Differential mechanism between Listeria monocytogenes strains with different virulence contaminating ready-to-eat sausages during the simulated gastrointestinal tract.

Listeria monocytogenes exhibits varying levels of pathogenicity when entering the host through contaminated food. However, little is known regarding the stress response and environmental tolerance mechanism of different virulence strains to host gastrointestinal (GI) stimuli. This study analyzed the differences in the survival and genes of stress responses among two strains of L. monocytogenes 10403S (serotype 1/2a, highly virulent strain) and M7 (serotype 4a, low-virulence strain) during simulated gastrointestinal digestion. The results indicated that L. monocytogenes 10403S showed greater acid and bile salt tolerance than L. monocytogenes M7, with higher survival rates and less cell deformation and cell membrane permeability during the in vitro digestion. KEGG analysis of the transcriptomes indicated that L. monocytogenes 10403S displayed significant activity in amino acid metabolism, such as glutamate and arginine, associated with acid tolerance. Additionally, L. monocytogenes 10403S demonstrated a higher efficacy in promoting activities that preserve bacterial cell membrane integrity and facilitate flagellar protein synthesis. These findings will contribute valuable practical insights into the tolerance distinctions among different virulence strains of L. monocytogenes in the GI environment.

Outbreak of Listeria monocytogenes in hospital linked to a fava bean product, Finland, 2015 to 2019.

Listeria monocytogenes (Lm) is a bacterium widely distributed in the environment. Listeriosis is a severe disease associated with high hospitalisation and mortality rates. In April 2019, listeriosis was diagnosed in two hospital patients in Finland. We conducted a descriptive study to identify the source of the infection and defined a case as a person with a laboratory-confirmed Lm serogroup IIa sequence type (ST) 37. Six cases with Lm ST 37 were notified to the Finnish Infectious Diseases Registry between 2015 and 2019. Patient interviews and hospital menus were used to target traceback investigation of the implicated foods. In 2021 and 2022, similar Lm ST 37 was detected from samples of a ready-to-eat plant-based food product including fava beans. Inspections by the manufacturer and the local food control authority indicated that the food products were contaminated with Lm after pasteurisation. Our investigation highlights the importance that companies producing plant-based food are subject to similar controls as those producing food of animal origin. Hospital menus can be a useful source of information that is not dependent on patient recall.

Rapid detection of the source of a Listeria monocytogenes outbreak in Switzerland through routine interviewing of patients and whole-genome sequencing.

AIMS OF THE STUDY: Listeriosis is a notifiable disease in Switzerland. In summer 2022, the Swiss Federal Office of Public Health noticed an increase in reports of listeriosis cases, indicating a possible ongoing outbreak. Here we present the approaches applied for rapidly confirming the outbreak, detecting the underlying source of infection and the measures put in place to eliminate it and contain the outbreak. METHODS: For close surveillance and early detection of outbreak situations with their possible sources, listeriosis patients in Switzerland are systematically interviewed about risk behaviours and foods consumed prior to the infection. Listeria monocytogenes isolates derived from patients in medical laboratories are sent to the National Reference Laboratory for Enteropathogenic Bacteria and Listeria, where they routinely undergo whole-genome sequencing. Interview and whole-genome sequencing data are continuously linked for comparison and analysis. RESULTS: In summer 2022, 20 patient-derived L. monocytogenes serotype 4b sequence type 388 strains were found to belong to an outbreak cluster (≤10 different alleles between neighbouring isolates) based on core genome multilocus sequence typing analysis. Geographically, 18 of 20 outbreak cases occurred in northeastern Switzerland. The median age of patients was 77.4 years (range: 58.1-89.7), with both sexes equally affected. Rolling analysis of the interview data revealed smoked trout from a local producer as a suspected infection source, triggering an on-site investigation of the production facility and sampling of the suspected products by the responsible cantonal food inspection team on 15 July 2022. Seven of ten samples tested positive for L. monocytogenes and the respective cantonal authority ordered a ban on production and distribution as well as a product recall. The Federal Food Safety and Veterinary Office released a nationwide public alert covering the smoked fish products concerned. Whole-genome sequencing analysis confirmed the interrelatedness of the L. monocytogenes smoked trout product isolates and the patient-derived isolates. Following the ban on production and distribution and the product recall, reporting of new outbreak-related cases rapidly dropped to zero. CONCLUSIONS: This listeriosis outbreak could be contained within a relatively short time thanks to identification of the source of contamination through the established combined approach of timely interviewing of every listeriosis patient or a representative and continuous molecular analysis of the patient- and food-derived L. monocytogenes isolates. These findings highlight the effectiveness of this well-established, joint approach involving the federal and cantonal authorities and the research institutions mandated to contain listeriosis outbreaks in Switzerland.

Selection of starter lactic acid bacteria capable of forming biofilms on wooden vat prototypes for their future application in traditional Sicilian goat's milk cheese making.

In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.

Growth, biofilm formation, and motility of Listeria monocytogenes strains isolated from food and clinical samples located in Shanghai (China).

Listeria monocytogenes is a common foodborne pathogen that frequently causes global outbreaks. In this study, the growth characteristics, biofilm formation ability, motility ability and whole genome of 26 L. monocytogenes strains isolated from food and clinical samples in Shanghai (China) from 2020 to 2022 were analyzed. There are significant differences among isolates in terms of growth, biofilm formation, motility, and gene expression. Compared with other sequence type (ST) types, ST1930 type exhibited a significantly higher maximum growth rate, the ST8 type demonstrated a stronger biofilm formation ability, and the ST121 type displayed greater motility ability. Furthermore, ST121 exhibited significantly high mRNA expression levels compared with other ST types in virulence genes mpl, fbpA and fbpB, the quorum sensing gene luxS, starvation response regulation gene relA, and biofilm adhesion related gene bapL. Whole-genome sequencing (WGS) analyses indicated the isolates of lineage I were mostly derived from clinical, and the isolates of lineage II were mostly derived from food. The motility ability, along with the expression of genes associated with motility (motA and motB), exhibited a significantly higher level in lineage II compared with lineage I. The isolates from food exhibited significantly higher motility ability compared with isolates from clinical. By integrating growth, biofilm formation, motility phenotype with molecular and genotyping information, it is possible to enhance comprehension of the association between genes associated with these characteristics in L. monocytogenes.

Genomic Approach of Listeria monocytogenes Strains Isolated from Deli-Meats in Mexico.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis worldwide. In México, L. monocytogenes has been identified as a hazard of deli-meats. However, the genomic analysis that supports the transmission of L. monocytogenes strains via deli-meats and its role as a source for virulence and resistance genes is lacking. Here, we present four high-quality genome drafts of L. monocytogenes strains isolated from deli-meats in Mexico. In silico typing was used to determine the serotype, lineage, clonal complexes (CC), and multilocus sequence (ST). Also, comparative genomics were performed to explore the diversity, virulence, mobile elements, antimicrobial resistant and stress survival traits. The genome sequence size of these strains measured 3.05 ± 0.07 Mb with a mean value of 37.9%G+C. All strains belonged to linage I, which was divided into two groups: 4b, CC2, ST1 (n = 3) and 1/2b, CC5, ST5 (n = 1). The pangenome and core genome contained 3493 and 2625 genes, respectively. The strains harbor the L. monocytogenes pathogenicity island-1 (LIPI-1) and the same multidrug resistance pattern (fosX, norB, mprF, lin) via in silico analysis. Comparative analysis delineated the genomes as essentially syntenic, whose genomic differences were due to phage insertion. These results expand what is known about the biology of the L. monocytogenes strains isolated from deli-meats in Mexico and warns of the risk that these strains belong to epidemic linage and harbor virulence genes linked to human disease.

Attenuation of biofilm and virulence factors of Pseudomonas aeruginosa by tetramethylpyrazine-gold nanoparticles.

Pseudomonas aeruginosa is often identified as the causative agent in nosocomial infections. Their adapted resistance makes them strong towards antimicrobial treatments. They protect and empower their survival behind strong biofilm architecture that works as their armor toward antimicrobial therapy. Additionally, P. aeruginosa generates virulence factors, contributing to chronic infection and recalcitrant phenotypic characteristics. The current study utilizes the benevolence of nanotechnology to develop an alternate technique to control the spreading of P. aeruginosa by limiting its biofilm and virulence development. This study used a natural compound, tetramethylpyrazine, to generate gold nanoparticles. Tetramethylpyrazine-gold nanoparticles (Tet-AuNPs) were presented in spherical shapes, with an average size of 168 ± 52.49 nm and a zeta potential of -12.22 ± 2.06 mV. The minimum inhibition concentration (MIC) of Tet-AuNPs that proved more than 90 % effective in inhibiting P. aeruginosa was 256 µg/mL. Additionally, it also shows antibacterial activities against Staphylococcus aureus (MIC, 256 µg/mL), Streptococcus mutans (MIC, 128 µg/mL), Klebsiella pneumoniae (MIC, 128 µg/mL), Listeria monocytogenes (MIC, 256 µg/mL), and Escherichia coli (MIC, 256 µg/mL). The sub-MIC values of Tet-AuNPs significantly inhibited the early-stage biofilm formation of P. aeruginosa. Moreover, this concentration strongly affected hemolysis, protease activity, and different forms of motilities in P. aeruginosa. Additionally, Tet-AuNPs destroyed the well-established mature biofilm of P. aeruginosa. The expression of genes linked with the biofilm formation and virulence in P. aeruginosa treated with sub-MIC doses of Tet-AuNPs was shown to be significantly suppressed. Gene expression studies support biofilm- and virulence-suppressing effects of Tet-AuNPs at the phenotypic level.

Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Revisiting old friends: updates on the role of two-component signaling systems in Listeria monocytogenes survival and pathogenesis.

Listeria monocytogenes is well recognized for both its broad resistance to stress conditions and its ability to transition from a soil bacterium to an intracellular pathogen of mammalian hosts. The bacterium's impressive ability to adapt to changing environments and conditions requires the rapid sensing of environmental cues and the coordinated response of gene products that enable bacterial growth and survival. Two-component signaling systems (TCSs) have been long recognized for their ability to detect environmental stimuli and transmit those signals into transcriptional responses; however, often the precise nature of the stimulus triggering TCS responses can be challenging to define. L. monocytogenes has up to 16 TCSs that have been recognized based on homology and included in this list are several whose functions remain poorly described. This review highlights the current understanding of the breadth and scope of L. monocytogenes TCS as relates to stress resistance and pathogenesis. Precise signals still often remain elusive, but the gene networks associated with TCSs are providing clues into possible functions.

Raw milk cheeses from Beira Baixa, Portugal-A contributive study for the microbiological hygiene and safety assessment.

Due to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g-1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g-1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production.

Comparison of real-time PCR and nested PCR based on the HlyA gene for the detection of Listeria monocytogenes. Application on cheese samples.

The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.

The ARIMA model approach for the biofilm-forming capacity prediction of Listeria monocytogenes recovered from carcasses.

The present study aimed to predict the biofilm-formation ability of L. monocytogenes isolates obtained from cattle carcasses via the ARIMA model at different temperature parameters. The identification of L. monocytogenes obtained from carcass samples collected from slaughterhouses was determined by PCR. The biofilm-forming abilities of isolates were phenotypically determined by calculating the OD value and categorizing the ability via the microplate test. The presence of some virulence genes related to biofilm was revealed by QPCR to support the biofilm profile genotypically. Biofilm-formation of the isolates was evaluated at different temperature parameters (37 °C, 22 °C, 4 °C and - 20 °C). Estimated OD values were obtained with the ARIMA model by dividing them into eight different estimation groups. The prediction performance was determined by performance measurement metrics (ME, MAE, MSE, RMSE, MPE and MAPE). One week of incubation showed all isolates strongly formed biofilm at all controlled temperatures except - 20 °C. In terms of the metrics examined, the 3 days to 7 days forecast group has a reasonable prediction accuracy based on OD values occurring at 37 °C, 22 °C, and 4 °C. It was concluded that measurements at 22 °C had lower prediction accuracy compared to predictions from other temperatures. Overall, the best OD prediction accuracy belonged to the data obtained from biofilm formation at -20 °C. For all temperatures studied, especially after the 3 days to 7 days forecast group, there was a significant decrease in the error metrics and the forecast accuracy increased. When evaluating the best prediction group, the lowest RMSE at 37 °C (0.055), 22 °C (0.027) and 4 °C (0.024) belonged to the 15 days to 21 days group. For the OD predictions obtained at -20 °C, the 15 days to 21 days prediction group had also good performance (0.011) and the lowest RMSE belongs to the 7 days to 15 days group (0.007). In conclusion, this study will guide in using indicator parameters to evaluate biofilm forming ability to predict optimum temperature-time. The ARIMA models integrated with this study can be useful tools for industrial application and risk assessment studies using different parameters such as pH, NaCl concentration, and especially temperature applied during food processing and storage on the biofilm-formation ability of L. monocytogenes.

Rapid Nucleic Acid Detection of Listeria monocytogenes Based on RAA-CRISPR Cas12a System.

Listeria monocytogenes (L. monocytogenes) is a food-borne pathogenic bacteria that frequently contaminates animal-derived food and low-temperature preserved food. Listeriosis caused by its infection has a high mortality rate and poses a serious threat to human health. Therefore, it is crucial to establish a sensitive, rapid and easy-to-operate technique. In this study, a Recombinase Aided Amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform was established for highly sensitive nucleic acid detection of L. monocytogenes. The established RAA-CRISPR/Cas12a showed high sensitivity and high specificity, with the sensitivity of 350 CFU/mL and 5.4 × 10-3 ng/µL for pure bacterial solution and genomic DNA, and good specificity for 5 strains of Listeria spp. and 14 strains of other common pathogenic bacteria. L. monocytogenes could be detected at an initial concentration of 2.3 CFU/25g within 2 h of enriching the beef in the food matrix, and this method could be applied to food samples that were easily contaminated with L. monocytogenes The results of RAA-CRISPR/Cas12a could be observed in 5 min, while the amplification was completed in 20-30 min. The speed and sensitivity of RAA-CRISPR/Cas12a were significantly higher than that of the national standard method. In conclusion, the RAA-CRISPR/Cas12a system established in this study has new application potential in the diagnosis of food-borne pathogens.

High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA.

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

Molecular detection of Listeria monocytogenes from different dairy and street food sources in North Karnataka, India.

BACKGROUND: Food-borne pathogen Listeria monocytogenes is abundantly present in nature and accountable for sporadic and epidemic cases of listeriosis in humans. The objective of this study was to screen common food sources for L. monocytogenes using biochemical and molecular methods to detect and characterise its toxin genes as well as for biofilm formation. METHODS: A total of 92 samples, comprising dairy and street food products, were randomly collected from various sources for this investigation. The collected samples were processed for biochemical and molecular methods to detect L. monocytogenes. Additionally, virulence factors associated genes, antibiogram profiles and biofilm formation related assays were determined. RESULTS: L. monocytogenes presence was confirmed using molecular detection methods targeting prs and lmo1030 genes, along with MALDI-TOF MS. Following 16 S rRNA sequencing, the identified Listeria species were further categorised into two groups. L. monocytogenes was detected in two (2.17%) food samples tested (L-23 and L-74). Multiplex PCR indicated the presence of seven virulence-related genes in L. monocytogenes isolates, i.e., inlA, inlB, prfA, iap, actA, plcB, and hlyA. In addition, 17 antibiotics were tested, whereby two isolates showed resistance to clindamycin and azithromycin, while one isolate (L-74) was also resistant to nalidixic acid, co-trimoxazole, ampicillin, norfloxacin, and cefotaxime. L-23 and L-74 isolates showed biofilm formation, especially at pH 8.6 and 37°C. CONCLUSIONS: Besides the demonstration of the presence of L. monocytogenes in some dairy and street food products, this study underscores the need to increase the standards of hygiene on the one hand and the importance of the surveillance of food-borne pathogens on the other.

Virulence properties of Listeria monocytogenes isolated from meat and meat contact surfaces in a slaughterhouse.

Listeria monocytogenes is a ubiquitous microorganism that is isolated from a variety of sources such as soil, water, decaying vegetation, sewage, animal feeds, silage, farm environments and food-processing environments. This study aimed to determine the prevalence, serogroups, biofilm formation, virulence factor genes, and genetic relationships of L. monocytogenes strains isolated from beef meat and meat contact surfaces obtained from a slaughterhouse in Burdur, Turkey. In this study, a total of 179 beef meat and meat contact surface samples were analyzed for the presence of L. monocytogenes by polymerase chain reaction (PCR). Out of a total of 179 beef meat and meat contact surface samples, 83 (46.37%) were found to be contaminated with L. monocytogenes, with the highest incidence (53.01%) occurring in beef meat. In the present study, most of the isolated strains belonged to serogroups IIB and IVB (lineage I). The L. monocytogenes strain also contained monoA-B, prfA, plcA, plcB, mpl, hlyA, actA, gtcA, dltA, Fri, flaA, InlA, InlC, InlJ, and iap genes. Biofilm formation was not determined in the tested samples at pH 5.5 and different temperatures (4°C, 10°C, 25°C, and 37°C). However, strong biofilm formation was observed in 6.45% (2/31) of the strains at pH 7.0 after 48 h incubation at 37°C, and in 3.22% (1/31) of the strains at pH 7.0 after 48 h incubation at 4°C and 10°C. Pulsed-field gel electrophoresis (PFGE) results showed that L. monocytogenes isolates were clonally related, and cross-contamination was present. In addition, PFGE results also revealed that AscI had more distinguishing power than the ApaI restriction enzyme. These results indicate that L. monocytogenes detected from meat and meat contact surfaces in the slaughterhouse pose a potential risk to public health.

Airborne signals of Pseudomonas fluorescens modulate swimming motility and biofilm formation of Listeria monocytogenes in a contactless coculture system.

Bacterial volatile compounds (BVCs) facilitate interspecies communication in socio-microbiology across physical barriers, thereby influencing interactions between diverse species. The impact of BVCs emitted from Pseudomonas on the biofilm formation characteristics of Listeria monocytogenes within the same ecological niche has been scarcely investigated under practical conditions of food processing. The objective of this study was to explore the motility and biofilm formation characteristics of L. monocytogenes under the impact of Pseudomonas BVCs. It was revealed that BVCs of P. fluorescens, P. lundensis, and P. fragi significantly promoted swimming motility of L. monocytogenes (P < 0.05). As evidenced by crystal violet staining, the L. monocytogenes biofilms reached a maximum OD570 value of approximately 3.78 at 4 d, which was 0.65 units markedly higher than that of the control group (P < 0.05). Despite a decrease in adherent cells of L. monocytogenes biofilms among the BVCs groups, there was a remarkable increase in the abundance of extracellular polysaccharides and proteins with 3.58 and 4.90 µg/cm2, respectively (P < 0.05), contributing to more compact matrix architectures, which suggested that the BVCs of P. fluorescens enhanced L. monocytogenes biofilm formation through promoting the secretion of extracellular polymers. Moreover, the prominent up-regulated expression of virulence genes further revealed the positive regulation of L. monocytogenes under the influence of BVCs. Additionally, the presence of BVCs significantly elevated the pH and TVB-N levels in both the swimming medium and biofilm broth, thereby exhibiting a strong positive correlation with increased motility and biofilm formation of L. monocytogenes. It highlighted the crucial signaling regulatory role of BVCs in bacterial interactions, while also emphasizing the potential food safety risk associated with the hitchhiking behavior of L. monocytogenes, thereby shedding light on advancements in control strategies for food processing.

Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing Escherichia coli and Listeria monocytogenes by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

Mechanistic insights into the alternative ribosome recycling by HflXr.

During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.

Airborne survival and stress response in Listeria monocytogenes across different growth temperatures.

In the food industry, ensuring food safety during transportation and storage is vital, with temperature regulation preventing spoilage. However, airborne contamination through foodborne pathogens remains a concern. Listeria monocytogenes, a psychrotolerant foodborne pathogen, has been linked to various foodborne outbreaks. Therefore, understanding how its airborne characteristics depend on the growth temperature is imperative. As a result, when the L. monocytogenes was floated in air for 30 and 60 min, the surviving population of 15 °C-grown L. monocytogenes that was suspended in air and attached on the surface was significantly higher than L. monocytogenes grown at 25°C and 37 °C. The fatty acid analysis revealed a significantly higher proportion of shorter chain fatty acids in L. monocytogenes grown at 15 °C compared to those grown at 37 °C. Under aerosolization, L. monocytogenes encountered osmotic and cold stresses regardless of their growth temperature. Transcriptomic analysis showed that stress response related genes, such as oxidative and cold stress response, as well as PTS system related genes were upregulated at 15 °C, resulting in the enhanced resistance to various stresses during aerosolization. These results provide insights into the different responses of aerosolized L. monocytogenes according to the different growth temperatures, highlighting a critical factor in preventing airborne cross-contamination.