A laboratory exercise for detecting microbial contaminants.

The development and evaluation of a 6-hours laboratory class, based on capillary electrophoresis (CE) and the detection of microbial contaminants, is described. It can be easily scaled up or down, to suit class sizes up to 188 and completed in a shorter time scale. CE uses narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. A laboratory exercise has been devised to illustrate how CE-based genetic analysis system processes DNA fragment analysis to detect three microbial contaminants. The protocol is relatively inexpensive and uses standard molecular biology reagents and equipment. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):279-284, 2018.

Lipidomics as an important key for the identification of beer-spoilage bacteria.

Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used for characterizing intact plasmalogen phospholipid molecules in beer-spoilage bacteria. Identification of intact plasmalogens was carried out using collision-induced dissociation and the presence of suitable marker molecular species, both qualitative and quantitative, was determined in samples containing the anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method had a limit of detection at 1 pg for the standard, i.e. 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and be linear in the range of four orders of magnitude from 2 pg to 20 ng. This technique was applied to intact plasmalogen extracts from the samples of contaminated and uncontaminated beer without derivatization and resulted in the identification of contamination of beer by Megasphaera and Pectinatus bacteria. The limit of detection was about 830 cells of anaerobic bacteria, i.e. bacteria containing natural cyclopropane plasmalogenes (c-p-19:0/15:0), which is the majority plasmalogen located in both Megasphaera and Pectinatus. The SIM ESI-MS method has been shown to be useful for the analysis of low concentration of plasmalogens in all biological samples, which were contaminated with anaerobic bacteria, e.g. juice, not only in beer. Significance and impact of the study: Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) using collision-induced dissociation was used to characterize intact plasmalogen phospholipid molecules in beer-spoilage anaerobic bacteria Megasphaera and Pectinatus. Using selected ion monitoring (SIM), this method has a detection limit of 1 pg for the standard 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine and is linear within four orders of magnitude (2 pg to 20 ng). The limit of detection was about 830 cells of bacteria containing natural cyclopropane plasmalogen (c-p-19:0/15:0). SIM ESI-MS method is useful for analyzing low concentrations of plasmalogens in biological samples contaminated with anaerobic bacteria, e.g. beer or juice.

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

Pectinatus sottacetonis sp. nov., isolated from a commercial pickle spoilage tank.

A strictly anaerobic, Gram-stain-negative, non-spore-forming, motile bacterium, designated strain FSRU B0405(T), was isolated from a commercial pickle spoilage tank and characterized by biochemical, physiological and molecular biological methods. Analyses of the 16S rRNA gene sequence of strain FSRU B0405(T) showed affiliation to the class Negativicutes in the phylum Firmicutes, with the closest relatives being the type strains of Pectinatus haikarae (96 %) and Pectinatus brassicae (95 %). In maximum-likelihood and neighbour-joining phylogenetic trees, strain FSRU B0405(T) clustered definitively (in 100 % of bootstrapped trees) within the genus Pectinatus, but not specifically with any characterized species within this genus. Strain FSRU B0405(T) was a slightly curved rod, varying from 3 to 30 µm in length, motile with a distinctive X-wise movement, having flagella only on the concave side of the cell. The isolate produced acetate and propionate from fructose and glucose as major metabolites similar to type strains of species of the genus Pectinatus. The major fatty acids were C11 : 0, C13 : 0, C15 : 0, C13 : 0 3-OH, C17 : 1 and C18 : 1ω11t. Strain FSRU B0405(T) differed from the pickle wastewater strain, Pectinatus brassicae TY(T), due to its lack of susceptibility to vancomycin, acetoin production, growth temperature range, acid production from adonitol, erythritol, glycerol, inositol, lactose, maltose, mannose, ribose, salicin, sorbitol, trehalose and xylitol and lack of hydrolysis of milk. Strain FSRU B0405(T) could be differentiated from other species of the genus Pectinatus both phenotypically and genetically. The results indicate that strain FSRU B0405(T) represents a novel species of the genus Pectinatus, for which the name Pectinatus sottacetonis sp. nov. is proposed. The type strain is FSRU B0405(T) ( = ATCC BAA-2501(T) = VTT E-113163(T)). An emended description of the genus Pectinatus is also provided.

Characterization of cucumber fermentation spoilage bacteria by enrichment culture and 16S rDNA cloning.

UNLABELLED: Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram-negative anaerobic bacteria supersede Gram-positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. PRACTICAL APPLICATION: An understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.

Pectinatus brassicae sp. nov., a Gram-negative, anaerobic bacterium isolated from salty wastewater.

A novel Gram-negative, non-spore-forming, strictly anaerobic, heterotrophic bacterium, strain TY(T), was isolated from salty pickle wastewater. Cells were rod-shaped with comb-like flagella, slightly curved and very variable in length. Optimal growth occurred at 28 °C and pH 6.5. Cells were resistant to up to 50 g NaCl l(-1). Strain TY(T) produced acid from glycerol, sucrose, glucose, fructose and mannitol. The main fermentation products from glucose were acetic and propionic acids. Tests for acid phosphatase and naphthol-AS-BI-phosphohydrolase activities were positive. The major fatty acids were C(14 : 0) DMA (18.7 %), C(15 : 0) (15.4 %), anteiso-C(18 : 1) (15.2 %), C(11 : 0) (13.3 %) and summed feature 5 (C(17 : 1)ω7c and/or C(17 : 2)) (11.0 %). The DNA G+C content was 35.9 mol%. 16S rRNA gene sequence-based phylogenetic analysis indicated that strain TY(T) represented a novel species of the genus Pectinatus (sequence similarity to other members of the genus ranged from 93.2 to 94.8 %). Based on its phenotypic, genotypic and phylogenetic characteristics, strain TY(T) is proposed to represent a novel species, named Pectinatus brassicae sp. nov. (type strain TY(T) = JCM 17499(T) = DSM 24661(T)).

Pectinatus portalensis nov. sp., a relatively fast-growing, coccoidal, novel Pectinatus species isolated from a wastewater treatment plant.

The genus Pectinatus is currently composed by two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis , both asociated with beer spoilage. This study describes a novel isolate (strain B6) retrieved from a wastewater treatment plant collecting residues from a large number of wineries. Based on similarity analysis of 16S rRNA gene sequences, strain B6 belongs to the genus Pectinatus . Strain B6 is a strict anaerobe like other Pectinatus species and it presents non-motile, coccoid cells showing a slight oval shape. Strain B6 shows marked physiological differences with other Pectinatus species both in fatty acid composition and carbon source utilization. The most abundant fatty acids found in strain B6 were 18:1 (42.8%) and 16:0 (18.3%) representing a total of over 61% of fatty acids in this microorganism while these fatty acids represented 41.3% in P. cerevisiiphilusT and 2.4% in P. frisingensisT of their total. Fatty acid 15:0 was not significant in strain B6 and represented 28.6% and 13.3% for P. cerevisiiphilusT and P. frisingensisT, respectively. Strain B6 showed a faster growth rate and higher optimum temperature than its relatives P. cerevisiiphilus and P. frisingensis . Strain B6, P. cerevisiiphilus and P. frisingensis could be clearly differentiated by acid production tests from substrates such as esculine and gluconate, and the lack of acid production from rhamnose and fucose among others. G+C mol% content in strain B6 is 36.5%. Based on genotypic and phenotypic differences, strain B6 is proposed as a novel Pectinatus species, P. portalensis nov. sp. Both strain B6 and the two described species of Pectinatus grow on beers and wines. These results provide insights about the origin and reservoirs of Pectinatus species and spoiling alcoholic beverages.