Lélia Chambel

Thirty years of viable but nonculturable state research: Unsolved molecular mechanisms

Abstract 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.

Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces

During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.

Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli

Aims:  To analyse viable but nonculturable (VBNC) state induction in Escherichia coli and resuscitation of VBNC suspensions in several conditions.

Listeria monocytogenes Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of L. monocytogenes but Is Absent from Many Field Isolates

ABSTRACT 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.

Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samples

The aim of this study was to develop a PCR‐based method of gene‐directed multiplex PCR to rapidly identify microcystins producing cyanobacteria, regardless of their taxa, that could be applied in routine freshwater monitoring. Instead of using the amplification of only one or two mcy gene fragments, a multiplex PCR that simultaneously amplifies mcyA‐cd, mcyAB, and mcyB fragments of the microcystin gene cluster was validated with DNA from 124 cyanobacterial isolates and applied in 37 environmental samples. The toxicological status of the isolates was assessed by high‐performance liquid chromatography also used as the “gold standard” for the evaluation of multiplex mcy genes‐based PCR, where a sensitivity of 92.3% and a specificity of 100% have been obtained. For the environmental samples, a rapid protocol for their direct use in the PCR reaction has been developed and, by using ELISA results as “gold standard” for the presence of microcystins in these samples, a sensitivity of 80% and a specificity of 100% were achieved, showing that this multiplex PCR test is a rapid, reliable, and economical way of assessing the microcystin‐producing potential of cyanobacteria in freshwaters, regardless of their taxa or microcystins variant produced.

MLVA16 Typing of Portuguese Human and Animal Brucella melitensis and Brucella abortus Isolates

To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16Orsay 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 MLVA16Orsay 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 MLVA16Orsay 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.

Characterization of two resuscitation promoting factors of Listeria monocytogenes

In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate growth of vegetative cells through lag phase reduction. Recently, it was suggested that proteins Lmo0186 and Lmo2522 of Listeria monocytogenes are equivalent to Rpf proteins based on their genomic context and conserved domain architecture. It was proposed that they have evolved through non-orthologous displacement of the Rpf domain found in actinobacteria. Here we present biological and biochemical data supporting a function of Lmo0186 and Lmo2522 as Rpfs. These proteins are collectively dispensable for growth but a lmo0186 lmo2522 double mutant exhibits an extended lag phase when diluted in minimal medium. This phenotype could be partially complemented by medium supplementation with fM to nM concentrations of purified hexahistidine-tagged versions of Lmo0186 and Lmo2522, showing that these proteins can stimulate growth. Gel filtration analysis and cross-linking experiments suggest that the recombinant proteins in solution are elongated monomers. Both proteins display muralytic activity against crude cell wall preparations and are active against an artificial lysozyme substrate. Our study thus supports the hypothesis that Lmo0186 and Lmo2522 are functional equivalents of actinobacteria Rpf proteins and represents the first characterization of two Rpf homologues from firmicutes.

Impact of discards of beam trawl fishing on the nematode community from the Tagus estuary (Portugal)

The impact of dead discards, originating from beam trawl fishing on the nematode community from the Tagus estuary was investigated in terms of vertical distribution of the dominant nematode groups. Sediment cores were collected from a mud-flat from the Tagus estuary. Crangoncrangon (Linnaeus, 1758) carcasses were added to the surface of the cores, simulating the settling of dead discards on the sediment. The vertical distribution of the dominant nematode groups was determined up to 4cm deep at four different moments in time post deposition (0, 2, 4 and 6h) and compared to control cores. The C.crangon addition to the sediment led to the formation of black spots and therefore oxygen depleted areas at the sediment surface. The Chromadora/Ptycholaimellus group, normally dominant at the surface layer, migrated downwards due to their high sensibility to toxic conditions. Sabatieria presented the opposite trend and became the dominant group at the surface layer. Since Sabatieria is tolerant to oxygen stressed conditions and high sulphide concentrations, we suggest that it migrated opportunistically towards an unoccupied niche. Daptonema, Metachromadora and Terschellingia did not show any vertical migration, reflecting their tolerance to anoxic and high sulphidic conditions. Our study showed that an accumulation of dead discards at the sediment surface might therefore alter the nematode community vertical distribution. This effect is apparently closely related to toxic conditions in the sediment, induced by the deposition of C.crangon at the sediment surface. These alterations might be temporal and reflect an adaptation of the nematode community to dynamic intertidal environments.

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.

Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.

ABSTRACT 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.