Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.

Cell Envelope Modifications Generating Resistance to Hop Beta Acids and Collateral Sensitivity to Cationic Antimicrobials in Listeria monocytogenes.

Hop beta acids (HBAs) are characteristic compounds from the hop plant that are of interest for their strong antimicrobial activity. In this work, we report a resistance mechanism against HBA in the foodborne pathogen Listeria monocytogenes. Using an evolution experiment, we isolated two HBA-resistant mutants with mutations in the mprF gene, which codes for the Multiple Peptide Resistance Factor, an enzyme that confers resistance to cationic peptides and antibiotics in several Gram-positive bacteria by lysinylating membrane phospholipids. Besides the deletion of mprF, the deletion of dltA, which mediates the alanylation of teichoic acids, resulted in increased HBA resistance, suggesting that resistance may be caused by a reduction in positive charges on the cell surface. Additionally, we found that this resistance is maintained at low pH, indicating that the resistance mechanism is not solely based on electrostatic interactions of HBA with the cell surface. Finally, we showed that the HBA-resistant mutants display collateral sensitivity to the cationic antimicrobials polymyxin B and nisin, which may open perspectives for combining antimicrobials to prevent resistance development.

Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,ß-Unsaturated Aldehydes in Listeria monocytogenes.

The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,ß-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,ß-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,ß-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field.

Hurdle Technology Approach to Control Listeria monocytogenes Using Rhamnolipid Biosurfactant.

This study evaluates the combination of mild heat with a natural surfactant for the inactivation of L. monocytogenes Scott A in low-water-activity (aw) model systems. Glycerol or NaCl was used to reduce the aw to 0.92, and different concentrations of rhamnolipid (RL) biosurfactant were added before heat treatment (60 °C, 5 min). Using glycerol, RL treatment (50-250 µg/mL) reduced bacterial population by less than 0.2 log and heat treatment up to 1.5 log, while the combination of both hurdles reached around 5.0 log reduction. In the NaCl medium, RL treatment displayed higher inactivation than in the glycerol medium at the same aw level and a larger synergistic lethal effect when combined with heat, achieving ≥ 6.0 log reduction at 10-250 µg/mL RL concentrations. The growth inhibition activity of RL was enhanced by the presence of the monovalent salts NaCl and KCl, reducing MIC values from >2500 µg/mL (without salt) to 39 µg/mL (with 7.5% salt). The enhanced antimicrobial activity of RL promoted by the presence of salts was shown to be pH-dependent and more effective under neutral conditions. Overall, results demonstrate that RL can be exploited to design novel strategies based on hurdle approaches aiming to control L. monocytogenes.

Selection and Development of Nontoxic Nonproteolytic Clostridium botulinum Surrogate Strains for Food Challenge Testing.

Clostridium botulinum causes severe foodborne intoxications by producing a potent neurotoxin. Challenge studies with this pathogen are an important tool to ensure the safety of new processing techniques and newly designed or modified foods, but they are hazardous and complicated by the lack of an effective selective counting medium. Therefore, this study aimed to develop selectable nontoxic surrogate strains for group II, or nonproteolytic, C. botulinum, which are psychotropic and hence of particular concern in mildly treated, refrigerated foods. Thirty-one natural nontoxic nonproteolytic strains, 16 of which were isolated in this work, were characterized in detail, revealing that 28 strains were genomically and phenotypically indistinguishable from toxic strains. Five strains, representing the genomic and phenotypic diversity of group II C. botulinum, were selected and successfully equipped with an erythromycin (Em) resistance marker in a defective structural phage gene without altering phenotypic features. Finally, a selective medium containing Em, cycloserine (Cs), gentamicin (Gm), and lysozyme (Ly) was developed, which inhibited the background microbiota of commercial cooked ham, chicken filet, and salami, but supported spore germination and growth of the Em-resistant surrogate strains. The surrogates developed in this work are expected to facilitate food challenge studies with nonproteolytic C. botulinum for the food industry and can also provide a safe alternative for basic C. botulinum research.

AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion.

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

The Natural Antimicrobial trans-Cinnamaldehyde Interferes with UDP-N-Acetylglucosamine Biosynthesis and Cell Wall Homeostasis in Listeria monocytogenes.

Trans-cinnamaldehyde (t-CIN), an antimicrobial compound from cinnamon essential oil, is of interest because it inhibits various foodborne pathogens. In the present work, we investigated the antimicrobial mechanisms of t-CIN in Listeria monocytogenes using a previously isolated yvcK::Himar1 transposon mutant which shows hypersensitivity to t-CIN. Time-lapse microscopy revealed that t-CIN induces a bulging cell shape followed by lysis in the mutant. Complementation with wild-type yvcK gene completely restored the tolerance of yvcK::Himar1 strain to t-CIN and the cell morphology. Suppressor mutants which partially reversed the t-CIN sensitivity of the yvcK::Himar1 mutant were isolated from evolutionary experiments. Three out of five suppression mutations were in the glmU-prs operon and in nagR, which are linked to the biosynthesis of the peptidoglycan precursor uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc). GlmU catalyzes the last two steps of UDP-GlcNAc biosynthesis and NagR represses the uptake and utilization of N-acetylglucosamine. Feeding N-acetylglucosamine or increasing the production of UDP-GlcNAc synthetic enzymes fully or partially restored the t-CIN tolerance of the yvcK mutant. Together, these results suggest that YvcK plays a pivotal role in diverting substrates to UDP-GlcNAc biosynthesis in L. monocytogenes and that t-CIN interferes with this pathway, leading to a peptidoglycan synthesis defect.

Microbiological Safety of Ready-to-Eat Foods in Hospital and University Canteens in Hanoi, Vietnam.

ABSTRACT: The aim of this study was to analyze and document the microbiological safety and quality of ready-to-eat foods in hospital and university canteens in Hanoi, Vietnam. In total, 420 ready-to-eat food products from 21 canteens were sampled in July 2018 and May 2019. The ratio of samples exceeding the unsatisfactory level for total plate count was 31%. Escherichia coli, Listeria, and Staphylococcus aureus were detected in 35 (8.3%), 99 (24%), and 46 (11%) samples, with 3, 10, and 0% exceeding the unsatisfactory level, respectively. The total plate count, Listeria, Bacillus cereus, E. coli, and S. aureus ranged from below detection limit to 5 × 109, 4.6 × 105, 6.2 × 103, 3.4 × 103, and 7.6 × 103 CFU/g, respectively. L. monocytogenes was isolated from 3 (0.7%) of 420 samples. In addition, 21 (5%) of 410 samples were contaminated with Salmonella. Overall, our data indicate frequent problems with the microbiological quality and safety of these canteen foods in Hanoi and provide a baseline measurement that will allow environmental health officers and food microbiologists to develop targeted intervention strategies to reduce the economic and public health risk associated with these foods.

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli.

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600-800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.

Evaluation of factors influencing the growth of non-toxigenic Clostridium botulinum type E and Clostridium sp. in high-pressure processed and conditioned tender coconut water from Thailand.

Bacterial spores survive high pressure processing (HPP). Group II Clostridium botulinum is an obligate anaerobe spore-forming pathogen that can produce the botulinum neurotoxin under refrigeration. This study assessed nontoxigenic type E C. botulinum and Group II Clostridium sp. growth in raw and HPP (550 MPa, 3 min, 10 °C) Thai coconut water (CCW; pH 5.2). No spore germination or growth occurred in HPP CCW inoculated with 105 CFU/ml after 61 days regardless of oxygen concentration (<0.5 - 11 mg/l) or storage temperature (4 and 20 °C). Spore concentration decreased by 3.0 ± 0.1 log CFU/ml in a worst-case scenario consisting of non-HPP filter-sterilized CCW (pH 7.0) under anoxic incubation at 30 °C during 61 days, suggesting spore germination followed by cellular death. Supplementing filter-sterilized CCW (pH 7.0) with selected germinants and free amino acids did not support spore development, but the addition of nutrient-rich laboratory media (TPGY broth) at low concentrations (6.25%) promoted growth, suggesting that a lack of nutrients prevents C. botulinum development in CCW. Further risk assessment will require evaluating other CCW varieties and toxin production.

Author Correction: Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combination of mild heat and plant essential oil constituents to inactivate resistant variants of Escherichia coli in buffer and in coconut water.

The growing demand for minimally processed foods with clean labels has stimulated research into mild processing methods and natural antimicrobials to replace intensive heating and conventional preservatives, respectively. However, we have previously demonstrated that repetitive exposure of some bacteria to mild heat or subinhibitory concentrations of essential oil constituents (EOCs) may induce the emergence of mutants with increased resistance to these treatments. Since the combination of mild heat with some EOCs has a synergistic effect on microbial inactivation, we evaluated the potential of such combinations against our resistant E. coli mutants. While citral, carvacrol and t-cinnamaldehyde synergistically increased heat inactivation (53.0 °C, 10 min) of the wild-type MG1655 suspended in buffer, only the combination with carvacrol (200 µl/l) was able to mitigate the increased resistance of all the mutants. Moreover, the combination of heat and carvacrol acted synergistically inactivating heat-resistant variants of E. coli O157:H7 (ATCC 43888). This combined treatment could synergistically achieve more than 5 log10 reductions of the most resistant mutants in coconut water, although the temperature had to be raised to 57.0 °C. Therefore, the combination of mild heat with carvacrol appears to hold promise for mild processing, and it is expected to counteract the development of heat resistance.

Identification of novel genes involved in high hydrostatic pressure resistance of Escherichia coli.

High hydrostatic pressure (HHP) is an interesting hurdle in minimal food processing that aims to synergistically combine different stresses to improve food microbiological safety and stability without compromising quality. For a proper understanding and design of hurdle technology, the cellular impact of the applied stresses on foodborne pathogens should be well-established. To study the mechanism of HHP-mediated cell injury and death, we screened for loss-of-function mutations in E. coli MG1655 that affected HHP sensitivity. More specifically, ca. 6000 random transposon insertion mutants were individually exposed to HHP, after which the phenotype of the most resistant or sensitive mutations was confirmed by de novo gene deletions in the parental strain. We found that disruption of rbsK, rbsR, hdfR and crl decreased HHP resistance, while disruption of sucC and sucD (encoding subunits of the succinyl-CoA synthetase) increased HHP resistance. More detailed study of the tricarboxylic acid cycle enzymes encoded by the sdhCDAB-sucABCD operon surprisingly showed that disruption of the sucA or sucB gene (encoding subunits of the 2-oxoglutarate dehydrogenase complex) notably decreased HHP survival. We also found that the increased HHP resistance of a ΔsucC and ΔsucD mutant was mediated by increased basal RpoS activity levels, although it did not correlate with their heat resistance. Our results reveal that compromising TCA cycle enzymes can profoundly affect HHP resistance in E. coli.

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219.

Clostridium botulinum is an anaerobic sporeforming bacterium that is notorious for producing a potent neurotoxin. Spores of C. botulinum can survive mild food processing treatments and subsequently germinate, multiply, produce toxin and cause botulism. Control of spore germination and outgrowth is therefore essential for the safety of mildly processed foods. However, little is known about the process of spore germination in group II C. botulinum (gIICb), which are a major concern in chilled foods because they are psychrotrophic. The classical model of spore germination states that germination is triggered by the binding of a germinant molecule to a cognate germinant receptor. Remarkably, unlike many other sporeformers, gIICb has only one predicted canonical germinant receptor although it responds to multiple germinants. Therefore, we deleted the gerBAC locus that encodes this germinant receptor to determine its role in germination. Surprisingly, the deletion did not affect germination by any of the nutrient germinants, nor by the non-nutrient dodecylamine. We conclude that one or more other, so far unidentified, germinant receptors must be responsible for nutrient induced germination in gIICb. Furthermore, the gerBAC locus was strongly conserved with intact open reading frames in 159 gIICb genomes, suggesting that it has nevertheless an important function.

RpoS-independent evolution reveals the importance of attenuated cAMP/CRP regulation in high hydrostatic pressure resistance acquisition in E. coli.

High hydrostatic pressure (HHP) processing is an attractive non-thermal alternative to food pasteurization. Nevertheless, the large inter- and intra-species variations in HHP resistance among foodborne pathogens and the ease by which they can acquire extreme resistance are an issue of increasing concern. Since RpoS activity has been considered as a central determinant in the HHP resistance of E. coli and its pathovars, this study probed for the potential of an E. coli MG1655 ΔrpoS mutant to acquire HHP resistance by directed evolution. Despite the higher initial HHP sensitivity of the ΔrpoS mutant compared to the wild-type strain, evolved lineages of the former readily managed to restore or even succeed wild-type levels of resistance. A number of these ΔrpoS derivatives were affected in cAMP/CRP regulation, and this could be causally related to their HHP resistance. Subsequent inspection revealed that some of previously isolated HHP-resistant mutants derived from the wild-type strain also incurred a causal decrease in cAMP/CRP regulation. cAMP/CRP attenuated HHP-resistant mutants also exhibited higher resistance to fosfomycin, a preferred treatment for STEC infections. As such, this study reveals attenuation of cAMP/CRP regulation as a relevant and RpoS-independent evolutionary route towards HHP resistance in E. coli that coincides with fosfomycin resistance.

Membrane fatty acid composition as a determinant of Listeria monocytogenes sensitivity to trans-cinnamaldehyde.

trans-Cinnamaldehyde, the major compound of cinnamon essential oil, is a potentially interesting natural antimicrobial food preservative. Although a number of studies have addressed its mode of action, the factors that determine bacterial sensitivity or tolerance to trans-cinnamaldehyde are poorly understood. We report the detailed characterization of a Listeria monocytogenes Scott A trans-cinnamaldehyde hypersensitive mutant defective in IlvE, which catalyzes the reversible transamination of branched-chain amino acids to the corresponding short-chain α-ketoacids. This mutant showed an 8.4 fold extended lag phase during growth in sublethal concentrations (4 mM), and faster inactivation in lethal concentrations of trans-cinnamaldehyde (6 mM). trans-Cinnamaldehyde hypersensitivity could be corrected by genetic complementation with the ilvE gene and supplementation with branched-chain α-ketoacids. Whole-cell fatty acid analyses revealed an almost complete loss of anteiso branched-chain fatty acids (BCFAs), which was compensated by elevated levels of unbranched saturated fatty acids and iso-BCFAs. Sub-inhibitory concentrations of trans-cinnamaldehyde induced membrane fatty acid adaptations predicted to reduce membrane fluidity, possibly as a response to counteract the membrane fluidizing effect of trans-cinnamaldehyde. These results demonstrate the role of IlvE in BCFA production and the role of membrane composition as an important determinant of trans-cinnamaldehyde sensitivity in L. monocytogenes.

Severely Heat Injured Survivors of E. coli O157:H7 ATCC 43888 Display Variable and Heterogeneous Stress Resistance Behavior.

Although minimal food processing strategies aim to eliminate foodborne pathogens and spoilage microorganisms through a combination of mild preservation techniques, little is actually known on the resistance behavior of the small fraction of microorganisms surviving an inimical treatment. In this study, the conduct of severely heat stressed survivors of E. coli O157:H7 ATCC 43888, as an indicator for the low infectious dose foodborne enterohemorrhagic strains, was examined throughout their resuscitation and outgrowth. Despite the fact that these survivors were initially sublethally injured, they were only marginally more sensitive to a subsequent heat treatment and actually much more resistant to a subsequent high hydrostatic pressure (HHP) shock in comparison with unstressed control cells. Throughout further resuscitation, however, their initial HHP resistance rapidly faded out, while their heat resistance increased and surpassed the initial heat resistance of unstressed control cells. Results also indicated that the population eventually emerging from the severely heat stressed survivors heterogeneously consisted of both growing and non-growing cells. Together, these observations provide deeper insights into the particular behavior and heterogeneity of stressed foodborne pathogens in the context of food preservation.

Identification of Genes Required for Growth of Escherichia coli MG1655 at Moderately Low pH.

The survival of some pathotypes of Escherichia coli in very low pH environments like highly acidic foods and the stomach has been well documented and contributes to their success as foodborne pathogens. In contrast, the ability of E. coli to grow at moderately low pH has received less attention, although this property can be anticipated to be also very important for the safety of mildly acidic foods. Therefore, the objective of this study was to identify cellular functions required for growth of the non-pathogenic strain E. coli MG1655 at low pH. First, the role of the four E. coli amino acid decarboxylase systems, which are the major cellular mechanisms allowing extreme acid survival, was investigated using mutants defective in each of the systems. Only the lysine decarboxylase (CadA) was required for low pH growth. Secondly, a screening of 8544 random transposon insertion mutants resulted in the identification of six genes affecting growth in LB broth acidified to pH 4.50 with HCl. Two of the genes, encoding the transcriptional regulator LeuO and the elongation factor P-ß-lysine ligase EpmA, can be linked to CadA production. Two other genes, encoding the diadenosine tetraphosphatase ApaH and the tRNA modification GTPase MnmE, have been previously implicated in the bacterial response to stresses other than low pH. A fifth gene encodes the LPS heptosyltransferase WaaC, and its mutant has a deep rough colony phenotype, which has been linked to reduced acid tolerance in earlier work. Finally, tatC encodes a secA-independent protein translocase that exports a few dozen proteins and thus is likely to have a pleiotropic phenotype. For mnmE, apaH, epmA, and waaC, de novo in frame deletion and genetic complementation confirmed their role in low pH growth, and these deletion mutants were also affected in growth in apple juice and tomato juice. However, the mutants were not affected in survival in gastric simulation medium at pH 2.5, indicating that growth at moderately low pH and survival of extremely low pH depend mostly on different cellular functions.

A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line.

The antimicrobial secondary metabolite kalimantacin (also called batumin) is produced by a hybrid polyketide/non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites. This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.

Stress-Induced Evolution of Heat Resistance and Resuscitation Speed in Escherichia coli O157:H7 ATCC 43888.

The development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution of Escherichia coli O157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 106-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits in E. coli ATCC 43888 that can compromise food safety. IMPORTANCE: In this investigation, we demonstrated that Escherichia coli O157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants of E. coli ATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be rapidly evolvable traits that can compromise the control of foodborne pathogens in minimal processing strategies, which rely on the efficacy of combined mild preservation stresses for food safety.