Functionality of the bacteriocin sakacin G produced by Lactobacillus curvatus ACU-1 on cooked sausages under industrial conditions.

Biopreservation is an alternative to prevent the growth of pathogens and reduce microbial spoilage in food based on the use of microorganisms and/or their metabolic products. The objective of this study was to determine the optimal mode of application and the effectiveness of cell-free supernatant (CFS) from Lactobacillus curvatus ACU-1, containing sakacin G, in Vienna-type sausages to control Listeria and spoilage flora. The functionality and the optimal dosage form between CFS, producing bacteria, a combination or concentrate of bacteriocin applied on Vienna-type sausages before and after stuffing the casings on an industrial scale were determined. Sakacin G was effective for the control of Listeria applied to the casing both before and after stuffing. The application of the antimicrobial on the ready sausages inhibits both lactic acid bacteria and mesophilic microorganisms from zero sampling time. The heat resistance of the bacteriocin in the food was verified under industrial manufacturing conditions. The antimicrobial activity of sakacin G was maintained throughout the period studied in all the conditions tested. In conclusion, the application of CFS containing bacteriocin is useful given both before and after casing stuffing; but the application prior to the stuffing is more practical for the process of elaboration.

Assessment of Listeria monocytogenes Surface Proteins Identified from Proteomics Analysis for Use as Diagnostic Biomarkers.

The Gram-positive bacterium Listeria monocytogenes is an important pathogen that causes a foodborne illness with a high percentage of fatalities. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the detection and isolation of this pathogen using antibody-based methods. Among a number of surface proteins identified by mass spectrometry in a previous proteomic study, six candidates (annotated as LMOf2365_0148, LMOf2365_0312, LMOf2365_0546, LMOf2365_1883, LMOf2365_2111, and LMOf2365_2742) were selected here for investigating their expression in the bacterial cells cultured in vitro by raising rabbit polyclonal antibodies (PAbs) against the recombinant form of each candidate. These protein candidates contained regions conserved among various L. monocytogenes isolates but variable in other Listeria species. LMOf2365_0148, an uncharacterized protein with a LPXTG motif accountable for covalent linkage to the cell wall peptidoglycan, exhibited a strong reaction signal from anti-LMOf2365_0148 PAb binding to the cell surface, as detected by immunofluorescence microscopy. Further study, through the generation of a panel of mouse monoclonal antibodies (MAbs) to the recombinant LMOf2365_0148, showed that one of the MAbs, M3686, reacted to bacterial isolates belonging to all three lineages of L. monocytogenes under Health Canada's standard enrichment culture conditions (MFHPB-07 and MFHPB-30). These results demonstrated the potential of using LMOf2365_0148 as a surface biomarker, in conjunction with specific MAbs developed here, for the isolation and detection of L. monocytogenes from foods and food processing environments. IMPORTANCE Strains of Listeria monocytogenes are differentiated serologically into at least 13 serotypes and grouped phylogenetically into 4 distinct lineages (I, II, III, and IV). No single monoclonal antibody (MAb) reported to date is capable of binding to the surface of L. monocytogenes strains representing all the serotypes. This study assessed the expression of six surface proteins selected from a previous proteomic study and demonstrated that surface protein LMOf2365_0148 has the greatest potential as a surface biomarker. A panel of 24 MAbs to LMOf2365_0148 were assessed extensively, revealing that one of the MAbs, M3686, reacted to a wide range of L. monocytogenes isolates (lineage I, II, and III isolates) grown under standard enrichment culture conditions and thus led to the conclusion that LMOf2365_0148 is a useful novel surface biomarker for identifying, detecting, and isolating the pathogen from food and environmental samples.

Hemolysin function of Listeria is related to biofilm formation: transcriptomics analysis.

Listeriolysin O (LLO) is the main virulence protein of Listeria monocytogenes (LM), that helps LM escape lysosomes. We previously found that the cellular immune response elicited by L.ivanovii (LI) is weaker than that elicited by LM. We speculated that this may be related to the function of ivanolysin O (ILO). Here, we constructed hemolysin gene deletion strain, LIΔilo, and a modified strain, LIΔilo::hly, in which ilo was replaced by hly. Prokaryotic transcriptome sequencing was performed on LI, LIΔilo, and LIΔilo::hly. Transcriptome differences between the three strains were compared, and genes and pathways with significant differences between the three strains were analyzed. Prokaryotic transcriptome sequencing results revealed the relationship of ilo to the ribosome, quorum sensing, and phosphotransferase system (PTS) pathways, etc. LIΔilo exhibited attenuated biofilm formation ability compared to LI. Biofilm formation was significantly recovered or even increased after replenishing hly. After knocking out ilo, the relative expression levels of some virulence genes, including sigB, prfA, actA, smcL, and virR, were up-regulated compared to LI. After replenishing hly, these genes were down-regulated compared to LIΔilo. The trend and degree of such variation were not completely consistent when cultured in media containing only monosaccharides or disaccharides. The results confirmed that hemolysin is related to some important biological properties of Listeria, including biofilm formation and virulence gene expression levels. This is the first comprehensive study on ILO function at the transcriptomic level and the first evidence of a relationship between Listeria hemolysin and biofilm formation.

Glucose Decoration on Wall Teichoic Acid Is Required for Phage Adsorption and InlB-Mediated Virulence in Listeria ivanovii.

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall teichoic acid [WTA]) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene liv1070, which encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention on the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting that the trade-off between phage resistance and virulence attenuation may be a general feature in the genus Listeria. IMPORTANCE Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants and occasionally in immunocompromised humans. Recent investigations revealed that in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently results in disconnection of one of the bacterium's major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria, and the host may be a feature common to the genus Listeria.

Structure and function of Listeria teichoic acids and their implications.

Teichoic acids (TAs) are the most abundant glycopolymers in the cell wall of Listeria, an opportunistic Gram-positive pathogen that causes severe foodborne infections. Two different structural classes of Listeria TA exist: the polyribitolphosphate-based wall teichoic acid (WTA) that is covalently anchored to the peptidoglycan, and the polyglycerolphosphate-based lipoteichoic acid (LTA) that is tethered to the cytoplasmic membrane. While TA polymers govern many important physiological processes, the diverse glycosylation patterns of WTA result in a high degree of surface variation across the species and serovars of Listeria, which in turn bestows varying effects on fitness, biofilm formation, bacteriophage susceptibility and virulence. We review the advances made over the past two decades, and our current understanding of the relationship between TA structure and function. We describe the various types of TA that have been structurally determined to date, and discuss the genetic determinants known to be involved in TA glycosylation. We elaborate on surface proteins functionally related to TA decoration, as well as the molecular and analytical tools used to probe TAs. We anticipate that the growing knowledge of the Listeria surface chemistry will also be exploited to develop novel diagnostic and therapeutic strategies for this pathogen.

Dissemination and conservation of cadmium and arsenic resistance determinants in Listeria and other Gram-positive bacteria.

Metal homeostasis in bacteria is a complex and delicate balance. While some metals such as iron and copper are essential for cellular functions, others such as cadmium and arsenic are inherently cytotoxic. While bacteria regularly encounter essential metals, exposure to high levels of toxic metals such as cadmium and arsenic is only experienced in a handful of special habitats. Nonetheless, Listeria and other Gram-positive bacteria have evolved an impressively diverse array of genetic tools for acquiring enhanced tolerance to such metals. Here, we summarize this fascinating collection of resistance determinants in Listeria, with special focus on resistance to cadmium and arsenic, as well as to biocides and antibiotics. We also provide a comparative description of such resistance determinants and adaptations in other Gram-positive bacteria. The complex coselection of heavy metal resistance and other types of resistance seems to be universal across the Gram-positive bacteria, while the type of coselected traits reflects the lifestyle of the specific microbe. The roles of heavy metal resistance genes in environmental adaptation and virulence appear to vary by genus, highlighting the need for further functional studies to explain the mystery behind the array of heavy metal resistance determinants dispersed and maintained among Gram-positive bacteria.

Mining and Statistical Modeling of Natural and Variant Class IIa Bacteriocins Elucidate Activity and Selectivity Profiles across Species.

Class IIa bacteriocin antimicrobial peptides (AMPs) are a compelling alternative to current antimicrobials because of potential specific activity toward antibiotic-resistant bacteria, including vancomycin-resistant enterococci. Engineering of these molecules would be enhanced by a better understanding of AMP sequence-activity relationships to improve efficacy in vivo and limit effects of off-target activity. Toward this goal, we experimentally evaluated 210 natural and variant class IIa bacteriocins for antimicrobial activity against six strains of enterococci. Inhibitory activity was ridge regressed to AMP sequence to predict performance, achieving an area under the curve of 0.70 and demonstrating the potential of statistical models for identifying and designing AMPs. Active AMPs were individually produced and evaluated against eight enterococcus strains and four Listeria strains to elucidate trends in susceptibility. It was determined that the mannose phosphotransferase system (manPTS) sequence is informative of susceptibility to class IIa bacteriocins, yet other factors, such as membrane composition, also contribute strongly to susceptibility. A broadly potent bacteriocin variant (lactocin DT1) from a Lactobacillus ruminis genome was identified as the only variant with inhibitory activity toward all tested strains, while a novel enterocin variant (DT2) from an Enterococcus faecium genome demonstrated specificity toward Listeria strains. Eight AMPs were evaluated for proteolytic stability to trypsin, chymotrypsin, and pepsin, and three C-terminal disulfide-containing variants, including divercin V41, were identified as compelling for future in vivo studies, given their high potency and proteolytic stability.IMPORTANCE Class IIa bacteriocin antimicrobial peptides (AMPs), an alternative to traditional small-molecule antibiotics, are capable of selective activity toward various Gram-positive bacteria, limiting negative side effects associated with broad-spectrum activity. This selective activity is achieved through targeting of the mannose phosphotransferase system (manPTS) of a subset of Gram-positive bacteria, although factors affecting this mechanism are not entirely understood. Peptides identified from genomic data, as well as variants of previously characterized AMPs, can offer insight into how peptide sequence affects activity and selectivity. The experimental methods presented here identify promising potent and selective bacteriocins for further evaluation, highlight the potential of simple computational modeling for prediction of AMP performance, and demonstrate that factors beyond manPTS sequence affect bacterial susceptibility to class IIa bacteriocins.

Comparison of predicted and impedance determined growth of Listeria innocua in complex food matrices.

Indirect impedance has been used for the detection and enumeration of bacteria, however there is limited data regarding the ability of the method to measure growth and inhibition of microorganisms in food in response to preservatives. The aim of this study was to evaluate the suitability of the technique to determine maximum growth rates of Listeria innocua (used as a surrogate for Listeria monocytogenes) in complex food matrices to which multiple preservative factors had been applied and assess the suitability of the data for use in predictive microbiology. Growth of L. innocua in laboratory medium (BHI broth) and two food matrices (zucchini purée and béarnaise sauce) under varying conditions of pH (5 & 5.3), water activity (0.93, 0.96 & 0.98) and acetic and propionic acid concentration (0, 1 & 2 mM) was monitored by the conductimetric Rapid Automated Bacterial Impedance Technology (R.A.B.I.T) system by means of CO2 emission for up to 120 h. Growth rates of L. innocua were determined for several conditions across the three test matrices and a good correlation between detection times and initial inoculum level was observed in most cases (R2 ≥ 0.82). However, growth of L. innocua was not detected in a large number of conditions and comparison of growth rates determined by indirect impedance to those determined by plate counts indicated that in general, the R.A.B.I.T. system under-estimated growth. This study demonstrates that there are limitations associated with the technology, and as a result the system may be unsuitable for measuring microbial growth rates in complex food matrices under the environmental conditions tested and within the time duration of the study.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.

Structural and thermodynamic insights into ß-1,2-glucooligosaccharide capture by a solute-binding protein in Listeria innocua.

ß-1,2-Glucans are bacterial carbohydrates that exist in cyclic or linear forms and play an important role in infections and symbioses involving Gram-negative bacteria. Although several ß-1,2-glucan-associated enzymes have been characterized, little is known about how ß-1,2-glucan and its shorter oligosaccharides (Sop n s) are captured and imported into the bacterial cell. Here, we report the biochemical and structural characteristics of the Sop n -binding protein (SO-BP, Lin1841) associated with the ATP-binding cassette (ABC) transporter from the Gram-positive bacterium Listeria innocua Calorimetric analysis revealed that SO-BP specifically binds to Sop n s with a degree of polymerization of 3 or more, with Kd values in the micromolar range. The crystal structures of SO-BP in an unliganded open form and in closed complexes with tri-, tetra-, and pentaoligosaccharides (Sop3-5) were determined to a maximum resolution of 1.6 Å. The binding site displayed shape complementarity to Sop n , which adopted a zigzag conformation. We noted that water-mediated hydrogen bonds and stacking interactions play a pivotal role in the recognition of Sop3-5 by SO-BP, consistent with its binding thermodynamics. Computational free-energy calculations and a mutational analysis confirmed that interactions with the third glucose moiety of Sop n s are significantly responsible for ligand binding. A reduction in unfavorable changes in binding entropy that were in proportion to the lengths of the Sop n s was explained by conformational entropy changes. Phylogenetic and sequence analyses indicated that SO-BP ABC transporter homologs, glycoside hydrolases, and other related proteins are co-localized in the genomes of several bacteria. This study may improve our understanding of bacterial ß-1,2-glucan metabolism and promote the discovery of unidentified ß-1,2-glucan-associated proteins.

Coiled-coil interactions mediate serine integrase directionality.

Serine integrases are bacteriophage enzymes that carry out site-specific integration and excision of their viral genomes. The integration reaction is highly directional; recombination between the phage attachment site attP and the host attachment site attB to form the hybrid sites attL and attR is essentially irreversible. In a recent model, extended coiled-coil (CC) domains in the integrase subunits are proposed to interact in a way that favors the attPxattB reaction but inhibits the attLxattR reaction. Here, we show for the Listeria innocua integrase (LI Int) system that the CC domain promotes self-interaction in isolated Int and when Int is bound to attachment sites. Three independent crystal structures of the CC domain reveal the molecular nature of the CC dimer interface. Alanine substitutions of key residues in the interface support the functional significance of the structural model and indicate that the same interaction is responsible for promoting integration and for inhibiting excision. An updated model of a LI Int•attL complex that incorporates the high resolution CC dimer structure provides insights that help to explain the unusual CC dimer structure and potential sources of stability in Int•attL and Int•attR complexes. Together, the data provide a molecular basis for understanding serine integrase directionality.

Increase in bacterial biosurfactant production by co-cultivation with biofilm-forming bacteria.

Considering that bacterial biosurfactants (BSFs) are released as secondary metabolites involved in biotic relations within mixed bacterial assemblages, the hypothesis that the co-cultivation of BSF producing bacteria with biofilm-forming strains would enhance BSF synthesis was tested. Environmental BSF producing strains of Bacillus licheniformis and Pseudomonas sp. were cultivated with reference biofilm-forming strains (Pseudomonas aeruginosa and Listeria innocua). BSF production and quorum-quenching effects were tested in solid media. Tensioactive and anionic BSFs were also quantified in cell-free extracts (CFEs). BSF production increased in co-cultures with inducer strains although this was not demonstrated by all screening methods. Increased concentrations of anionic BSF were detected in CFEs of co-cultures in which Pseudomonas aeruginosa was included as inducer, which is in accordance with the observation of larger halos in cetyl trimethylammonium bromide-methylene blue agar. The results demonstrate that co-cultivation positively affects the efficiency of BSF production and that higher production yields may be attained by selecting convenient inducer partners in designed consortia. SIGNIFICANCE AND IMPACT OF THE STUDY: The high production cost of biosurfactants (BSFs) still represents a major limitation to the industrial use of these otherwise advantageous alternatives to chemical surfactants. This work demonstrates that the co-cultivation of consortia of biosurfactant-producer and biofilm-forming bacteria enhances BSF production and may contribute to the cost-effectiveness of biosurfactant-based products.

Listeria innocua Dps as a nanoplatform for bioluminescence based photodynamic therapy utilizing Gaussia princeps luciferase and zinc protoporphyrin IX.

Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490 nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent.

Bacteriocin production by Lactobacillus plantarum ST16Pa in supplemented whey powder formulations.

Whey, the main by-product of the dairy industry, is frequently disposed of in the environment without any treatment due to the high cost of this process. Alternatively, whey can be used as a medium to culture lactic acid bacteria and produce value-added products such as bacteriocins. In this work, we attempted to improve bacteriocin production by Lactobacillus plantarum ST16Pa in a whey powder formulation supplemented with additional sources of carbon, nitrogen, and vitamin B12 at different levels and varying the agitation intensity according to a Plackett-Burman experimental design. Only the addition of tryptone positively influenced the production of this bacteriocin. The results allowed us to identify a supplemented whey formulation, comprising 150 g/L of whey total solids plus 10 g/L of tryptone and soybean extract, whose fermentation by Lb. plantarum ST16Pa in shake flasks under agitation at 150 rpm led to a cell-free supernatant with an antimicrobial activity against Listeria innocua 6a CLIST 2865 (inhibition zone of 13.23 mm) close to that previously obtained in de Man, Rogosa and Sharpe medium by other authors. These results are significant considering that the same strain cultured in cheese whey did not previously display any antimicrobial activity.

Structural and functional diversity in Listeria cell wall teichoic acids.

Wall teichoic acids (WTAs) are the most abundant glycopolymers found on the cell wall of many Gram-positive bacteria, whose diverse surface structures play key roles in multiple biological processes. Despite recent technological advances in glycan analysis, structural elucidation of WTAs remains challenging due to their complex nature. Here, we employed a combination of ultra-performance liquid chromatography-coupled electrospray ionization tandem-MS/MS and NMR to determine the structural complexity of WTAs from Listeria species. We unveiled more than 10 different types of WTA polymers that vary in their linkage and repeating units. Disparity in GlcNAc to ribitol connectivity, as well as variable O-acetylation and glycosylation of GlcNAc contribute to the structural diversity of WTAs. Notably, SPR analysis indicated that constitution of WTA determines the recognition by bacteriophage endolysins. Collectively, these findings provide detailed insight into Listeria cell wall-associated carbohydrates, and will guide further studies on the structure-function relationship of WTAs.

Metabolic profiling of listeria rhombencephalitis in small ruminants by 1 H high-resolution magic angle spinning NMR spectroscopy.

Listeria rhombencephalitis is caused by infection with Listeria monocytogenes and is associated with a high mortality rate in humans and ruminants. Little is known about the metabolic changes associated with neurolisteriosis in particular and infectious central nervous system (CNS) diseases in general. The purpose of our study was to investigate the metabolic changes associated with listeria rhombencephalitis in small ruminants (goats and sheep) as a model for inflammatory CNS disease by 1 H high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) spectroscopy of brain biopsies obtained from the brainstem and thalamus. Statistical analysis revealed distinct differences in the metabolic profile of brainstem biopsies, the primary location of listeria rhombencephalitis with moderate or severe inflammatory changes. N-Acetylaspartate (NAA), N-acetylaspartylglutamate, choline, myo-inositol and scyllo-inositol were decreased, and glycine, phosphocholine, taurine and lactate were increased, in the diseased group (n = 13) in comparison with the control group (n = 12). In the thalamus, which showed no or only mild inflammatory changes in the majority of animals, no statistically significant metabolic changes were observed. However, trends for metabolic alterations were partly the same as those found in the brainstem, including NAA, choline and lactate. This may be an indicator of metabolic changes occurring in the early stages of the disease. Therefore, further research with a larger number of animals is needed to evaluate the presence of subtle metabolic changes associated with mild inflammatory changes in the thalamus. In conclusion, 1 H HR-MAS NMR investigation of listeria rhombencephalitis identified brain metabolite changes, offering new insights into the disease pathophysiology.

Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly.

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.

Isotopologue profiling of the listerial N-metabolism.

The nitrogen (N-) sources and the relative contribution of a nitrogenous nutrient to the N-pool of the gram-positive pathogen Listeria monocytogenes are largely unknown. Therefore, (15) N-isotopologue profiling was established to study the N-metabolism of L. monocytogenes. The pathogen was grown in a defined minimal medium supplemented with potential (15) N-labeled nutrients. The bacteria were harvested and hydrolysed under acidic conditions, and the resulting amino acids were analysed by GC-MS, revealing (15) N-enrichments and isotopomeric compositions of amino acids. The differential (15) N-profiles showed the substantial and simultaneous usage of ammonium, glutamine, methionine, and, to a lower extent, the branched-chain amino acids valine, leucine, and isoleucine for anabolic purposes, with a significant preference for ammonium. In contrast, arginine, histidine and cysteine were directly incorporated into proteins. L. monocytogenes is able to replace glutamine with ethanolamine or glucosamine as amino donors for feeding the core N-metabolism. Perturbations of N-fluxes caused by gene deletions demonstrate the involvement of ethanolamine ammonia lyase, and suggest a role of the regulator GlnK of L. monocytogenes distinct from that of Escherichia coli. The metabolism of nitrogenous nutrients reflects the high flexibility of this pathogenic bacterium in exploiting N-sources that could also be relevant for its proliferation during infection.

Genetic Stability and Evolution of the sigB Allele, Used for Listeria Sensu Stricto Subtyping and Phylogenetic Inference.

Sequencing of single genes remains an important tool that allows the rapid classification of bacteria. Sequencing of a portion of sigB, which encodes a stress-responsive alternative sigma factor, has emerged as a commonly used molecular tool for the initial characterization of diverse Listeria isolates. In this study, evolutionary approaches were used to assess the validity of sigB allelic typing for Listeria For a data set of 4,280 isolates, sigB allelic typing showed a Simpson's index of diversity of 0.96. Analyses of 164 sigB allelic types (ATs) found among the 6 Listeriasensu stricto species, representing these 4,280 isolates, indicate that neither frequent homologous recombination nor positive selection significantly contributed to the evolution of sigB, confirming its genetic stability. The molecular clock test provided evidence for unequal evolution rates across clades; Listeria welshimeri displayed the lowest sigB diversity and was the only species in which sigB evolved in a clocklike manner, implying a unique natural history. Among the four L. monocytogenes lineages, sigB evolution followed a molecular clock only in lineage IV. Moreover, sigB displayed a significant negative Tajima D value in lineage II, suggesting a recent population bottleneck followed by lineage expansion. The absence of positive selection along with the violation of the molecular clock suggested a nearly neutral mechanism of Listeriasensu strictosigB evolution. While comparison with a whole-genome sequence-based phylogeny revealed that the sigB phylogeny did not correctly reflect the ancestry of L. monocytogenes lineage IV, the availability of a large sigB AT database allowed accurate species classification.IMPORTANCEsigB allelic typing has been widely used for species delineation and subtyping of Listeria However, an informative evaluation of this method from an evolutionary perspective was missing. Our data indicate that the genetic stability of sigB is affected by neither frequent homologous recombination nor positive selection, which supports that sigB allelic typing provides reliable subtyping and classification of Listeria sensu stricto strains. However, multigene data are required for accurate phylogeny reconstruction of Listeria This study thus contributes to a better understanding of the evolution of sigB and confirms the robustness of the sigB subtyping system for Listeria.

Biomarkers of oxidative damage in bacteria for the assessment of sanitation efficacy in lettuce wash water.

In the fresh produce industry, validation of sanitation efficacy is critical to prevent cross-contamination of produce. The current validation approaches are either based on time-consuming plate counting assays or indirect measurements of chemical properties of wash water. In the study, the focus was to identify biomarkers that can provide direct assessment of oxidative damage in bacteria upon exposure to sanitizers in the presence of fresh produce and correlation of these oxidative biomarkers with logarithmic inactivation of bacteria. Two endogenous bacterial biomarkers, protein carbonylation and thiol oxidation, were evaluated for assessing oxidative damage in Escherichia coli O157:H7 and Listeria innocua during sanitation of pre-cut lettuce leaves with NaOCl or H2O2. Results show that NaOCl treatment was more effective than H2O2 for oxidation of both the intracellular thiols and protein carbonylation in the selected strains. Statistical analysis of the measurements illustrates that oxidation of the intracellular thiol induced by NaOCl or H2O2 was correlated with logarithmic reduction of E. coli O157:H7 and L. innocua. In contrast, changes in the protein carbonylation content were not correlated with reduction in bacterial cell viability. In summary, these results provide a novel approach to validate sanitation efficacy for the fresh produce industry.