Effects of direct-fed Bacillus subtilis and Bacillus licheniformis on production performance and milk fatty acid profile in dairy cows.

The aim of the study was to determine the effect of a Bacillus-based direct-fed microbial on performance of mid-lactating Holstein dairy cows and on their milk fatty acid composition. Six multiparous cows fitted with a rumen cannula were used in a randomized replicated crossover design. Cows received 200 g/d of either whey powder as a control or BioPlus 2B (Chr. Hansen), a commercial direct-fed microbial providing Bacillus subtilis and Bacillus licheniformis, representing a daily dose of 6.4 × 1011 cfu, and using whey powder as a carrier. The 2 experimental periods lasted 14 d and were separated by a 7-d washout interval. Samples were collected on d 0, 13, and 14 of each period. Data from d 0 were used as covariate. Significance was declared at P ≤ 0.05 and tendency at 0.05

Navigating Microbiological Food Safety in the Era of Whole-Genome Sequencing.

The epidemiological investigation of a foodborne outbreak, including identification of related cases, source attribution, and development of intervention strategies, relies heavily on the ability to subtype the etiological agent at a high enough resolution to differentiate related from nonrelated cases. Historically, several different molecular subtyping methods have been used for this purpose; however, emerging techniques, such as single nucleotide polymorphism (SNP)-based techniques, that use whole-genome sequencing (WGS) offer a resolution that was previously not possible. With WGS, unlike traditional subtyping methods that lack complete information, data can be used to elucidate phylogenetic relationships and disease-causing lineages can be tracked and monitored over time. The subtyping resolution and evolutionary context provided by WGS data allow investigators to connect related illnesses that would be missed by traditional techniques. The added advantage of data generated by WGS is that these data can also be used for secondary analyses, such as virulence gene detection, antibiotic resistance gene profiling, synteny comparisons, mobile genetic element identification, and geographic attribution. In addition, several software packages are now available to generate in silico results for traditional molecular subtyping methods from the whole-genome sequence, allowing for efficient comparison with historical databases. Metagenomic approaches using next-generation sequencing have also been successful in the detection of nonculturable foodborne pathogens. This review addresses state-of-the-art techniques in microbial WGS and analysis and then discusses how this technology can be used to help support food safety investigations. Retrospective outbreak investigations using WGS are presented to provide organism-specific examples of the benefits, and challenges, associated with WGS in comparison to traditional molecular subtyping techniques.

Genomic Features of Environmental and Clinical Vibrio parahaemolyticus Isolates Lacking Recognized Virulence Factors Are Dissimilar.

Vibrio parahaemolyticus is a bacterial pathogen that can cause illness after the consumption or handling of contaminated seafood. The primary virulence factors associated with V. parahaemolyticus illness are thermostable direct hemolysin (TDH) and Tdh-related hemolysin (TRH). However, clinical strains lacking tdh and trh have recently been isolated, and these clinical isolates are poorly understood. To help understand the emergence of clinical tdh- and trh-negative isolates, a genomic approach was used to comprehensively compare 4 clinical tdh- and trh-negative isolates with 16 environmental tdh- and trh-negative isolates and 34 clinical isolates positive for tdh or trh, or both, with the objective of identifying genomic features that are unique to clinical tdh- and trh-negative isolates. The prevalence of pathogenicity islands (PAIs) common to clinical isolates was thoroughly examined in each of the clinical tdh- and trh-negative isolates. The tdh PAI was not present in any clinical or environmental tdh- and trh-negative isolates. The trh PAI was not present in any environmental isolates; however, in clinical tdh- and trh-negative isolate 10-4238, the majority of the trh PAI including a partial trh1 gene was present, which resulted in reclassification of this isolate as a tdh-negative and trh-positive isolate. In the other clinical tdh- and trh-negative isolates, neither the trh gene nor the trh PAI was present. We identified 862 genes in clinical tdh- and trh-negative isolates but not in environmental tdh- and trh-negative isolates. Many of these genes are highly homologous to genes found in common enteric bacteria and included genes encoding a number of chemotaxis proteins and a novel putative type VI secretion system (T6SS) effector and immunity protein (T6SS1). The availability of genome sequences from clinical V. parahaemolyticus tdh- and trh-negative isolates and the comparative analysis may help provide an understanding of how this pathotype is able to survive in vivo during clinical illness.

Draft Whole-Genome Sequences of 14 Vibrio parahaemolyticus Clinical Isolates with an Ambiguous K Serogroup.

Vibrio parahaemolyticus is a bacterial pathogen responsible for mild to severe gastroenteritis, wound infections, and septicemia resulting from the ingestion or handling of raw or undercooked contaminated seafood. Here, we report the draft whole-genome sequences and annotations of 14 Canadian V. parahaemolyticus clinical isolates that were serologically identified as K group II using polyvalent antisera but were not specifically K serogrouped using monovalent antisera.

An active atmospheric methane sink in high Arctic mineral cryosols.

Methane (CH4) emission by carbon-rich cryosols at the high latitudes in Northern Hemisphere has been studied extensively. In contrast, data on the CH4 emission potential of carbon-poor cryosols is limited, despite their spatial predominance. This work employs CH4 flux measurements in the field and under laboratory conditions to show that the mineral cryosols at Axel Heiberg Island in the Canadian high Arctic consistently consume atmospheric CH4. Omics analyses present the first molecular evidence of active atmospheric CH4-oxidizing bacteria (atmMOB) in permafrost-affected cryosols, with the prevalent atmMOB genotype in our acidic mineral cryosols being closely related to Upland Soil Cluster α. The atmospheric (atm) CH4 uptake at the study site increases with ground temperature between 0 °C and 18 °C. Consequently, the atm CH4 sink strength is predicted to increase by a factor of 5-30 as the Arctic warms by 5-15 °C over a century. We demonstrate that acidic mineral cryosols are a previously unrecognized potential of CH4 sink that requires further investigation to determine its potential impact on larger scales. This study also calls attention to the poleward distribution of atmMOB, as well as to the potential influence of microbial atm CH4 oxidation, in the context of regional CH4 flux models and global warming.

A mineralogical characterization of biogenic calcium carbonates precipitated by heterotrophic bacteria isolated from cryophilic polar regions.

Precipitation of calcium carbonate (CaCO3(s) ) can be driven by microbial activity. Here, a systematic approach is used to identify the morphological and mineralogical characteristics of CaCO3(s) precipitated during the heterotrophic growth of micro-organisms isolated from polar environments. Focus was placed on establishing mineralogical features that are common in bioliths formed during heterotrophic activity, while in parallel identifying features that are specific to bioliths precipitated by certain microbial phylotypes. Twenty microbial isolates that precipitated macroscopic CaCO3(s) when grown on B4 media supplemented with calcium acetate or calcium citrate were identified. A multimethod approach, including scanning electron microscopy, high-resolution transmission electron microscopy, and micro-X-ray diffraction (µ-XRD), was used to characterize CaCO3(s) precipitates. Scanning and transmission electron microscopy showed that complete CaCO3(s) crystal encrustation of Arthrobacter sp. cells was common, while encrustation of Rhodococcus sp. cells did not occur. Several euhedral and anhedral mineral formations including disphenoid-like epitaxial plates, rhomboid-like aggregates with epitaxial rhombs, and spherulite aggregates were observed. While phylotype could not be linked to specific mineral formations, isolates tended to precipitate either euhedral or anhedral minerals, but not both. Three anhydrous CaCO3(s) polymorphs (calcite, aragonite, and vaterite) were identified by µ-XRD, and calcite and aragonite were also identified based on TEM lattice-fringe d value measurements. The presence of certain polymorphs was not indicative of biogenic origin, although several mineralogical features such as crystal-encrusted bacterial cells, or casts of bacterial cells embedded in mesocrystals are an indication of biogenic origin. In addition, some features such as the formation of vaterite and bacterial entombment appear to be linked to certain phylotypes. Identifying phylotypes consistent with certain mineralogical features is the first step toward discovering a link between these crystal features and the precise underlying molecular biology of the organism precipitating them.

Methanogen community composition and rates of methane consumption in Canadian High Arctic permafrost soils.

Increasing permafrost thaw, driven by climate change, has the potential to result in organic carbon stores being mineralized into carbon dioxide (CO2) and methane (CH4) through microbial activity. This study examines the effect of increasing temperature on community structure and metabolic activity of methanogens from the Canadian High Arctic, in an attempt to predict how warming will affect microbially controlled CH4 soil flux. In situ CO2 and CH4 flux, measured in 2010 and 2011 from ice-wedge polygons, indicate that these soil formations are a net source of CO2 emissions, but a CH4 sink. Permafrost and active layer soil samples were collected at the same sites and incubated under anaerobic conditions at warmer temperatures, with and without substrate amendment. Gas flux was measured regularly and indicated an increase in CH4 flux after extended incubation. Pyrosequencing was used to examine the effects of an extended thaw cycle on methanogen diversity and the results indicate that in situ methanogen diversity, based on the relative abundance of the 16S ribosomal ribonucleic acid (rRNA) gene associated with known methanogens, is higher in the permafrost than in the active layer. Methanogen diversity was also shown to increase in both the active layer and permafrost soil after an extended thaw. This study provides evidence that although High Arctic ice-wedge polygons are currently a sink for CH4, higher arctic temperatures and anaerobic conditions, a possible result of climate change, could result in this soil becoming a source for CH4 gas flux.

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b.

AIMS: This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes. METHODS AND RESULTS: Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immunofluorescence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes, five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. CONCLUSIONS: The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b. SIGNIFICANCE AND IMPACT OF THE STUDY: These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.