Viable but nonculturable (VBNC) cells were recognized 30 years ago; and despite decades of research on the topic, most results are disperse and apparently incongruous. Since its description, a huge controversy arose regarding the ecological significance of this state: is it a degradation process without real significance for bacterial life cycles or is it an adaptive strategy of bacteria to cope with stressful conditions? In order to solve the molecular mechanisms of VBNC state induction and resuscitation, researchers in the field must be aware and overcome common issues delaying research progress. In this review, we discuss the intrinsic characteristic features of VBNC cells, the first clues on what is behind the VBNC state’s induction, the models proposed for their resuscitation and the current methods to prove not only that cells are in VBNC state but also that they are able to resuscitate.

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene andrpoC1phylogenies. A highly congruent association was observed between STTR- and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon–Wiener (J′) indices a high diversity was observed within all species, withPlanktothrix agardhiishowing the lowest diversity values (D=0.83;J′=0.88) andAphanizomenon flos-aquaethe highest ones (D=J′=0.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR) fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO₂, chitosan, copper, H₂O₂, and Grape Juice Medium), aroma-related activities (resistance to 5, 5′, 5′′-trifluor-d, l-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities) as well as other relevant technological proprieties (protease activity and biogenic amines production). M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM) and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.

To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16_Orsay assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16_Orsay showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the “Americas” (17%) and “East Mediterranean” (83%) groups. No isolate belonged to the “West Mediterranean” group. Eighty-five percent of the human isolates (39 in 46) fit in the “East-Mediterranean” group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16_Orsay provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.