Streptococcus dysgalactiae ssp. dysgalactiae in Norwegian bovine dairy herds: Risk factors, sources, and genomic diversity.

Despite the importance of Streptococcus dysgalactiae ssp. dysgalactiae (SDSD) as an udder pathogen, the reservoir and epidemiological characteristics of this bacterium are largely unexplored. The aims of this study were to investigate risk factors for SDSD intramammary infections (SDSD-IMI) in Norwegian bovine dairy herds, identify sources of SDSD on animals and in the environment, and elucidate the genetic diversity of SDSD isolates. Data from herd recordings and a questionnaire were used to investigate herd-level risk factors for SDSD-IMI in 359 freestall dairy herds. Seven herds with a suspected high prevalence of SDSD-IMI were visited to sample extramammary sources (e.g., skin, wounds, mucous membranes, and freestall environment). Bacterial isolates were whole-genome sequenced to investigate the distribution of SDSD genotypes within herds and to assess the phylogenetic relationship between SDSD isolates from 27 herds across Norway. Risk factors for high incidence of SDSD-IMI in freestall dairy herds were related to housing, including closed flooring in alleys and rubber mats in cubicle bases. Parlor milking was also a risk factor compared with automatic milking systems. From herd visits, a considerable proportion of extramammary samples were SDSD positive, particularly from wounds and skin of the animals and the cubicle bases. Samples from mucous surfaces (nostrils, rectum, and vagina) and water troughs were least frequently positive. Eight multilocus sequence types (ST) were identified among the sequenced isolates from 27 herds, and phylogenetic analyses revealed 8 clades corresponding to ST. No significant association was identified between sampling site (milk, body sites, and environment) and ST. In 4 of 6 herds from which 5 or more isolates were available, one ST dominated and was found in milk and extramammary samples. One ST (ST453) was found in 15 of 27 herds, which implies that this is a widely distributed and possibly a bovine-adapted strain. Findings in this study suggest that SDSD is a cow-adapted opportunist with potential for contagious transmission, and that the freestall environment is likely to play a role in transmission between cows.

Feeding concentrates with different protein sources to high-yielding, mid-lactation Norwegian Red cows: Effect on cheese ripening.

Soybean meal is one of the most important protein sources in concentrate feeds for dairy cows. The objective of the present study was to provide knowledge on the effects of using a novel yeast microbial protein source (Candida utilis) in concentrate feed for dairy cows on the production and quality of a Gouda-type cheese. Forty-eight Norwegian Red dairy cows in early to mid lactation were fed a basal diet of grass silage, which was supplemented with 3 different concentrate feeds. The protein source of the concentrates was based on conventional soybean meal (SBM), novel yeast (C. utilis; YEA), or barley (BAR; used as negative control because barley has a lower protein content). The experiment was carried out for a period of 10 wk, with the first 2 wk as an adaptation period where all dairy cows were fed grass silage and the SBM concentrate. The cows were then randomly allocated to 1 of the 3 different compound feeds: SBM, yeast, or barley. Cheeses were made during wk 8 and 9 of the experiment, with 4 batches of cheese made from milk from each of the 3 groups. The cheeses made from milk from cows fed SBM concentrate (SBM cheese) had a higher content of dl-pyroglutamic acid and free amino acids than the other cheeses, indicating a faster ripening in the SBM cheeses. Despite these differences, the sensory properties, the microbiota, and the Lactococcus population at 15 wk of ripening were not significantly different between the cheeses. This experiment showed that although the raw materials used in the concentrate feed clearly influenced the ripening of the cheeses, this did not affect cheese quality. Yeast (C. utilis) as a protein source in concentrate feed for dairy cows can be used as a replacement for soybean meal without compromising the quality of Norwegian Gouda-type cheeses.

Insect frass in stored cereal products as a potential source of Lactobacillus sanfranciscensis for sourdough ecosystem.

AIM: Insect frass samples were collected from Drosophila melanogaster, Plodia interpunctella, Rhyzopertha dominica, Sitophilus granarius, Sitophilus oryzae, Sitophilus zeamais, Tribolium confusum and Tribolium castaneum to elucidate if they can be the origin of Type I sourdough micro-organisms (Lactobacillus sanfranciscensis and Candida milleri). METHODS AND RESULTS: Selective enrichments were carried out to isolate lactic acid bacteria (LBA) and yeast. A metagenetic analysis, targeted on bacterial 16S rRNA gene and fungal ITS region, was performed by using Illumina MiSeq protocol. In cultivation conditions, Lactococcus garvieae and Rhodotorula mucilaginosa were the most frequently species among LAB and yeasts respectively. The Next Generation Sequencing approach revealed that Enterobacteriaceae, Pseudomonadacae and Bacillaceae were the dominating taxa, accounting for 61% of the bacterial community. Lactobacillus genus showed a relative abundance of only 0·36%, but L. sanfranciscensis proved to be the species most frequent between lactobacilli and predominant in faecal samples of T. castaneum and T. confusum larvae. The core fungal microbiota was constituted by Saccharomycetales, Pleosporaceae and Nectriaceae that attained the 51% of recognized OTUs. While the most abundant yeast genus was Candida (17·1%), sequences belonging to C. milleri were not found. CONCLUSIONS: Frass released by the insects of stored cereal products can be the natural reservoir of L. sanfranciscensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Insect dejections are potential sources of novel strains for controlled bakery productions.

Potential of Lactobacillus curvatus LFC1 to produce slits in Cheddar cheese.

Defects in Cheddar cheese resulting from undesired gas production are a sporadic problem that results in significant financial losses in the cheese industry. In this study, we evaluate the potential of a facultatively heterofermentative lactobacilli, Lactobacillus curvatus LFC1, to produce slits, a gas related defect in Cheddar cheese. The addition of Lb. curvatus LFC1 to cheese milk at log 3 CFU/ml resulted in the development of small slits during the first month of ripening. Chemical analyses indicated that the LFC1 containing cheeses had less galactose and higher levels of lactate and acetate than the control cheeses. The composition the cheese microbiota was examined through a combination of two culture independent approaches, 16S rRNA marker gene sequencing and automated ribosomal intergenic spacer analysis; the results indicated that no known gas producers were present and that high levels of LFC1 was the only significant difference between the cheese microbiotas. A ripening cheese model system was utilized to examine the metabolism of LFC1 under conditions similar to those present in cheeses that exhibited the slit defect. The combined cheese and model system results indicate that when Lb. curvatus LFC1 was added to the cheese milk at log 3 CFU/ml it metabolized galactose to lactate, acetate, and CO2. For production of sufficient CO2 to result in the formation of slits there needs to be sufficient galactose and Lb. curvatus LFC1 present in the cheese matrix. To our knowledge, facultatively heterofermentative lactobacilli have not previously been demonstrated to result in gas-related cheese defects.

Application of ARISA to assess the influence of salt content and cation type on microbiological diversity of Cheddar cheese.

UNLABELLED: The structure and dynamics of microbial populations play a significant role during cheese manufacture and ripening. Therefore, fast and accurate methods for identification and characterization of the microbial populations are of fundamental importance to the cheese industry. In this study, we investigate the application of the automated ribosomal intergenic spacer analysis (ARISA) for the assessment of the microbial dynamics in cheeses differing in salt cation level and type. We developed a database of the observed and theoretical length of the 16S-23S intergenic spacer of common lactic acid bacteria (LAB) found in cheese and used the database to describe the structure and dynamics of microbial populations during ripening. Salt content and cation concentration did not significantly influence the overall bacteria structure, except that lower salt levels resulted in enhanced starter survival. Presence of nonstarter LAB was detected by ARISA and denaturing gradient gel electrophoresis (DGGE) after 3 months for all the cheeses analysed. ARISA used as fingerprinting method, proved to be a rapid and inexpensive technique for the discrimination of LAB in cheese and demonstrated higher resolution and performance in comparison with DGGE. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial communities play important roles during cheese making and ripening, hence rapid inexpensive methods to characterize this microbiota are of great interest to both academic and industrial scientists. The application of automated ribosomal intergenic spacer analysis (ARISA) was used to examine the microbial ecology of Cheddar cheese differing in salt level and type. ARISA is well suited to the analysis of the microbial ecology of cheese during ripening. Additionally, the results confirm that salt concentration influences starter culture survival in the cheese matrix, while significant differences were not observed in the nonstarter lactic acid bacteria.

Metabolism of milk fat globule membrane components by nonstarter lactic acid bacteria isolated from cheese.

The objective of this study was to investigate how components present in the milk fat globule membrane (MFGM) may be used for growth and survival by cheese-ripening lactobacilli. This was achieved by analyzing metabolites produced during incubation on appropriate media. The lactobacilli investigated were able to utilize components from the MFGM throughout a 24-d incubation period. We observed an apparent connection between the higher proteolytic activity of Lactobacillus paracasei INF448 and its ability to grow in the MFGM media after depletion of readily available sugars. All the studied strains produced large amounts of acetate when grown on an acylated aminosugar, presumably from deacetylation of the monosaccharides. Growth of Lb. plantarum INF15D on D-galactose resulted in a metabolic shift, expressed as different fates of the produced pyruvate, compared with growth on the other monosaccharides. For Lb. plantarum INF15D, the presence of D-galactose also seemed to initiate degradation of some amino acids known to take part in energy production, specifically Arg and Tyr.

Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis.

The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands.

AIM: To investigate the application of high-resolution melt (HRM) analysis for rapid species-level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. METHODS AND RESULTS: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR-DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. CONCLUSION: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. SIGNIFICANCE AND IMPACT OF THE STUDY: HRM profiling is suggested as an additional approach for identification of DGGE bands.