Resilience in the Face of Uncertainty: Sigma Factor B Fine-Tunes Gene Expression To Support Homeostasis in Gram-Positive Bacteria.

Gram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor σ(B) σ(B) has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus Recent insight from next-generation-sequencing results indicates that σ(B)-dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of σ(B) to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of σ(B).

Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption.

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA.

Increased spread and replication efficiency of Listeria monocytogenes in organotypic brain-slices is related to multilocus variable number of tandem repeat analysis (MLVA) complex.

BACKGROUND: Listeria (L.) monocytogenes causes fatal infections in many species including ruminants and humans. In ruminants, rhombencephalitis is the most prevalent form of listeriosis. Using multilocus variable number tandem repeat analysis (MLVA) we recently showed that L. monocytogenes isolates from ruminant rhombencephalitis cases are distributed over three genetic complexes (designated A, B and C). However, the majority of rhombencephalitis strains and virtually all those isolated from cattle cluster in MLVA complex A, indicating that strains of this complex may have increased neurotropism and neurovirulence. The aim of this study was to investigate whether ruminant rhombencephalitis strains have an increased ability to propagate in the bovine hippocampal brain-slice model and can be discriminated from strains of other sources. For this study, forty-seven strains were selected and assayed on brain-slice cultures, a bovine macrophage cell line (BoMac) and a human colorectal adenocarcinoma cell line (Caco-2). They were isolated from ruminant rhombencephalitis cases (n = 21) and other sources including the environment, food, human neurolisteriosis cases and ruminant/human non-encephalitic infection cases (n = 26). RESULTS: All but one L. monocytogenes strain replicated in brain slices, irrespectively of the source of the isolate or MLVA complex. The replication of strains from MLVA complex A was increased in hippocampal brain-slice cultures compared to complex C. Immunofluorescence revealed that microglia are the main target cells for L. monocytogenes and that strains from MLVA complex A caused larger infection foci than strains from MLVA complex C. Additionally, they caused larger plaques in BoMac cells, but not CaCo-2 cells. CONCLUSIONS: Our brain slice model data shows that all L. monocytogenes strains should be considered potentially neurovirulent. Secondly, encephalitis strains cannot be conclusively discriminated from non-encephalitis strains with the bovine organotypic brain slice model. The data indicates that MLVA complex A strains are particularly adept at establishing encephalitis possibly by virtue of their higher resistance to antibacterial defense mechanisms in microglia cells, the main target of L. monocytogenes.

Pyrrolizidine alkaloids and tropane alkaloids in milk samples from individual dairy farms of the German federal states of Bavaria and Schleswig-Holstein.

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020-2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC-ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number.

Impact of the Revision of European Food Hygiene Legislation and the Introduction of Convenience-based Food on Food Safety in the German Military.

The implementation of the European Food Regulation in the German military started in 2003 and was fully implemented in 2006. In addition, in 2003 the German military introduced the concept of using convenience-based foods targeted to improve the safety of food served to the troops. The aim of this study was to evaluate the impact of these changes on food safety and the occurrence of food-borne disease outbreaks in the German military. For this purpose, data from a total of 517 food-borne outbreaks that occurred between 1995 and 2019 in the responsible areas of the German military both within the country and abroad were subjected to a retrospective analysis. As a result, a significant decrease (p = 2.47 × 10-5) in the number of the food-borne outbreak was observed in the second observation period (2003-2019) compared to the first period (1995-2002). Food groups often found contaminated with pathogens were desserts and prepared dishes (first period), fresh produce, soups, and sauces (second period). Bacillus cereus, Enterobacteriaceae, Salmonella spp., and Staphylococcus aureus were dominant pathogens isolated from suspected foods during disease outbreaks in both periods, however, the absolute number of isolates reduced significantly in the second period. Therefore it can be concluded that the implementation of European food hygiene regulations together with the introduction of convenience-based foods had a significant positive impact on food safety in the German military.

Atypical H-type bovine spongiform encephalopathy in a cow born after the reinforced feed ban on meat-and-bone meal in Europe.

The significance of atypical bovine spongiform encephalopathies (BSE) in cattle for controlling the BSE epidemic is poorly understood. Here we report a case of atypical H-type BSE in a cow born after the implementation of the reinforced feed ban in Europe. This supports an etiology of H-type BSE unrelated to that of classical BSE.

Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis.

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

Asymptomatic Carriage of Listeria monocytogenes by Animals and Humans and Its Impact on the Food Chain.

Humans and animals can become asymptomatic carriers of Listeria monocytogenes and introduce the pathogen into their environment with their feces. In turn, this environmental contamination can become the source of food- and feed-borne illnesses in humans and animals, with the food production chain representing a continuum between the farm environment and human populations that are susceptible to listeriosis. Here, we update a review from 2012 and summarize the current knowledge on the asymptomatic carrier statuses in humans and animals. The data on fecal shedding by species with an impact on the food chain are summarized, and the ways by which asymptomatic carriers contribute to the risk of listeriosis in humans and animals are reviewed.

Draft Genome Sequence of a Salmonella enterica subsp. enterica Serotype Choleraesuis Strain Isolated from the Pulp of Muskmelons.

Salmonella enterica subsp. enterica serotype Choleraesuis is a foodborne pathogen with zoonotic potential. We report the draft genome sequence and a closed plasmid sequence from a plant-internalized S. Choleraesuis strain that was isolated from the pulp of a Spanish Galia melon purchased from a German supermarket in 2015.

Growth Potential of Listeria monocytogenes in Three Different Salmon Products.

Cold smoked salmon and sushi salmon have been implicated in outbreaks of listeriosis. We performed challenge tests and a durability study with Listeria monocytogenes on different salmon products to determine the growth potential of this important food-borne pathogen. Data from the challenge test showed a significant growth potential of L. monocytogenes on all of the tested salmon products, with faster growth in sushi salmon than in cold smoked salmon. In identical products that were naturally contaminated at low levels, the durability study did not confirm a high growth potential, possibly due to interactions with competing microflora. The injection of sodium lactate (NaL) at a high concentration (30%) into cold smoked salmon significantly reduced the growth potential of L. monocytogenes. In addition to good manufacturing practices, the injection of higher concentrations of NaL may therefore be a useful additional hurdle to prevent growth of L. monocytogenes to high numbers in the tested salmon products.

Growth potential of Listeria monocytogenes in twelve different types of RTE salads: Impact of food matrix, storage temperature and storage time.

Listeriosis is a food borne disease associated with high hospitalization and fatality rates; in 2014, EU member states reported 2194 cases with 98.9% hospitalization rates and 210 fatalities. Proper risk analysis and the development of effective food safety strategies critically depend on the knowledge of the growth characteristics of L. monocytogenes on the product in question. Ready-to-eat (RTE) salads present a challenge in this context due to the absence of a heat treatment step before consumption. This study provides challenge-test based data of the growth characteristics of L. monocytogenes on twelve RTE salads. The food matrix, storage time and storage temperature were factors with a significant impact on the growth of L. monocytogenes. While most tested salads permitted a significant increase of L. monocytogenes in at least one of the tested conditions, no growth was observed on celeriac, carrot and corn salad products. There was a considerable increase in growth at 8 °C compared to 5 °C. Our data indicate that the reduction of the storage temperature at retail level to 5 °C and product shelf life could help mitigate the risk of L. monocytogenes in RTE salads.

Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

Listeria monocytogenes can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of L. monocytogenes isolated from a meat processing facility in Switzerland by serotyping, multi locus sequence typing (MLST) typing and whole genome sequencing. We then analyzed their ability to form biofilms and their resistance to the disinfectants benzalkonium chloride (BC) and peracetic acid (PAA). The genotyping results of the strains showed that several clonal populations of L. monocytogenes belonging to CC9, CC204 and CC121 had persisted in this meat processing facility for at least four years. All of the strains showed biofilm forming capacity comparable to a known high biofilm forming strain. Known efflux pumps for BC were present in CC204, CC9 (brcABC) and CC121 (qacH) strains, while strains from other CC showed very low minimal inhibitory concentrations (MICs) for BC. For PAA, minimal bactericidal concentrations of 1.2⁻1.6% for 20 min and minimal inhibitory concentrations between 0.1 and 0.2% were observed. These values were close to or above the recommended concentration for use (0.5⁻1%), suggesting that PAA might be ineffective at controlling L. monocytogenes in this and potentially other meat processing facilities.

Identification of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate.

Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG (isopropyl-ß-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3 Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance.IMPORTANCE Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a "highest priority critically important antimicrobial for human medicine" (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

Global Transcriptional Response of Three Highly Acid-Tolerant Field Strains of Listeria monocytogenes to HCl Stress.

Tolerance to acid is of dual importance for the food-borne pathogen Listeria monocytogenes: acids are used as a preservative, and gastric acid is one of the first defenses within the host. There are considerable differences in the acid tolerance of strains. Here we present the transcriptomic response of acid-tolerant field strains of L. monocytogenes to HCl at pH 3.0. RNAseq revealed significant differential expression of genes involved in phosphotransferase systems, oxidative phosphorylation, cell morphology, motility, and biofilm formation. Genes in the acetoin biosynthesis pathway were upregulated, suggesting that L. monocytogenes shifts to metabolizing pyruvate to acetoin under organic acid stress. We also identified the formation of cell aggregates in microcolonies as a potential relief strategy. A motif search within the first 150 bp upstream of differentially expressed genes identified a novel potential regulatory sequence that may have a function in the regulation of virulence gene expression. Our data support a model where an excess of intracellular H+ ions is counteracted by pumping H+ out of the cytosol via cytochrome C under reduced activity of the ATP synthase. The observed morphological changes suggest that acid stress may cause cells to aggregate in biofilm microcolonies to create a more favorable microenvironment. Additionally, HCl stress in the host stomach may serve as (i) a signal to downregulate highly immunogenic flagella, and (ii) as an indicator for the imminent contact with host cells which triggers early stage virulence genes.

Surviving host - and food relevant stresses: phenotype of L. monocytogenes strains isolated from food and clinical sources.

The aim of this study was to compare the phenotype of 40 strains of L. monocytogenes under food and host relevant stress conditions. The strains were chosen to represent food and clinical isolates and to be equally distributed between the most relevant clonal complexes for clinical and food isolates (CC1 and CC6 vs CC121 and CC9), plus one group of eight strains of rare clonal complexes. Human-associated CC1 had a faster maximal growth rate than the other major complexes, and the lag time of CC1 and CC6 was significantly less affected by the addition of 4% NaCl to the medium. Food-associated CC9 strains were hypohemolytic compared to other clonal complexes, and all strains found to be resistant to increased concentrations of benzalkonium chloride belonged to CC121 and were positive for Tn6188 carrying the qacH gene. Lactic acid affected the survival of L. monocytogenes more than HCl, and there was a distinct, strain specific pattern of acid tolerant and sensitive strains. Strains from CC6 and human clinical isolates are less resilient under acid stress than those from other complexes and from food. One strain isolated from a human patient exhibited significant growth defects across all conditions.

Growth potential of Listeria monocytogenes in six different RTE fruit products: impact of food matrix, storage temperature and shelf life.

We tested the growth potential of Listeria monocytogenes on six RTE fruit products at low (4°C at the factory followed by 8°C retail/home storage) and abusive (4°C followed by 12°C) storage temperatures. Sliced coconut and fresh cut cantaloupe, as well as a fruit mix containing diced pineapple, cantaloupe, apples and grapes supported the growth of L. monocytogenes with a growth potential δ>0.5 log CFU/g over six days. Mangoes, a mix of diced kiwi, cantaloupe and pineapple as well as a mix of diced pineapple, mango, grapefruit, kiwi and pomegranate did not support a growth potential that exceeded 0.5 log CFU/g over six days. The growth potential of L. monocytogenes correlated significantly with the pH; no product with a pH below 4 showed a significant growth potential of L. monocytogenes. Time after inoculation was also a significant predictor of the growth potential, while the fruit type and storage temperature were not.

The Listeria monocytogenes Bile Stimulon under Acidic Conditions Is Characterized by Strain-Specific Patterns and the Upregulation of Motility, Cell Wall Modification Functions, and the PrfA Regulon.

Listeria monocytogenes uses a variety of transcriptional regulation strategies to adapt to the extra-host environment, the gastrointestinal tract, and the intracellular host environment. While the alternative sigma factor SigB has been proposed to be a key transcriptional regulator that facilitates L. monocytogenes adaptation to the gastrointestinal environment, the L. monocytogenes' transcriptional response to bile exposure is not well-understood. RNA-seq characterization of the bile stimulon was performed in two L. monocytogenes strains representing lineages I and II. Exposure to bile at pH 5.5 elicited a large transcriptomic response with ~16 and 23% of genes showing differential transcription in 10403S and H7858, respectively. The bile stimulon includes genes involved in motility and cell wall modification mechanisms, as well as genes in the PrfA regulon, which likely facilitate survival during the gastrointestinal stages of infection that follow bile exposure. The fact that bile exposure induced the PrfA regulon, but did not induce further upregulation of the SigB regulon (beyond that expected by exposure to pH 5.5), suggests a model where at the earlier stages of gastrointestinal infection (e.g., acid exposure in the stomach), SigB-dependent gene expression plays an important role. Subsequent exposure to bile induces the PrfA regulon, potentially priming L. monocytogenes for subsequent intracellular infection stages. Some members of the bile stimulon showed lineage- or strain-specific distribution when 27 Listeria genomes were analyzed. Even though sigB null mutants showed increased sensitivity to bile, the SigB regulon was not found to be upregulated in response to bile beyond levels expected by exposure to pH 5.5. Comparison of wildtype and corresponding ΔsigB strains newly identified 26 SigB-dependent genes, all with upstream putative SigB-dependent promoters.

Whole-Genome Shotgun Sequencing of Three Listeria monocytogenes Strains Isolated from a Ready-to-Eat Salad-Producing Facility in Switzerland.

Ready-to-eat (RTE) raw foods harbor the risk of transmitting Listeria monocytogenes from the environment to the consumer. We isolated three strains from a facility producing RTE salad. These strains were used to perform challenge tests on different RTE salad products. Here, we present the shotgun genome sequences of all three of these strains.

Whole-Genome Sequences of Six Listeria monocytogenes Strains Isolated from Food.

Here, we report the whole-genome sequences of six Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA.