Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions.

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.

Adaptive evolution of Salmonella Typhimurium LT2 exposed to carvacrol lacks a uniform pattern.

Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Comparative study on the impact of equally stressful environmental sporulation conditions on thermal inactivation kinetics of B. subtilis spores.

Control of bacterial spores continues to be one of the main challenges for the food industry due to their wide dissemination and extremely high resistance to processing methods. Furthermore, the large variability in heat resistance in spores that contaminate foods makes it difficult to establish general processing conditions. Such heterogeneity not only derives from inherent differences among species and strains, but also from differences in sporulation environments that are generally ignored in spores encountered in foods. We evaluated heat inactivation kinetics and the thermodependency of resistance parameters in B. subtilis 168 spores sporulated at adverse temperatures, water activity (aw), and pH, applying an experimental approach that allowed us to quantitatively compare the impact of each condition. Reduction of incubation temperature from the optimal temperature dramatically reduced thermal resistance, and it was the most influential factor, especially at the highest treatment temperatures. These spores were also more sensitive to chemicals presumably acting in the inner membrane. Reducing sporulation aw increased heat resistance, although the magnitude of that effect depended on the solute and the treatment temperature. Thus, changes in sporulation environments varied 3D100°C values up to 10.4-fold and z values up to 1.7-fold, highlighting the relevance of taking such a source of variability into account when setting heat processing conditions. UV-C treatment and sodium hypochlorite efficiently inactivated all spore populations, including heat-resistant ones produced at low aw.

Dynamics of high hydrostatic pressure resistance development in RpoS-deficient Escherichia coli.

High hydrostatic pressure (HHP) treatment is one of the most widely accepted non-thermal food processing methods, but HHP-resistance development in pathogenic or spoilage bacteria might compromise the safety and stability of HHP-treated foods. Charting the possible routes and mechanisms of HHP resistance development in foodborne bacteria is therefore essential to anticipate or prevent the appearance of resistant variants. While upregulation of the RpoS-governed general stress response is a well-established route for increased HHP resistance in Escherichia coli, previous work revealed that mutations causing attenuated cAMP/CRP activity or aggregation-prone TnaA variants can evolve to overcome the HHP-hypersensitivity of an E. coli ΔrpoS mutant. In this study, further directed evolution and genetic analysis approaches allowed us to demonstrate that both kinds of mutants tend to co-emerge and compete with each other in E. coli ΔrpoS populations evolving towards HHP resistance, because of the higher HHP resistance of cAMP/CRP mutants and the faster growth rate of the TnaA mutants. Moreover, closer scrutiny of evolving populations revealed RpoS, cAMP/CRP and TnaA independent routes of HHP resistance development, based on downregulation of YegW or RppH activity.

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.

Carvacrol Selective Pressure Allows the Occurrence of Genetic Resistant Variants of Listeria monocytogenes EGD-e.

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety.

Gene Erosion Can Lead to Gain-of-Function Alleles That Contribute to Bacterial Fitness.

Despite our extensive knowledge of the genetic regulation of heat shock proteins (HSPs), the evolutionary routes that allow bacteria to adaptively tune their HSP levels and corresponding proteostatic robustness have been explored less. In this report, directed evolution experiments using the Escherichia coli model system unexpectedly revealed that seemingly random single mutations in its tnaA gene can confer significant heat resistance. Closer examination, however, indicated that these mutations create folding-deficient and aggregation-prone TnaA variants that in turn can endogenously and preemptively trigger HSP expression to cause heat resistance. These findings, importantly, demonstrate that even erosive mutations with disruptive effects on protein structure and functionality can still yield true gain-of-function alleles with a selective advantage in adaptive evolution.

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.

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.

Construction and validation of the Tn5-PLtetO-1-msfGFP transposon as a tool to probe protein expression and localization.

In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a PLtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the PLtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame "sandwich" msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.

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.

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.

Impact of high hydrostatic pressure on bacterial proteostasis.

High hydrostatic pressure (HHP) is an important factor that limits microbial growth in deep-sea ecosystems to specifically adapted piezophiles. Furthermore, HHP treatment is used as a novel food preservation technique because of its ability to inactivate pathogenic and spoilage bacteria while minimizing the loss of food quality. Disruption of protein homeostasis (i.e. proteostasis) as a result of HHP-induced conformational changes in ribosomes and proteins has been considered as one of the limiting factors for both microbial growth and survival under HHP conditions. This work therefore reviews the effects of sublethal (≤100MPa) and lethal (>100MPa) pressures on protein synthesis, structure, and functionality in bacteria. Furthermore, current understanding on the mechanisms adopted by piezophiles to maintain proteostasis in HHP environments and responses developed by atmospheric-adapted bacteria to protect or restore proteostasis after HHP exposure are discussed.

Intracellular movement of protein aggregates reveals heterogeneous inactivation and resuscitation dynamics in stressed populations of Escherichia coli.

Inactivation of bacterial pathogens is of critical importance in fields ranging from antimicrobial therapy to food preservation. The efficacy of an antimicrobial treatment is often experimentally determined through viable plate counts that inherently provide a poor focus on the mechanisms and distribution of (sub)lethal injury and subsequent inactivation or resuscitation behavior of the stressed cells, which are increasingly important features for the proper understanding and design of inactivation strategies. In this report, we employ a live cell biology approach focusing on the energy-dependent motion of intracellular protein aggregates to investigate the heterogeneity within heat stressed Escherichia coli populations. As such, we were able to identify differential dynamics of cellular resuscitation and inactivation that are impossible to distinguish using more traditional approaches. Moreover, our data indicate the existence of late-resuscitating cells that remain physiologically active and are able to persist in the presence of antibiotics before resuscitation.

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.

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.

Modeling optimal process conditions for UV-heat inactivation of foodborne pathogens in liquid foods.

The combination of ultraviolet radiation and heat (UV-H treatment) has been demonstrated as a promising strategy to overcome the limited UV germicidal effect in fruit juices. Nonetheless, there are so far no data regarding the efficacy of the combined process for the inactivation of bacterial foodborne pathogens in other liquid foods with different pH and composition. In this investigation, the optimum UV-H processing conditions for the inactivation of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and S. aureus in chicken and vegetable broth, in addition to juices, were determined. From these data models that accurately predict the most advantageous UV-H treatment temperature and the expected synergistic lethal effect from UV and heat resistance data separately were constructed. Equations demonstrated that the optimum UV-H treatment temperature mostly depended on heat resistance, whereas the maximum synergistic lethal effect also was affected by the UV resistance of the microorganism of concern in a particular food.

Influence of dimethyl dicarbonate on the resistance of Escherichia coli to a combined UV-Heat treatment in apple juice.

Commercial apple juice inoculated with Escherichia coli was treated with UV-C, heat (55°C) and dimethyl dicarbonate - DMDC (25, 50, and 75 mg/L)-, applied separately and in combination, in order to investigate the possibility of synergistic lethal effects. The inactivation levels resulting from each treatment applied individually for a maximum treatment time of 3.58 min were limited, reaching 1.2, 2.9, and 0.06 log10 reductions for UV, heat, and DMDC (75 mg/L), respectively. However, all the investigated combinations resulted in a synergistic lethal effect, reducing the total treatment time and UV dose, with the synergistic lethal effect being higher when larger concentrations of DMDC were added to the apple juice. The addition of 75 mg/L of DMDC prior to the combined UV-C light treatment at 55°C resulted in 5 log10 reductions after only 1.8 min, reducing the treatment time and UV dose of the combined UV-Heat treatment by 44%.

Effect of pressure-induced changes in the ionization equilibria of buffers on inactivation of Escherichia coli and Staphylococcus aureus by high hydrostatic pressure.

Survival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (ΔV°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ΔV° values are approximately 5.0 to 7.0 cm(3) mol(-1), than in phosphate or dimethyl glutarate (DMG), whose ΔV° values are about -25 cm(3) mol(-1). However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na2SO4 and CaCl2) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na2SO4 than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes.