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A total of 1797 imported food samples collected during 2018 to 2020 were investigated for Listeria monocytogenes. Antibiotic susceptibility tests and whole genome sequencing analysis were performed for the obtained isolates. The overall prevalence of L. monocytogenes was 5.62 %; the highest prevalence was observed for pork (13.65 %), followed by fish (6.25 %), sheep casing (6.06 %), chicken (3.61 %), and beef (2.06 %). Geographical differences in prevalence were also observed for pork. Resistance to oxacillin (39.33 %) and clindamycin (16.85 %) was common, whereas resistance rates for other antibiotics were relatively low, ranging from 0 % to 6.74 %. Pork and fish isolates showed resistance to more antibiotics than beef isolates. Tetracycline and chloramphenicol resistance phenotypes strongly correlated with genotypes. The predominant serogroup was 1/2a, 3a, at 44.44 %, while the percentages of three other serogroups were similar and relatively lower, from 17.28 % to 19.75 %. Significant genetic differences were observed among lineage I and II isolates. LIPI-3 was carried by 19.75 % (16/81) of isolates and LIPI-4 by 6.17 % (5/81); all were lineage I. The stress survival island was present in 31.03 % (9/29) of lineage I and 83.02 % (44/53) of lineage II. Benzalkonium chloride tolerance genes were carried by 10.34 % (3/29) of lineage I and 23.08 % (12/52) of lineage II isolates. A total of 25 sequence types (STs) were identified, among which one was novel; ST9 and ST121 were the most prevalent. Disparate distribution of STs among food types was observed, and geographical and food related characteristics were also found for some STs. Hypervirulent STs, such as ST1, ST4 and ST6, belonged to 4b,4e,4e; carried LIPI-3 and/or LIPI-4; and some even were ECI or ECII; while only one carried SSI or BC tolerance genes. In contrast, hypo-virulent STs such as ST9 and ST121 carried SSI and BC tolerance genes, while none had LIPI-3/LIPI-4. Certain STs were detected frequently from a particular food of a particular country for a long time, indicating more attention should be given to these special persistent isolates. These findings are valuable for source tracking, prevention and control of L. monocytogenes in the global food chain. • Prevalence and antibiotic resistance of L. monocytogenes in food samples differed by geographical region and food type. • Virulence and stress tolerance gene differed among lineages and sequence types of L. monocytogenes. • Distinct traits were observed for L. monocytogenes isolates from a specific food/country. • Hypervirulent strains of L. monocytogenes were found. [ABSTRACT FROM AUTHOR]

The bacterial heat shock response in foodborne pathogens is caused by exposure to higher temperatures which poses a great threat to food safety because it can undermine food processing interventions and host defense. The study assessed the heat and acid resistance of Cronobacter sakazakii following heat shock (53 °C for 15min). Inactivation curves of the heat-shocked and non-shocked C. sakazakii cells at four temperatures (56, 58, 60, and 62 °C) and simulated gastric fluid (SGF, pH 3.0) were examined and fitted with the log-linear model and the Weibull model. The inactivation parameters obtained on the basis of the Weibull model showed that heat shock significantly (p < 0.05) increased the values of δ (time to reach 1 log reduction) and t 3d (time to reach 3 log reduction) under thermal and acid inactivation. The results proved that heat shock provided C. sakazakii direct protection from a more adverse heat challenge and cross-protection from SGF, i.e. there was a heat shock response. Results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that seven protein species showed enhanced expression, while four protein species showed decreased expression in the heat-shocked compared to the non-heat-shocked C. sakazakii cells. Quantitative real-time reverse transcriptase PCR (RT-qPCR) revealed upregulation of six stress related genes, ibpA , ibpB (both encoding molecular chaperons), Hsp15 (encoding heat shock protein), Hsp20 (encoding small heat-shock protein), HspQ (encoding proteases) and rpoS (encoding stationary phase sigma factor), following heat shock treatment. In addition, heat shock induced an increase proportion of saturated fatty acids (SFA), cyclic fatty acids (CFA) and the ratio of saturated fatty acids to unsaturated fatty acids (SFA/USFA), whereas reducing the proportion of unsaturated fatty acids (USFA). Consequently, establishment of inactivation models of C. sakazakii could provide data support for quantitative microbial risk assessment (QMRA). Exploration of enhanced resistance mechanisms might provide clues for prevention and control of contamination by heat-shocked C. sakazakii. • Survival curves of heat-shocked and non-shocked C. sakazakii cells could be described with the Weibull model. • Heat-shock treatment greatly increased thermal resistance of C. sakazakii. • Heat-shock cross-protected C. sakazakii against simulated gastric fluid (pH 3.0). • Heat-shock altered the protein profile, stress-related gene expression and fatty acids composition of C. sakazakii. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is recognized as a significant foodborne pathogen, capable of causing listeriosis in humans, which is a global public health concern. This pathogen is particularly dangerous for pregnant women, as it can lead to invasive listeriosis in fetuses and neonates, posing a significant threat to both maternal and fetal health. Therefore, establishing suitable in vitro and in vivo models for L . monocytogenes placenta infection, as well as analyzing and exploring the infection process and its pathogenic mechanism, are important approaches to prevent and control L . monocytogenes infection in mothers and infants. In this study, we reviewed the in vitro and in vivo placental models used for studying the infection of L . monocytogenes in maternal and infant, summarized and discussed the advantages and limitations of each model, and explored the potential of in vitro cell models and organoids for the study of L . monocytogenes infection. This paper aims to support the study of the infection pathway and pathogenesis of listeriosis and provide scientific references for the prevention and control of L . monocytogenes infection.

Aims: Disinfectants such as benzalkonium chloride (BC), extensively used in animal farms and food-processing industries, contribute to the development of adaptive and cross-resistance in foodborne pathogens, posing a serious threat to food safety and human health. The purpose of this study is to explore whether continuous exposure of Salmonella enterica serovar 1,4,[5],12:i:- (S. 1,4,[5],12:i:-) to sublethal concentrations of BC could result in acquired resistance to this agent and other environmental stresses (e.g. antibiotics, heat, and acid)., Methods and Results: BC tolerance increased in all tested strains after exposure to gradually increasing concentrations of BC, with increases in minimum inhibitory concentrations between two and sixfold. The survival rate of BC-adapted strains was significantly (P < 0.05) higher than that of their wild-type (non-adapted) counterparts in lethal concentrations of BC. In addition, significant reductions (P < 0.05) in zeta potential were observed in BC-adapted strains compared to wild-type ones, indicating that a reduction in cell surface charge was a cause of adaptative resistance. More importantly, two BC-adapted strains exhibited increased antibiotic resistance to levofloxacin, ceftazidime, and tigecycline, while gene mutations (gyrA, parC) and antibiotic efflux-related genes (acrB, mdsA, mdsB) were detected by genomic sequencing analysis. Moreover, the tolerance of BC-adapted strains to heat (50, 55, and 60°C) and acid (pH 2.0, 2.5) was strain-dependent and condition-dependent., Conclusions: Repeated exposure to sublethal concentrations of BC could result in the emergence of BC- and antibiotic-resistant S. 1,4,[5],12:i:- strains., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Listeria monocytogenes (LM) is a food-borne pathogen that can cause listeriosis. Pregnant women are main target population of listeriosis due to pregnancy-associated immune deficiency and unique intracellular infection ability of LM to non-phagocytic cells. LM can cross the placental barrier and cause significant harm to the fetus, including premature birth, miscarriage and even stillbirth. The role of placenta-specific virulence factors is particularly important for researchers to understand how it crosses the placental barrier and infects the fetus during LM infection. This review started by describing the listeriosis in pregnant women, followed by summarizing the advances in understanding the LM vertical transplacental infection and the mechanism of LM colonization in the placenta. Finally, recent advances in identifying placenta-specific virulence factors involved in LM infections were presented, with the aim to facilitate the control of LM transplacental infection and the improvement of food safety.

Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein gene system can limit the horizontal gene transfer, thereby effectively preventing the invasion of foreign gene elements such as bacteriophages. CRISPR arrays of different bacteria are diverse. Based on the differences in the CRISPR system, this review summarizes the application of CRISPR in food-borne pathogen evolution analysis, detection and typing, virulence and antibiotic resistance in recent years. We also address bacterial detection typing method developed based on the characteristics of CRISPR arrays and the association of CRISPR with virulence and drug resistance of food-borne pathogens. The shortcomings of CRISPR in evolution, detection and typing, virulence and resistance applications are analyzed. In addition, we suggest standardizing CRISPR typing methods, improving and expanding the CRISPR database of pathogenic bacteria, and further exploring the co-evolution relationship between phages and bacteria, to provide references for further exploration of CRISPR functions.

Strain variability is one of the most important factors to influence the accuracy of foodborne pathogens risk assessment, such as Listeria monocytogenes, Salmonella spp. Strain-to-strain variation is defined as the inherent differences among identically treated strains of the same microbial species. The differences cannot be eliminated by changing test methods or improving test protocols. This review addresses presently related studies of strain variability. Based on the effect of strain variability on the outcome of risk assessment, we summarize sources of variabilities in food chain, strain phenotypic variabilities and the methods to integrate strain variability in growth and inactivation into predictive modelling, and indicate the inadequacies in the study of strain variability. We suggest further study the mechanism of strain variability, expand the comparison of variability among different sources, and integrate the variability of gene expression, protein and cell metabolism into the predictive modelling.

Quorum sensing (QS) plays a major role in the outbreak mechanism of foodborne diseases caused by food poisoning and food spoilage. QS affects the formation of cell membrane and pathogenicity ofpathogenic bacteria. Through the in-depth understanding of QS molecules of food-borne pathogens, we describe here the types of signal molecules produced by Gram-negative and Gram-positive bacteria, and the differences in QS molecules. Meanwhile, we introduce the detection of QS molecules by different technologies. According to the influence of QS on food, we propose also future research needs for the control of foodborne pathogenic bacteria.

Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs). This work presents an innovative screening method, known as a bacterial macroarray, generated by contact printing of hybridoma cell supernatant samples on a nitrocellulose (NC) membrane initially coated with fluorescein isothiocyanate (FITC)-labeled bacteria. Given that bacterial fixation will be influenced by complex bacterial surface structures, we selected both gram-positive bacteria ( Staphylococcus aureus and Listeria monocytogenes) and gram-negative bacteria ( Escherichia coli O157:H7 and Cronobacter sakazakii) to optimize the fixation conditions for binding to the NC membrane, such as the aperture of the NC membrane, the concentration of bacteria, the dosage of glycerin in the spotting buffer, and the fixation time and temperature. As a result, we found that a better bacterial macroarray could be developed when the spotting buffer, containing 10 11 CFU mL -1 of FITC-labeled bacteria and 15% (V/V) glycerol, was spotted onto a 0.45 µm NC membrane with an incubation of 2 h at 37 °C. Finally, we verified the stability and specificity of the prepared bacterial macroarray by detecting cell cultures with the addition of two MAbs ( Escherichia coli O157:H7 MAb E7 and Cronobacter sakazakii MAb 1E9) to simulate the screening experiments. Here, we describe a bacterial macroarray to efficiently screen the targeted antibody-secreted hybridomas.

Although the level of reactive oxygen species (ROS) is altered upon the formation of bacterial biofilm, the relationship between ROS alteration and biofilm formation is still unclear. The aim of the present study is to use Listeria monocytogenes (L. monocytogenes) as a model organism to examine whether ROS have an effect on the biofilm formation. After eliminating ROS by treatment with NAD(P)H oxidase inhibitor Diphenyleneiodonium chloride (DPI) or scavenging reagents N-acetylcysteine (NAC), the biofilm formation of L. monocytogenes was examined. Our data demonstrate that DPI and NAC induced-reduction of ROS enhances the biofilm formation in L. monocytogenes without affecting bacterial growth and activity. These data provide the evidence that ROS produced by L. monocytogenes inhibit the biofilm formation., (Copyright © 2018. Published by Elsevier Ltd.)

Despite the results from zebrafish challenged model have demonstrated that Listeria monocytogenes (Lm) has strong adjuvant effects when this attenuated pathogenic bacteria is viewed as aquaculture vaccine vector, the underlying mechanism is not clear and extensive investigations are required. To further explore the potential of Lm in the field of aquaculture vaccine, zebrafish embryonic fibroblast cell line (ZF4) was used to evaluate the invasion ability of Lm. The data from cellular level showed that Lm had the lower invasion tendentiousness in ZF4 cells while bacterial invasion capacity was compared between zebrafish embryos cell line and human intestinal epithelial cell line. In ZF4 cells, there is no significant difference in bacterial invasion capacity between wild strain EGD-e and double-deleted strain ΔactA/inlB, which suggested that this attenuated effect was not showed in zebrafish cells. In addition, translation analysis indicated that the expressions of CD4 and CD8a in ZF4 cells increased after 2-h infection of the two Lm strains. These results further demonstrated that Lm presented multiple advantages including lower pathogenicity and antigen presentation when attenuated stain was viewed as aquaculture vaccine vector., (Copyright © 2018 Elsevier Ltd. All rights reserved.)