A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Inhibition of Virulence Gene Expression in Salmonella Dublin, Escherichia coli F5 and Clostridium perfringens Associated With Neonatal Calf Diarrhea by Factors Produced by Lactic Acid Bacteria During Fermentation of Cow Milk.

Diarrhea is a major health problem in neonatal and young calves worldwide. It can be caused by a variety of infectious agents, including the bacteria Salmonella enterica serovar Dublin (S. Dublin), enterotoxigenic Escherichia coli (ETEC), and Clostridium perfringens. Preventive alternatives to antibiotic treatment should be identified. As a first step toward this, the aim of the current study was to examine whether cell-free supernatants from cow milk fermented by lactic acid bacteria affects virulence-gene expression in strains of S. Dublin, ETEC E. coli F5 and C. perfringens. pH-neutralized, cell-free, spent medium of milk (nCFSM) fermented by 61 different lactic acid bacteria (LAB) and non-LAB starter cultures belonging to 17 genera was assayed for their effect on expression of important virulence factors (S. Dublin hilA, ssrB, ssaG, flhD, prgI, fliC; ETEC E. coli F5 fanC, estA, fim41a; C. perfringens cpa), when the bacteria were grown in the nCFSM. Screening was done using either a promoter-reporter expression system or RT-qPCR. nCFSM from Bifidobacterium longum BL-15955 and Limosilactobacillus reuteri LR-33016 downregulated the expression of fanC, fim41a and estA genes in the four tested ETEC E. coli F5 strains without affecting their growth, while mainly B. longum BL-15955 downregulated expression of cpa in the four tested strains of C. perfringens. nCFSM from the mixed cultures; NU-TRISH® BY-Mild (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Bifidobacterium BL-15954) and COMBO4 (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), as well as Lactobacillus helveticus CNRZ32 downregulated the tested virulence genes in the three tested strains of S. Dublin. To enable possible downregulation of the expression of virulence genes in all three target bacteria simultaneously, nCFSM was prepared from NU-TRISH® By-Mild in combination with B. longum BL-15955 (i.e. a four-strain combination). The nCFSM from this combination downregulated the virulence genes expression in all the three species. In the future, NU-TRISH® By-Mild and B. longum BL-15955 in combination could potentially be used for prevention of neonatal calf diarrhea caused by S. Dublin, E. coli F5, and C. perfringens, reducing the need for antimicrobial treatment, however, field studies are needed to prove that.

Cold-shock proteins affect desiccation tolerance, biofilm formation and motility in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen whose biofilm formation and desiccation tolerance may contribute to its survival in the food industry. L. monocytogenes possesses three cold-shock domain family proteins (CspA, CspB and CspD) known to be essential for adaptation against various food-relevant stress conditions including cold growth. The role of Csps in desiccation tolerance and biofilm formation was investigated in csp mutants as well as twenty-one other wild-type (WT) strains. Mutants with a single (ΔcspA) or multiple (ΔcspAB, ΔcspAD and ΔcspABD) deletions of csp genes, in a desiccation sensitive WT background (L. monocytogenes EGD-e) were immotile and exhibited an elevated desiccation tolerance compared to the parent strain. However, deletion of cspA in the more desiccation resistant food and outbreak related L. monocytogenes strains 568 and 08-5578 had no impact on desiccation tolerance although compared to the parent strains the mutants were also immotile. A correlation between lower motility and higher desiccation tolerance was observed among the 20 WT strains (Spearman rank correlation, rs = -0.56, p = 0.01), although exceptions occurred indicating that multiple factors influence the diverse desiccation tolerance among L. monocytogenes strains. Expression of cspA was upregulated in WT EGD-e, 568 and 08-5578 strains after desiccation for seven days, while the 568 and 08-5578 ΔcspA mutants expressed elevated levels of cspD and cspB (>30 fold higher) compared to their WTs. This indicates that upregulation of the other csps compensates for the deleted cspA gene. Although biofilm formation was improved in all EGDe csp mutants relative to the WT strain, the opposite was observed for 568 and 08-5578 WT strains and their cspA deletion mutants. Only motile strains formed biofilm in the peg lid assay but a significant negative correlation (rs = -0.60, p = 0.01) was seen between higher motility and higher biofilm formation of WT strains. In conclusion, the survival of L. monocytogenes strains in the food processing environment may depend on the control of motility, which is a necessity for biofilm formation but disadvantageous for desiccation survival.

A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments.

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

Initial Transcriptomic Response and Adaption of Listeria monocytogenes to Desiccation on Food Grade Stainless Steel.

The foodborne pathogen Listeria monocytogenes survives exposure to a variety of stresses including desiccation in the food industry. Strand-specific RNA sequencing was applied to analyze changes in the transcriptomes of two strains of L. monocytogenes (Lm 568 and Lm 08-5578) during desiccation [15°C, 43% relative humidity (RH)] on food grade stainless steel surfaces over 48 h to simulate a weekend with no food production. Both strains showed similar survival during desiccation with a 1.8-2 Log CFU/cm2 reduction after 48 h. Analysis of differentially expressed (DE) genes (>twofold, adjusted p-value <0.05) revealed that the initial response to desiccation was established after 6 h and remained constant with few new genes being DE after 12, 24, and 48 h. A core of 81 up- and 73 down-regulated DE genes were identified as a shared, strain independent response to desiccation. Among common upregulated genes were energy and oxidative stress related genes e.g., qoxABCD (cytochrome aa3) pdhABC (pyruvate dehydrogenase complex) and mntABCH (manganese transporter). Common downregulated genes related to anaerobic growth, proteolysis and the two component systems lmo1172/lmo1173 and cheA/cheY, which are involved in cold growth and flagellin production, respectively. Both strains upregulated additional genes involved in combatting oxidative stress and reactive oxygen species (ROS), including sod (superoxide dismutase), kat (catalase), tpx (thiol peroxidase) and several thioredoxins including trxAB, lmo2390 and lmo2830. Osmotic stress related genes were also upregulated in both strains, including gbuABC (glycine betaine transporter) and several chaperones clpC, cspA, and groE. Significant strain differences were also detected with the food outbreak strain Lm 08-5578 differentially expressing 1.9 × more genes (726) compared to Lm 568 (410). Unique to Lm 08-5578 was a significant upregulation of the expression of the alternative transcription factor σB and its regulon. A number of long antisense transcripts (lasRNA) were upregulated during desiccation including anti0605, anti0936, anti1846, and anti0777, with the latter controlling flagellum biosynthesis and possibly the downregulation of motility genes observed in both strains. This exploration of the transcriptomes of desiccated L. monocytogenes provides further understanding of how this bacterium encounters and survives the stress faced when exposed to dry conditions in the food industry.