Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (a w) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650-0.605 a w. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a w). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 a w for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a w for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life

Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes

In the last two decades major changes have occurred in how microbial ecologists study microbial communities. Limitations associated with traditional culture-based methods have pushed for the development of culture-independent techniques, which are primarily based on the analysis of nucleic acids. These methods are now increasingly applied in food microbiology as well. This review presents an overview of current community profiling techniques with their (potential) applications in food and food-related ecosystems. We critically assessed both the power and limitations of these techniques and present recent advances in the field of food microbiology attained by their application. It is unlikely that a single approach will be universally applicable for analyzing microbial communities in unknown matrices. However, when screening samples for well-defined species or functions, techniques such as DNA arrays and real-time PCR have the potential to overtake current culture-based methods. Most importantly, molecular methods will allow us to surpass our current culturing limitations, thus revealing the extent and importance of the `non-culturable¿ microbial flora that occurs in food matrices and production

Multiplex detection and identification of pathogenic Legionella species and associated hosts using DNA array technology

Legionnaires’ disease is a sporadic or endemic disease caused by legionellae, which are Gram-negative bacteria that are ubiquitous inhabitants of aquatic environments. Out of more than 50 Legionella species, Legionella pneumophila, and more in particular L. pneumophila serogroup (sg) 1, is reported as the most common cause of legionellosis. Nevertheless, other serogroups and other Legionella species are increasingly associated with human disease, including L. dumoffii, L. micdadei, L. longbeachae and L. feeleii. Consequently, in order to minimize health risks it is important to detect not only L. pneumophila, but also other Legionella species. Furthermore, correct identification of the species is relevant for epidemiological studies and for the identification of sources of infection. Accurate detection and identification of Legionella using conventional methods is complicated by their growth requirements, their ability to enter a viable non-culturable state, their association with protozoan hosts and the occurrence of Legionella within biofilms. These drawbacks can be circumvented by molecular detection methods, especially by those based on the detection of nucleic acids. Currently, polymerase chain reaction (PCR)-based DNA array technology is one of the most suitable techniques to detect multiple organisms in a single assay, even if they differ in only a single to a few bases in the nucleotide sequence that is targeted. As a result, DNA arrays have become highly attractive for several applications. Recently we developed a macrophage infectivity potentiator (mip) gene-based DNA array for the simultaneous detection and identification of a comprehensive set of Legionella species, including all species associated with human disease (about 20 species), and one of its protozoan hosts, Hartmanella vermiformis. Each diagnosis can be achieved within 36 hours of sampling based on an objective technique utilizing an array of specific DNA fragments. To validate the DNA array, environmental samples out of water distribution systems were collected from different companies/buildings and analyzed with both classical techniques and the developed DNA array. Although results from the DNA array were generally corroborated by the more classical techniques, the DNA array enables a more rapid, sensitive and more specific Legionella detection in the water samples. Moreover, taking into account the unlimited expanding possibilities of DNA arrays to include detector oligonucleotides for other and more microorganisms as well as for other biomarkers, this technique has the potential for studying population dynamics and ecology of several target populations in a single assay. To our knowledge, this is the first study that emphasizes multiplex detection and identification of several Legionella species and their associated hosts, revealing essential information for legionellosis risk assessment in one single assay.status: publishe

Protective effect of hop β-acids on microbial degradation of thick juice during storage

Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. However, thick juice storage commonly faces problems due to microbial degradation. In this study the value of a commercial alkaline solution of hop β-acids (HBA) was assessed for prevention of microbial degradation of thick juice. The antimicrobial effect of different concentrations of HBA against juice degradation was tested in a pilot-scale thick juice storage experiment. Thick juice degradation, indicated as a decrease in pH, was generally accompanied by an increase in the count of fastidious bacteria (FB) on Columbia Agar with Sheep Blood (CAwSB), which were mainly identified as Tetragenococcus halophilus. Addition of HBA delayed juice acidification and the development of T. halophilus in a concentration-dependent manner. The susceptibility of T. halophilus to HBA was determined by plating degraded thick juice (> 105 CFU/ml) on CAwSB plates with different concentrations of HBA (0–160 ppm). None of the HBA concentrations tested reduced the number of FB colonies formed, but increasing HBA concentrations extended the lag time of colony formation. In conclusion, HBA can prolong the storage life of thick juice in the sugar industry, although degradation cannot be eliminated.status: publishe

Molecular characterization of the microflora during malting to enhance malt quality and processability

Background - Optimization of malt quality with regard to a better lautering performance is a critical factor in the malting and brewing industry. Since malt quality is inversely related to the concentration of endosperm cell wall components such as arabinoxylans, management of cell wall degrading microorganisms may result in enhanced processing. An essential step in microflora management during malting requires the understanding of the microbial community structure as well as the population dynamics in the processing. Objectives – The objective of this study was to characterize the microbial (bacterial) communities in different industrial malting settings and relate these to malt quality and processability. Methods and Conclusions - Both culture-dependent and culture-independent molecular techniques were used to characterize the bacterial communities in different industrial malting settings, generating malt of different quality. 16S ribosomal DNA-targeted Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, supported by sequencing of clone libraries, were used to screen and compare the microbial communities at different stages of the malting process, i.e. from barley up to kilned malt. In addition, emphasis is put on the xylanase producing microflora. Apart from isolation and identification, the diversity of the xylanase genes was explored by amplifying xylanase genes from the glycosyl hydrolase (GH) family 10 and 11. Specific patterns associated with high-quality malt will be further investigated, aiming at obtaining cultures important for the malt quality. Ultimately, this study may result in the discovery of promising, process-borne microorganisms which may enhance malt processability, or the malting and brewing process in general.status: publishe

Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp. halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov.

Most bacteria recovered so far from sugar thick juice during storage represent strains of the species Tetragenococcus halophilus. Recently, several Gram-positive, non-motile, non-sporeforming cocci with other physiological and genetic traits were isolated from sugar thick juice samples from different origins. In this study, representative isolates were investigated using a polyphasic taxonomic approach. The 16S rRNA gene sequence similarity between these isolates and their closest relative, Tetragenococcus muriaticus, was 97.4 %. The level of DNA–DNA relatedness between isolate T1T, representing the newly found Tetragenococcus isolates, and T. muriaticus was 57 %. Isolate T1T had a DNA G+C content of 36.7 mol%. Phylogenetic data and genomic and phenotypic features demonstrated that the isolates represent a novel species, for which the name Tetragenococcus osmophilus sp. nov. is proposed with T1T as the type strain (5LMG 26041T 5DSM 23765T). Additionally, T. halophilus isolates from high-salt and highsugar environments showed clear differences in several physiological and genetic characteristics like RAPD fingerprints and 16S rRNA gene sequences. DNA–DNA hybridizations, however, showed 79 to 80% relatedness between osmophilic and halophilic T. halophilus isolates, demonstrating that the different strains belong to the same species. Based on the phenotypic and genotypic differences observed, as well as the different origins of the strains and the industrial relevance of thick juice degradation, two subspecies of T. halophilus are described in this manuscript: T. halophilus subsp. halophilus subsp. nov. for the strains isolated from salt media and T. halophilus subsp. flandriensis subsp. nov. for the strains isolated from sugar-rich environments, which were first isolated in Flanders, Belgium. The type strains for the subspecies are IAM 1676T (5LMG 11490T 5DSM 20339T) and T5T (5LMG 26042T 5DSM 23766T), respectively