii on the ability of C. jejuni to colonise chickens was determined. 3. Two doses of L. johnsonii given a week apart led to a reduction in C. jejuni colonisation in the caecal contents, but this biocontrol seemed reliant upon a high level of initial colonisation by the probiotic. 4. The microbial composition in the chicken gut was significantly altered by the probiotic treatment, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene amplicons. 5. Together these results demonstrate the potential of this probiotic strain to be tested further as a competitive exclusion agent in poultry against C. jejuni.

Purpose: Patients with advanced cancer frequently experience anxiety, depression and poor quality of life (QOL), as well as physical symptoms such as fatigue and weakness. Physical exercise has potential to help control these symptoms but the optimal training prescription is still not clear. We performed a study comparing medical Qigong (QG) and standard endurance and strength training (SET) in patients with advanced stage non-small cell lung (NSCLC) and gastrointestinal (GI) cancers., Methods: A randomized, cross-over study was performed in patients with advanced NSCLC and GI cancers receiving or eligible for chemotherapy. Patients received supervised QG or SET twice-weekly for 6 weeks. Psychological functioning, QOL, symptoms and physical functioning were assessed before and after each intervention period., Results: Nineteen patients completed both interventions. Comparing interventions revealed no difference between QG and SET on change in anxiety or depression scores or QOL. However, SET treatment was better at improving perceived strength (P = 0.05) and walking distance (P = 0.02). The order in which interventions were performed had a significant impact on the improvement in certain symptoms (sleep quality, breathlessness, P < 0.05), QOL (P = 0.01) and walking distance (P = 0.008). In all cases, the beneficial effects of the exercise interventions were markedly reduced during the second interval., Conclusions: QG and SET are equivalent in their impact on many aspects of psychological function in cancer patients. However, SET leads to greater improvements in exercise capacity and helps reduce some symptoms. The reduction in beneficial effect of SET on exercise function when offered as the second intervention is a new finding that warrants further study.

Purpose: Docetaxel is the first line chemotherapy currently used to treat patients with symptomatic metastatic castration resistant prostate cancer. Although it provides survival benefits, it is associated with significant side effects. Novel therapeutic options are needed for patients with metastatic castration resistant prostate cancer and an approach is combining docetaxel with chemosensitizing agents. Metformin has been shown to improve the survival of patients with breast, lung and endometrial cancer receiving chemotherapy, and enhance chemotherapeutic efficacy in breast cancer and colon cancer cells. However, to our knowledge the chemosensitizing effect of metformin in prostate cancer has not been explored. Therefore, the hypothesis for our study was that diabetic patients with metastatic castration resistant prostate cancer who were administered metformin during docetaxel chemotherapy would have improved prostate cancer specific and overall survival., Materials and Methods: This retrospective cohort study used data from several Ontario administrative health care databases. Men older than 65 years diagnosed with metastatic castration resistant prostate cancer and treated with docetaxel were stratified into groups based on diabetes status and use of antidiabetic medications. We evaluated the effect of metformin use with docetaxel on prostate cancer specific survival and overall survival using Kaplan-Meier survival curves, the log rank test and multivariate Cox proportional HRs., Results: Survival curves showed that metformin use with docetaxel did not improve prostate cancer specific survival (p = 0.9562) or overall survival (p = 0.9927). HRs showed no significant effect of metformin use with docetaxel on prostate cancer specific survival (HR = 0.96, p = 0.66) or overall survival (HR = 0.94, p = 0.39)., Conclusions: Metformin use during docetaxel chemotherapy did not significantly improve prostate cancer specific or overall survival in diabetic patients with metastatic castration resistant prostate cancer. This study indicates that metformin may not be an effective chemosensitizer for metastatic castration resistant prostate cancer., (Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The complete genome sequence of Desulfovibrio piger FI11049 was determined. The genome consists of a single circular chromosome of 2,807,531 bp encoding seven rRNA operons, 76 tRNA genes, and 2,535 coding genes., (Copyright © 2017 Wegmann et al.)

Bacterial microcompartments (BMCs) are proteinaceous organelles that are found in a broad range of bacteria and are composed of an outer shell that encases an enzyme cargo representing a specific metabolic process. The outer shell is made from a number of different proteins that form hexameric and pentameric tiles, which interact to allow the formation of a polyhedral edifice. We have previously shown that the Citrobacter freundii BMC associated with 1,2-propanediol utilization can be transferred into Escherichia coli to generate a recombinant BMC and that empty BMCs can be formed from just the shell proteins alone. Herein, a detailed structural and proteomic characterization of the wild type BMC is compared to the recombinant BMC and a number of empty BMC variants by 2D-gel electrophoresis, mass spectrometry, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Specifically, it is shown that the wild type BMC and the recombinant BMC are similar in terms of composition, size, shape and mechanical properties, whereas the empty BMC variants are shown to be smaller, hollow and less malleable.

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression., (© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Prostate cancer is the second most frequently diagnosed cancer in the world. Localized disease can be effectively treated with radiation therapy or radical prostatectomy. However, advanced prostate cancer is more difficult to treat and if metastatic, is incurable. There is a need for more effective therapy for advanced prostate cancer. One potential target is the cancer stem cell (CSC). CSCs have been described in several solid tumors, including prostate cancer, and contribute to therapeutic resistance and tumor recurrence. Metformin, a common oral biguanide used to treat type 2 diabetes, has been demonstrated to have anti-neoplastic effects. Specifically, metformin targets CSCs in breast cancer, pancreatic cancer, glioblastoma and colon cancer. Metformin acts directly on the mitochondria to inhibit oxidative phosphorylation and reduce mitochondrial ATP production. This forces tumor cells to compensate by increasing the rate of glycolysis. CSCs rely heavily on mitochondrial oxidative phosphorylation for energy production. The glycolytic switch results in an energy crisis in these cells. Metformin could be used to exploit this metabolic weakness in CSCs. This would increase CSC sensitivity to conventional cancer therapies, circumventing treatment resistance and enhancing treatment efficacy. This review will explore the characteristics of prostate CSCs, their role in tumor propagation and therapeutic resistance and the role of metformin as a potential prostate CSC sensitizer to current anticancer therapies.

Background: The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens., Results: Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses., Conclusions: This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiX(L)) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiX(L) in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiX(L). From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 μg L(-1)). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials., (Copyright © 2014. Published by Elsevier B.V.)

Clostridium perfringens is frequently found in food and the environment and produces potent toxins that have a negative impact on both human and animal health and particularly on the poultry industry. Lactobacillus johnsonii FI9785, isolated from the chicken gastrointestinal tract, has been demonstrated to exclude Cl. perfringens in poultry. We have investigated the interaction of wild-type Lact. johnsonii FI9785 or an engineered strain expressing a cell wall-hydrolysing endolysin with Cl. perfringens in vitro, using a batch culture designed to simulate human gastrointestinal tract conditions. Co-culture experiments indicated that acid production by Lact. johnsonii is important in pathogen control. The co-culture of the endolysin-secreting Lact. johnsonii with Cl. perfringens showed that the engineered strain had the potential to control the pathogen, but the ability to reduce Cl. perfringens numbers was not consistent. Results obtained indicate that survival of high numbers of Lact. johnsonii will be essential for effective pathogen control. Significance and impact of the study: The bacterium Lactobacillus johnsonii FI9785 reduces numbers of the pathogen Clostridium perfringens in vitro. Biocontrol was improved by engineering the strain to produce and export a cell wall-hydrolysing endolysin, but good survival of the producer strain is essential. The production of bacteriophage endolysins by commensal bacteria has the potential to improve competitive exclusion of pathogens in the gastrointestinal tract., (© 2014 The Society for Applied Microbiology.)

The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.

Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.