This study aimed to identify factors that influence the development of biofilm by
Listeria monocytogenes strains and to determine the extent to which biofilm production protects against quaternary ...ammonium compound (QAC) disinfectant challenge. A total of 95
L. monocytogenes strains were studied and biofilm production was assessed as a function of incubation temperature, media pH, strain origin, serotype, and environmental persistence status. Attachment and biofilm development (inferred by the level of attached biomass) were measured
in vitro using a colourimetric 96-well microtitre plate method in nutritive media (Brain–Heart Infusion). Increased biofilm production correlated with increasing temperature and the most acidic, or most alkaline, growth conditions tested. Clinical and environmental (food factory) strains were observed to increase biofilm production at higher and lower incubation temperatures respectively, independent of their rate of planktonic growth. Serotype 1/2a strains produced significantly more biofilm. Biofilm maturity, rather than strain, was correlated with resistance to QAC. Carbohydrate containing exopolymeric material could not be detected in the biofilm of representative strains, and no correlation between strains recovered as persistent food factory contaminants and biofilm production was identified. Although limited to
in vitro inference based on the assay system used, our results suggest that environmental conditions determine the level of biofilm production by
L. monocytogenes strains, independent of the rate of planktonic growth, and that this may manifest from selection pressures to which a given strain grows optimally.
► Variation in biofilm production may be reduced in strains of common origin. ► Prolific biofilm producing strains show rapid adhesion. ► Disinfectant resistance is related to biofilm maturity. ► EPS in biofilms of
L. monocytogenes are non-polysaccharide. ► Biofilm does not characterise persistent
L. monocytogenes.
Abstract
MPYS (also known as STING, MITA, and TMEM173) is a type I IFN stimulator that is essential for host defense against DNA virus infection and appears important in defense against certain ...bacteria. The in vivo significance and mechanisms by which MPYS mediates host defense against nonviral pathogens are unknown. Using an MPYS-deficient mouse (Tmem173 ), we determined that, distinct from the IFNAR−/− mice, MPYS deficiency leads to increased bacterial burden in the liver upon Listeria monocytogenes infection. The increase was correlated with the diminished MCP-1 and MCP-3 chemokine production and decreased blood and liver Ly6Chi monocyte frequency. We further demonstrate that MPYS-deficient Ly6Chi monocytes are intrinsically defective in migration to the liver. Lastly, adoptive transfer of wild-type Ly6Chi monocyte into MPYS-deficient mice decreases their liver bacterial burden. Our findings reveal a novel in vivo function of MPYS that is distinct from its role in activating type I IFN production.
CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a ...protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
During 2015-2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was ...compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages. WGS-based typing should replace PFGE as the primary typing method for L. monocytogenes.
The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, ...altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.
Significance The small nucleotide cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) recently emerged as a ubiquitous signaling molecule among bacteria, with essential roles in both bacterial physiology and host–pathogen interactions. Bacterial mutants with abnormal c-di-AMP levels exhibit growth and virulence defects, reflecting the importance of regulating c-di-AMP synthesis and degradation for normal signal transduction and adaptation to changing environments. Previously documented phosphodiesterases hydrolyze c-di-AMP via the DHH-DHHA1 domain, but they are not present in all c-di-AMP synthesizing species. We identified a previously unrecognized class of His-Asp -domain phosphodiesterases that are widespread across several taxonomic groups. Furthermore, for the bacterial pathogen Listeria monocytogenes , phosphodiesterase mutants exhibit enhanced host inflammation, growth defects inside host cells, and significantly attenuated virulence in a murine model of infection.
The objective of this work was to investigate the effect of stress conditions frequently encountered in food-associated environments on virulence-associated characteristics of eight strains of ...Listeria monocytogenes. Strains were grown at low (11 °C, cold stress) and optimal (37 °C) temperatures and in high NaCl concentrations (6% NaCl, 11 °C; cold-osmotic stress) and tested for their ability to invade the human intestinal epithelial Caco-2 cells. Results demonstrate that the correlation between exposure to cold stress and increased invasion phenotype is strain-dependent as strains investigated exhibited different behaviours, i.e. exposure to cold stress conditions resulted in a significant increase of invasion levels in five out of the eight strains tested, when compared to growth under optimal conditions. On the other hand, when these cold-adapted cells were subsequently submitted to high salt concentrations and low temperature, their enhanced ability to invade Caco-2 was lost. Surprisingly, saturated fatty acids (SFA) and branched chain fatty acids (BCFA) decreased when L. monocytogenes were exposed to stress conditions as opposed to what has been observed in other studies, therefore highlighting that further studies will need to deepen in the understanding of the lipid metabolism of these strains. The effect of stress conditions on the survival of three selected L. monocytogenes strains through an in vitro gastrointestinal (GI) tract digestion model was further investigated. The exposure to cold-osmotic stress increased the survival of one strain through the GI tract.
•Stress conditions on virulence traits of L. monocytogenes investigated.•Growth at 11 °C resulted in a significant increase of invasiveness in five strains.•Correlation between cold stress and increased invasiveness was strain-dependent.•Subsequent exposure to cold-osmotic stress resulted in reduced invasiveness.•SFA and BCFA decreased when L. monocytogenes were exposed to stress conditions.
Abstract Listeria monocytogenes consists of at least 4 evolutionary lineages (I, II, III, and IV) with different but overlapping ecological niches. Most L. monocytogenes isolates seem to belong to ...lineages I and II, which harbor the serotypes more commonly associated with human clinical cases, including serotype 1/2a (lineage II) and serotypes 1/2b and 4b (lineage I). Lineage II strains are common in foods, seem to be widespread in the natural and farm environments, and are also commonly isolated from animal listeriosis cases and sporadic human clinical cases. Most human listeriosis outbreaks are associated with lineage I isolates though. In addition, a number of studies indicate that, in many countries, lineage I strains are overrepresented among human isolates, as compared to lineage II strains. Lineage III and IV strains on the other hand are rare and predominantly isolated from animal sources. The apparent differences in the distribution of strains representing the L. monocytogenes lineages has lead to a number of studies aimed at identifying phenotypic differences among the different lineages. Interestingly, lineage II isolates seem to carry more plasmids than lineage I isolates and these plasmids often confer resistance to toxic metals and possibly other compounds that may be found in the environment. Moreover, lineage II isolates seem to be more resistant to bacteriocins than lineage I isolates, which probably confers an advantage in environments where bacteriocin-producing organisms are abundant. A large number of lineage II isolates and strains have been shown to be virulence-attenuated due to premature stop codon mutations in inlA and mutations in prfA . A subset of lineage I isolates carry a listeriolysin S hemolysin, which is not present in isolates belonging to lineages II, III, or IV. While lineage II isolates also show higher recombination rates than lineage I isolates, possibly facilitating adaptation of lineage II strains to diverse environments, lineage I isolates are clonal and show a low prevalence of plasmids and IS elements, suggesting that lineage I isolates may have mechanisms that limit the acquisition of foreign DNA by horizontal gene transfer. Diversifying selection has also been shown to have played an important role during evolution of the L. monocytogenes lineages and during divergence of L. monocytogenes from the non-pathogenic species L. innocua . Overall evidence thus suggests that the 4 L. monocytogenes lineages identified so far represent distinct ecologic, genetic, and phenotypic characteristics, which appear to affect their ability to be transmitted through foods and to cause human disease. Further insights into the ecology, evolution, and characteristics of these lineages will thus not only provide an improved understanding of the evolution of this foodborne pathogen, but may also facilitate improved control of foodborne listeriosis.
The objectives of this study were 1) to evaluate the impact of two industrial disinfectants on the viability of Listeria monocytogenes populations in biofilm and 2) to investigate the viability state ...of L. monocytogenes cells present on contact surfaces in the smoked salmon processing environment. In the first step, we cultured mono species and mixed species biofilms containing L. monocytogenes on stainless steel or polyvinyl chloride (PVC) at 8 °C for 48h. The biofilms were then exposed to quaternary ammonium- and hydrogen peroxide-based disinfectants. Residual total populations of L. monocytogenes were measured by qPCR, and viable culturable (VC) cell populations were quantified using standard microbiological culture-based techniques and by a quantitative PCR (qPCR) assay coupled with a propidium monoazide treatment. Decreases in VC populations and the appearance of viable but non culturable (VBNC) populations were observed in response to treatment with the disinfectants. An 8 month sampling campaign in 4 smoked salmon processing plants was also carried out to detect L. monocytogenes in environmental samples. VBNC cells were detected mainly after the cleaning and disinfection operations. This study showed that industrial disinfectants did not inactivate all L. monocytogenes cells on inert surfaces. The presence of VBNC populations of L. monocytogenes in the smoked salmon processing environment is a public health concern.
•Disinfectants based on hydrogen peroxide or quaternary ammonium induced the VBNC state of L. monocytogenes in biofilm.
Listeria monocytogenes is a gram‐positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in ...the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria‐derived cyclic‐di‐AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic‐di‐AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.
Synopsis
The innate immune system detects microbes and induces protective and pathological responses. Here, it is shown that bacterial DNA is the molecule‐triggering expression of interferon β during infection with Listeria monocytogenes in human myeloid cells and that this is dependent on the DNA sensors IFI16 and cGAS.
Bacterial DNA is the molecule stimulating IFNβ expression during L. monocytogenes infection in human cells
Listeria‐induced IFNβ expression correlates with bacteriolysis in the macrophage cytosol, and bacterial DNA associates with IFI16 and cGAS
Expression of IFNβ in Listeria‐infected cells proceeds through a pathway dependent on IFI16, cGAS and STING
While bacterium‐derived cyclic‐di‐AMP triggers interferon responses in murine cells, Listeria monocytogenes‐infected human myeloid cells utilize an innate immune sensory pathway that directly recognizes bacterial DNA.
•We describe the most recent findings in comparative pan-genomics of L. monocytogenes.•The epidemiology of L. monocytogenes is described in humans and foods.•We illustrate the surveillance systems of ...the European Union and United States.•Application of molecular techniques is a core tool in epidemiological investigation.
Listeria monocytogenes is an opportunistic food-borne pathogen responsible for listeriosis, a disease associated with high mortality rates. L. monocytogenes causes invasive syndromes and case-fatality can be as high as 30%, in specific high-risk population groups such as the elderly, immuno-compromised individuals, fetuses and newborns. Acquisition of the disease is mainly due to consumption of contaminated (predominantly ready-to-eat) food. We aimed to provide a state-of-the-art collection of different likely evolutionary models, based on recombination and positive selection, and the phylogenetic relationship between lineages of L. monocytogenes and between them and other Listeria species. We described the most recent findings in comparative pan-genomics, considering the core and accessory genome in relation to virulence and adaptation to different environments. Finally, this review illustrates L. monocytogenes epidemiology and transmission in humans, foods and animals, the surveillance systems of the European Union and United States and the application of molecular techniques as a core tool in epidemiological investigation.
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