Investigating Transcriptomic Induction of Resistance and/or Virulence in Listeria monocytogenes Cells Surviving Sublethal Antimicrobial Exposure

The potential transcriptomic induction of resistance and/or virulence in two L. monocytogenes strains belonging to the most frequent listeriosis-associated serovars (i.e., 1/2a and 4b), following their sublethal antimicrobial exposure, was studied through qPCR determination of the relative expression of 10 selected related genes (i.e., groEL, hly, iap, inlA, inlB, lisK, mdrD, mdrL, prfA, and sigB). To induce sublethal stress, three common antimicrobials (i.e., benzalkonium chloride, thymol, and ampicillin) were individually applied for 2 h at 37 °C against stationary phase cells of each strain, each at a sublethal concentration. In general, the expression of most of the studied genes remained either stable or was significantly downregulated following the antimicrobial exposure, with some strain-specific differences to be yet recorded. Thymol provoked downregulation of most of the studied genes, significantly limiting the expression of 6/10 and 4/10 genes in the strains of ser. 1/2a and ser. 4b, respectively, including those coding for the master regulators of stress response and virulence (SigB and PrfA, respectively), in both strains. At the same time, the two genes coding for the invasion internalin proteins (InlA and InlB), with crucial role in the onset of L. monocytogenes pathogenesis, were both importantly upregulated in ser. 4b strain. The results obtained increase our knowledge of the stress physiology of L. monocytogenes under certain sublethal antimicrobial conditions that could be encountered within the food chain and in clinical settings, and may assist in better and more effective mitigation strategies.

Phenotypic and genotypic characterization of Staphylococcus aureus isolated from pork sausage

The objective of this study was to characterize the genotype and phenotype of S. aureus isolates from pork sausages. Fifty samples of pork sausages were collected, counts of coagulase-positive Staphylococcus were made and isolates were obtained to identify S. aureus species. In the isolates, the presence of genes sea, seb, sec, and sed was surveyed, the methicillin-resistance was assessed and the production of biofilm in Congo red agar, stainless steel, polyethylene, glass, and pork casing was tested. The capacity of biofilm formation was assessed after the exposure to sublethal stress. Of the samples tested, 12% had counts superior to what is permitted by the legislation. S. aureus was isolated in 44% of the samples. Of these, 54% had only the gene sed and 32% had genes sec and sed, 73% were classified as methicillin-resistant S. aureus (MRSA). Of the MRSA isolates, 62% had only gene sed and 35% had both genes found in this study. Regarding the biofilm formation in Congo red agar, 68% of S. aureus isolates were considered as biofilm formers. After undergoing the sublethal stress, most of the biofilm former isolates proceeded to form biofilm and the non-biofilm former isolates responded in a distinct manner. The condition in which the sublethal stress greatly induced the biofilm formation was the cold. Biofilm production was observed only in the stainless steel and pork casing in 71% and 57% of the isolates tested, respectively. Thus, we stress the importance of implementing good manufacturing practices within the industry to control microbial contamination and biofilm formation.

Preconditioning of Caenorhabditis elegans to Anoxic Insult by Inactivation of Cholinergic, GABAergic, and Muscle Activity

For most metazoans, oxygen deprivation leads to cell dysfunction and if severe, death. Sublethal stress prior to a hypoxic or anoxic insult (“preconditioning”) can protect cells from subsequent oxygen deprivation. The molecular mechanisms by which sublethal stress can buffer against a subsequent toxic insult and the role of the nervous system in the response are not well understood. We studied the role of neuronal activity preconditioning to oxygen deprivation in C. elegans. Animals expressing the histamine gated chloride channels (HisCl1) in select cell populations were used to temporally and spatially inactivate the nervous system or tissue prior to an anoxic insult. We find that inactivation of the nervous system for 3 hours prior to the insult confers resistance to a 48-hour anoxic insult in 4th-stage larval animals. Experiments show that this resistance can be attributed to loss of activity in cholinergic and GABAergic neurons as well as in body wall muscles. These observations indicate that the nervous system activity can mediate the organism’s response to anoxia.

Trophic structure modulates community rescue following acidification

Community rescue occurs when a community that experiences lethal stress persists only through the spread of rare types, either genotypes or species, resistant to the stress. Rescue interacts with trophic structure because physical stress experienced by a focal assemblage within the community may also be experienced by its predators and prey. In general, trophic structure will facilitate rescue only when a stress has a less severe effect on a focal assemblage than on its predators. In other circumstances, when stress affects prey or has only a weak effect on predators, trophic structure is likely to hamper rescue. We exposed a community of phytoplankton and zooplankton derived from a natural lake to acidification in outdoor mesocosms large enough to support trophically complex communities. Rescue of the phytoplankton from severe acidification was facilitated by prior exposure to sublethal stress, confirming previous results from microcosm experiments. Even communities that have previously been less highly stressed were eventually rescued, however, because their zooplankton predators were more sensitive to acidification and became extinct. Our experiment shows how community rescue following severe stress is modulated by the differential effect of the stress relative to trophic level.

Community rescue in experimental phytoplankton communities facing severe herbicide pollution

Evolutionary rescue occurs when adaptation prevents local extinction in deteriorating environments. Laboratory experiments with microorganisms have shown that the likelihood of evolutionary rescue is greatest in large populations that have previously experienced sublethal doses of stress. To assess this result in natural communities, we conducted a mesocosm experiment with semi-natural phytoplankton communities exposed to glyphosate, a widely used herbicide. We tested whether community biomass and pre-exposure to sublethal stress would facilitate community rescue after severe contamination. Exposure to sublethal stress, but not community biomass, facilitated rescue significantly–even though it led to biodiversity loss. Furthermore, glyphosate had modest effects on community composition, suggesting that community resistance to glyphosate was primarily driven by changes in resistance within taxa, not by community turnover. Our results expand the scope of community evolutionary rescue theory to complex ecosystems and confirm that prior stress exposure is a key predictor of rescue.