ABSTRACT
In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.
Subject(s)
Flow Cytometry , Listeria monocytogenes , Microbial Viability , Listeria monocytogenes/growth & development , Listeria monocytogenes/physiology , Flow Cytometry/methods , Food Microbiology/methods , Fluorescent Dyes/chemistry , Staining and Labeling/methods , Cell Membrane/metabolismABSTRACT
Foodborne pathogens are responsible for foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can be entered in a viable but nonculturable (VBNC) state. VBNC cells are characterized by an active metabolism and a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples and thus may pose a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method combining propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.
Subject(s)
Azides , Food Microbiology , Microbial Viability , Propidium , Real-Time Polymerase Chain Reaction , Food Microbiology/methods , Azides/chemistry , Propidium/analogs & derivatives , Real-Time Polymerase Chain Reaction/methods , Bacteria/genetics , Bacteria/isolation & purification , Foodborne Diseases/microbiology , Microscopy, Fluorescence/methods , HumansABSTRACT
Properly using controllable atmospheric containers can facilitate investigations of the survival abilities and physiological states of key and emerging-foodborne pathogens under recreated applicable food processing environmental conditions. Notably, saturated salt solutions can efficiently control relative humidity in airtight containers. This chapter describes a practical experimental setup, with necessary prerequisites for exposing foodborne pathogens to simulated and relevant food processing environmental conditions. Subsequent analyses for studying cell physiology will also be suggested.
Subject(s)
Food Handling , Food Microbiology , Food Handling/methods , Foodborne Diseases/microbiology , Microbial Viability , Bacteria/growth & development , HumansABSTRACT
Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.
Subject(s)
Bacillus thuringiensis , Bacillus , Disinfectants , Glutaral , Spores, Bacterial , Disinfectants/pharmacology , Spores, Bacterial/drug effects , Bacillus/drug effects , Bacillus/physiology , Glutaral/pharmacology , Bacillus thuringiensis/drug effects , Bacillus thuringiensis/physiology , Microbial Sensitivity Tests , o-Phthalaldehyde/pharmacology , Bacillus cereus/drug effects , Microbial Viability/drug effects , Disinfection/methodsABSTRACT
Recent advancements in modeling suggest that microbial inactivation in leafy greens follows a nonlinear pattern, rather than the simple first-order kinetics. In this study, we evaluated 17 inactivation models commonly used to describe microbial decline and established the conditions that govern microbial survival on leafy greens. Through a systematic review of 65 articles, we extracted 530 datasets to model the fate of Shiga toxin-producing Escherichia coli O157:H7 on leafy greens. Various factor analysis methods were employed to evaluate the impact of identified conditions on survival metrics. A two-parameter model (jm2) provided the best fit to most of both natural and antimicrobial-induced persistence datasets, whereas the one-parameter exponential model provided the best fit to less than 20% of the datasets. The jm2 model (adjusted R2 = .89) also outperformed the exponential model (adjusted R2 = .58) in fitting the pooled microbial survival data. In the context of survival metrics, the model averaging approach generated higher values than the exponential model for >4 log reduction times (LRTs), suggesting that the exponential model may be overpredicting inactivation at later time points. The random forest technique revealed that temperature and inoculum size were common factors determining inactivation in both natural and antimicrobial-induced die-offs.. The findings show the limitations of relying on the first-order survival metric of 1 LRT and considering nonlinear inactivation in produce safety decision-making.
Subject(s)
Escherichia coli O157 , Escherichia coli O157/drug effects , Food Microbiology , Vegetables/microbiology , Microbial Viability , Plant Leaves/microbiology , Plant Leaves/chemistryABSTRACT
The preservation of microorganisms is pivotal in microbiological practice. Currently, cryopreservation is assumed to be an effective and inexpensive approach for the storage of microorganisms, including bacteria. The key point of cryopreservation is optimal cryoprotectant selection. In the present study, different cryoprotectant compositions were tested for long-term storage of 15 Enterobacterales bacterial strains at - 20 °C. The survival rates of the bacterial strains were evaluated in four different cryoprotectant solutions containing 70% glycerin only (cryoprotectants 1 and 4), 10% dimethyl sulfoxide (DMSO) with 70% glycerin (cryoprotectant 2), and 10% DMSO (cryoprotectant 3). In addition, cryoprotectants 1 and 2 contained peptone and yeast extract as nutritional supplements. The general survival rates of the bacterial strains were evaluated after 12 months of storage. After 12 months, the survival rates of the different cryoprotectants were as follows: cryoprotectant 1-88.87%; cryoprotectant 2-84.85%; cryoprotectant 3-83.50%; and cryoprotectant 4-44.81%. Thus, the composition of cryoprotectant 1 (70% glycerin with nutrient supplements) was optimal for preserving 15 tested strains of the order Enterobacterales. Despite these findings, the biochemical properties of the tested strains changed after cryopreservation for 12 months in the presence of 1 or 3 cryoprotectants. Alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation. We assume that the composition of cryoprotectant 1 can be optimal for storing the order Enterobacterales at - 20 °C. However, further investigations are needed to elucidate the problem of cryopreservation and to support our assumption.
Subject(s)
Cryopreservation , Cryoprotective Agents , Enterobacteriaceae , Microbial Viability , Cryoprotective Agents/pharmacology , Cryopreservation/methods , Microbial Viability/drug effects , Enterobacteriaceae/drug effects , Enterobacteriaceae/growth & development , Dimethyl Sulfoxide/pharmacology , Glycerol/pharmacologyABSTRACT
Acinetobacter species such as A. venetianus and A. guillouiae have been studied for various biotechnology applications, including bioremediation of recalcitrant and harmful environmental contaminants, as well as bioengineering of enzymes and diagnostic materials. Bacteria used in biotechnology are often combined with other microorganisms in mixtures to formulate efficacious commercial products. However, if the mixture contained a closely related Acinetobacter pathogen such as A. baumannii (Ab), it remains unclear whether the survival and virulence of Ab would be masked or augmented. This uncertainty poses a challenge in ensuring the safety of such biotechnology products, since Ab is one of the most significant pathogens for both hospital and community -acquired infections. This research aimed to investigate the growth and virulence of Ab within a mixture of 11 bacterial species formulated as a mock microbial mixture (MM). Growth challenges with environmental stressors (i.e., temperature, pH, sodium, iron, and antibiotics) revealed that Ab could thrive under diverse conditions except in the presence of ciprofloxacin. When cultured alone, Ab exhibited significantly more growth in the presence of almost all the environmental stressors than when it was co-incubated with the MM. During the exposure of A549 lung epithelial cells to the MM, Ab growth was stimulated compared to that in standard mammalian culture media. Cytotoxicity caused by Ab was suppressed in the presence of the MM. Lymphocytes were significantly reduced in mice exposed to Ab with or without MM via intravenous injection. The levels of the splenic cytokines IL-1α, IL-1ß, MCP-1, and MIP-1α were significantly reduced 24 h after exposure to Ab + MM. This study demonstrated that the presence of the MM marginally but significantly reduced the growth and virulence of Ab, which has implications for the safety of mixtures of microorganisms for biotechnological applications. Furthermore, these findings expand our understanding of the virulence of Ab during host-pathogen interactions.
Subject(s)
Acinetobacter Infections , Acinetobacter baumannii , Animals , Acinetobacter baumannii/pathogenicity , Acinetobacter baumannii/growth & development , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/genetics , Virulence , Mice , Humans , Acinetobacter Infections/microbiology , A549 Cells , Anti-Bacterial Agents/pharmacology , Female , Cytokines/metabolism , Microbial Viability/drug effectsABSTRACT
The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.
Subject(s)
Biofilms , Candida albicans , Plasma Gases , Streptococcus mutans , Biofilms/drug effects , Biofilms/growth & development , Plasma Gases/pharmacology , Candida albicans/physiology , Candida albicans/drug effects , Streptococcus mutans/drug effects , Streptococcus mutans/physiology , Dental Caries/microbiology , Dental Caries/therapy , Lacticaseibacillus casei/physiology , Humans , Microbial Viability/drug effects , Microscopy, Confocal , Cold TemperatureABSTRACT
Photodynamic inactivation is an emerging antimicrobial treatment that can be enhanced by employing exogenous photosensitizers to eradicate foodborne pathogens. This study investigated a novel combinatory strategy to eradicate Listeria monocytogenes using blackthorn fruit peel (BFP) and blue light (BL). Extracts of BFP were characterized in terms of polyphenolic content, individual constituents, and antioxidant and antimicrobial activity. The concentration of phenolic compounds and antioxidant activity were both found to be determinants of antimicrobial activity. It was further speculated that flavonols, predominantly quercetin and rutin, were responsible for the activity of BFP against L. monocytogenes. A combination of BFP and BL resulted in a rapid inactivation of the pathogen by up to 4 log CFU/mL at 58.5 J/cm2, corresponding to 15 min BL illumination. Flow cytometry analysis revealed that the bacterial cells lost activity and suffered extensive membrane damage, exceeding 90% of the population. After photosensitizing L. monocytogenes with the BFP constituents quercetin and rutin, a 1.3-log reduction was observed. When applied together, these compounds could inflict the same damaging effect on cells as they did individually when effects were added. Therefore, the results indicate that BFP represents a natural source of (pro-)photosensitizers, which act additively to create inactivation effects. This study may help identify more effective plant-based photosensitizers to control L. monocytogenes in food-related applications.
Subject(s)
Fruit , Light , Listeria monocytogenes , Photosensitizing Agents , Plant Extracts , Polyphenols , Listeria monocytogenes/drug effects , Listeria monocytogenes/radiation effects , Listeria monocytogenes/growth & development , Polyphenols/pharmacology , Plant Extracts/pharmacology , Plant Extracts/chemistry , Fruit/chemistry , Fruit/microbiology , Photosensitizing Agents/pharmacology , Crataegus/chemistry , Anti-Bacterial Agents/pharmacology , Antioxidants/pharmacology , Quercetin/pharmacology , Microbial Viability/drug effects , Microbial Viability/radiation effects , Blue LightABSTRACT
This study aimed to assess the impact of adaptation of ten strains of O157:H7 and non-O157 Escherichia coli to low pH (acid shock or slow acidification) and the effects of this exposure or not on the resistance of E. coli strains to UV radiation in orange juice (pH 3.5). The acid-shocked cells were obtained through culture in tryptic soy broth (TSB) with a final pH of 4.8, which was adjusted by hydrochloric, lactic, or citric acid and subsequently inoculated in orange juice at 4 °C for 30 days. No significant differences (p > 0.05) in survival in orange juice were observed between the serotypes O157:H7 and non-O157:H7 for acid-shocked experiments. After slow acidification, where the cells were cultured in TSB supplemented with glucose 1% (TSB + G), a significant increase (p < 0.05) in survival was observed for all strains evaluated. The D-values (radiation dose (J/cm2) necessary to decrease the microbial population by 90%) were determined as the inverse of the slopes of the regressions (k) obtained by plotting log (N/N0). The results show that among the strains tested, E. coli O157:H7 (303/00) and O26:H11 were the most resistant and sensitive strains, respectively. According to our results, the method of acid adaptation contributes to increasing the UV resistance for most of the strains tested.
Subject(s)
Adaptation, Physiological , Citrus sinensis , Escherichia coli O157 , Fruit and Vegetable Juices , Ultraviolet Rays , Escherichia coli O157/radiation effects , Escherichia coli O157/growth & development , Escherichia coli O157/drug effects , Fruit and Vegetable Juices/microbiology , Fruit and Vegetable Juices/analysis , Citrus sinensis/microbiology , Citrus sinensis/chemistry , Hydrogen-Ion Concentration , Escherichia coli/radiation effects , Escherichia coli/drug effects , Acids/pharmacology , Colony Count, Microbial , Food Microbiology , Microbial Viability/radiation effects , Microbial Viability/drug effects , Food IrradiationABSTRACT
BACKGROUNDS: Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis, secretes a multitude of proteins that modulate the host's immune response to ensure its own persistence. The region of difference (RD) genes encoding proteins play key roles in TB immunity and pathogenesis. Nevertheless, the roles of the majority of RD-encoded proteins remain to be elucidated. OBJECTS: To elucidate the role of Rv2652c located in RD13 in Mtb on bacterial growth, bacterial survival, and host immune response. METHODS: We constructed the strain MS_Rv2652c which over-expresses Mtb RD-encoding protein Rv2652c in M. smegmatis (MS), and compared it with the wild strain in the bacterial growth, bacterial survival, virulence of Rv2652c, and determined the effect of MS_Rv2652c on host immune response in macrophages. RESULTS: Rv2652c protein is located at cell wall of MS_Rv2652c strain and also an integral component of the Mtb H37Rv cell wall. Rv2652c can enhance the resistance of recombinant MS to various stressors. Moreover, Rv2652c inhibits host proinflammatory responses via modulation of the NF-κB pathway, thereby promoting Mtb survival in vitro and in vivo. CONCLUSION: Our data suggest that cell wall protein Rv2652c plays an important role in creating a favorable environment for bacterial survival by modulating host signals and could be established as a potential TB drug target.
Subject(s)
Bacterial Proteins , Macrophages , Mycobacterium tuberculosis , Mycobacterium tuberculosis/immunology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Animals , Mice , Macrophages/immunology , Macrophages/microbiology , Macrophages/metabolism , Tuberculosis/immunology , Tuberculosis/microbiology , Humans , Host-Pathogen Interactions/immunology , Virulence , Mycobacterium smegmatis/immunology , Microbial Viability/immunology , NF-kappa B/metabolism , Mice, Inbred C57BL , Cell Wall/immunology , Cell Wall/metabolismABSTRACT
Arguably, the greatest threat to bacteria is phages. It is often assumed that those bacteria that escape phage infection have mutated or utilized phage-defence systems; however, another possibility is that a subpopulation forms the dormant persister state in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defence systems more time to clear the phage infection. Here, we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages and sequencing their genomes. We found that bacteria in plaques can escape phage attack both by mutation (i.e. become resistant) and without mutation (i.e. become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g. causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially growing cells. Furthermore, our results seem general in that phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.
Subject(s)
Bacteriophages , Bacteriophages/genetics , Bacteriophages/physiology , Microbial Viability/drug effects , Mutation , Bacteria/virology , Bacteria/genetics , Bacteria/drug effects , Genome, Viral , Pseudomonas aeruginosa/virology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/geneticsABSTRACT
Biofilms are highly resistant to antimicrobials, often causing chronic infections. Combining antimicrobials with low-frequency ultrasound (LFU) enhances antimicrobial efficiency, but little is known about the underlying mechanisms. Biofilm physical characteristics, which depend on factors such as growth conditions and age, can have significant effects on inactivation efficiency. In this study, we investigated the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, with and without LFU treatment. The biofilms were grown under low and high fluid shear to provide different characteristics. Low-shear biofilms exhibited greater thickness, roughness, and porosity and lower density, compared to high-shear biofilms. The biofilm matrix of the high-shear biofilms had a three times higher protein-to-polysaccharide ratio, suggesting greater biofilm stiffness. This was supported by microrheology measurements of biofilm creep compliance. For the low-shear biofilms without LFU, the viability of the biofilms in their inner regions was largely unaffected by the antibiotic after a 2-hour treatment. However, when tobramycin was combined with LFU, the inactivation for the entire biofilm increased to 80% after 2 h. For the high-shear biofilms without LFU, higher LFU intensities were needed to achieve similar inactivation results. Microrheology measurements revealed that changes in biofilm inactivation profiles were closely related to changes in biofilm mechanical properties. Modeling suggests that LFU changes antibiotic diffusivity within the biofilm, probably due to a "decohesion" effect. Overall, this research suggests that biofilm physical characteristics (e.g., compliance, morphology) are linked to antimicrobial efficiency. LFU weakens the biofilm while increasing its diffusivity for antibiotics.
Subject(s)
Anti-Bacterial Agents , Biofilms , Pseudomonas aeruginosa , Tobramycin , Biofilms/drug effects , Biofilms/growth & development , Anti-Bacterial Agents/pharmacology , Tobramycin/pharmacology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiology , Microbial Sensitivity Tests , Microbial Viability/drug effects , Ultrasonic WavesABSTRACT
In mammals, enteric salmonellas can use tetrathionate (ttr), formed as a by-product from the inflammatory process in the intestine, as electron acceptor in anaerobic respiration, and it can fuel its energy metabolism by degrading the microbial fermentation product 1,2-propanediol. However, recent studies have shown that this mechanism is not important for Salmonella infection in the intestine of poultry, while it prolongs the persistence of Salmonella at systemic sites in this species. In the current study, we show that ΔttrApduA strains of Salmonella enterica have lower net survival within chicken-derived HD-11 macrophages, as CFU was only 2.3% (S. Enteritidis ΔttrApduA), 2.3% (S. Heidelberg ΔttrApduA), and 3.0% (S. Typhimurium ΔttrApduA) compared to wild-type strains after 24 h inside HD-11 macrophage cells. The difference was not related to increased lysis of macrophages, and deletion of ttrA and pduA did not impair the ability of the strains to grow anaerobically. Further studies are indicated to determine the reason why Salmonella ΔttrApduA strains survive less well inside macrophage cell lines.
Subject(s)
Chickens , Macrophages , Salmonella enterica , Macrophages/microbiology , Macrophages/immunology , Macrophages/metabolism , Animals , Chickens/microbiology , Salmonella enterica/genetics , Cell Line , Gene Deletion , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Salmonella Infections, Animal/microbiology , Salmonella Infections, Animal/immunology , Microbial Viability/geneticsABSTRACT
Cold plasma (CP) technology is a promising alternative to thermal treatments for the microbial decontamination of foods with low-water activity. The aim of this work is study the application of low-pressure CP (0.35 mbar) for the inactivation of Bacillus cereus in a soybean powder matrix using O2 and synthetic air as ionizing gases. The parameters tested were an input power of 100, 200 and 300 W and an exposure time of 10 to 30 min. The excited reactive species formed were monitored by optical emission spectroscopy, and survival data were analyzed using the Weibull mathematical model. Treatments with both gases were effective in inactivating B. cereus. Air plasma resulted in a maximum 3.71-log reduction in bacterial counts at 300 W and 30 min, while O2 plasma showed the strongest inactivation ability, achieving levels higher than 5 log cycles at 300 W and > 25 min. This is likely due to the strong antimicrobial activity of oxygen-derived radicals together with carbon monoxide as an oxidation by-product. In addition, the Weibull distribution function accurately modeled the inactivation of B. cereus. Cold plasma technology is a promising approach for the decontamination of bacteria in low-water activity foods.
Subject(s)
Bacillus cereus , Food Microbiology , Glycine max , Microbial Viability , Oxygen , Plasma Gases , Water , Bacillus cereus/drug effects , Bacillus cereus/growth & development , Plasma Gases/pharmacology , Water/chemistry , Glycine max/microbiology , Glycine max/chemistry , Food Microbiology/methods , Powders , Air , Colony Count, MicrobialABSTRACT
Biofilm formation enhances bacterial survival and antibiotic tolerance, but the underlying mechanisms are incompletely understood. Here, we show that biofilm growth is accompanied by a reduction in bacterial energy metabolism and membrane potential, together with metabolic exchanges between the inner and outer regions in biofilms. More specifically, nutrient-starved cells in the interior supply amino acids to cells in the periphery, while peripheral cells experience a decrease in membrane potential and provide fatty acids to interior cells. Fatty acids facilitate the repair of starvation-induced membrane damage in inner cells and enhance their survival in the presence of antibiotics. Thus, metabolic exchanges between inner and outer cells contribute to survival of the nutrient-starved inner cells and contribute to antibiotic tolerance within the biofilm.
Subject(s)
Anti-Bacterial Agents , Biofilms , Fatty Acids , Biofilms/growth & development , Biofilms/drug effects , Fatty Acids/metabolism , Anti-Bacterial Agents/pharmacology , Microbial Viability/drug effects , Energy Metabolism , Membrane Potentials , Amino Acids/metabolismABSTRACT
Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S. Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S. Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S. Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S. Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines (IL-1ß and IL-8) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S. Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.
Subject(s)
Bacterial Outer Membrane Proteins , Epithelial Cells , Macrophages , Salmonella typhi , Typhoid Fever , Salmonella typhi/pathogenicity , Salmonella typhi/immunology , Salmonella typhi/genetics , Humans , Macrophages/microbiology , Macrophages/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Outer Membrane Proteins/immunology , Epithelial Cells/microbiology , Epithelial Cells/immunology , Typhoid Fever/immunology , Typhoid Fever/microbiology , Immunomodulation , Cytokines/metabolism , Cytokines/immunology , Microbial Viability , Interleukin-8/metabolism , Interleukin-1beta/metabolism , Interleukin-1beta/immunology , Cell LineABSTRACT
The BCG vaccines on the market have employed a Mycobacterium bovis (M. bovis) sub-strains derived from the initial strain. To date, there has been no recommendation regarding the sub-strains with the highest effectiveness when administered to humans. Because it remains the standard for Tuberculosis treatment, the quality of the BCG vaccine must be verified. The viability test is one of the parameters for BCG vaccine quality control. The culture method has become the gold standard for viability testing with various testing media. The present study aimed to evaluate the performance of Lowenstein Jensen (LJ) and Ogawa media for the viability test of Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains of M. bovis in the BCG vaccine. The number of culturable particles of each sub-strain in the BCG vaccine was estimated and statistically evaluated using the t-test. The colonies of the Pasteur 1173P2 have characteristics; tended to clump on both mediums with tiny, rough, and pale yellow/cream colors. Although the colony character of the Russian (Moscow) - 384 generally has similar feature, it did not cluster and had a smooth texture. In terms of growth rate, LJ and Ogawa media performed similarly for Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains. Maximum growth is reached by the fifth week. The culturable particles of Pasteur P1173P2 sub-strains did not differ between mediums. Whereas the growth of the Russian (Moscow) - 384 sub-strains was statistically better on Ogawa media. The results of this study reveal that the performance of the media used for determining the number of culturable particles is based on the sub-strains of M. bovis present in the BCG vaccine.
Subject(s)
BCG Vaccine , Culture Media , Mycobacterium bovis , Microbial Viability , HumansABSTRACT
Although thermosonication (TS) treatment has been widely used in food sterilization, the viable but non-culturable (VBNC) of bacteria with TS treatment has still concerned potential food safety and public health. The molecular mechanism of VBNC status of bacteria with TS treatment is not clearly known. Therefore, in this study, we used Shewanella putrefaciens, which was a common putrefactive bacteria in aquatic products, to study the VBNC state of bacteria with TS treatment. Firstly, our results revealed that S. putrefaciens still could enter the VBNC state after TS treatments: 50 kHz, 300 W, 30 min ultrasonic treatment and 70 °C heating; Subsequently, we found the VBNC state of S. putrefaciens can resist the damage of TS treatment, such as cell wall break, DNA degradation, etc; Finally, four-dimensional data-independent acquisition-based proteomics showed that under VBNC state, S. putrefaciens upregulated functional proteins to resist TS treatment, such as: ribosomal proteins to accelerate the synthesis of stress proteins to counteract TS treatments, ornithine decarboxylase SpeF and MraY to repair TS treatment-induced damage, etc. Meanwhile, S. putrefaciens downregulates metabolic and transport functional proteins such as dehydrogenase to reduce the metabolism. Importantly, among those proteins, the ribosomal transcriptional regulatory protein family, such as rpsB, etc, may be the key proteins for S. putrefaciens entering VBNC state. This finding can provide some new strategies for preventing VBNC status of bacteria with TS treatment, such as: inhibition of key proteins, etc.
Subject(s)
Shewanella putrefaciens , Shewanella putrefaciens/metabolism , Shewanella putrefaciens/physiology , Sonication , Microbial Viability , Hot Temperature , Bacterial Proteins/metabolismABSTRACT
Food crops around the world are commonly contaminated with Aspergillus flavus, which can produce the carcinogenic mycotoxin aflatoxin B1 (AFB1). The objective of this study is to test an X-ray irradiation sterilization method for studying AFB1 in contaminated maize samples in the laboratory. Maize that had been naturally contaminated with 300 ppb AFB1 by the growth of aflatoxigenic A. flavus was ground and then irradiated at 0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. A. flavus was quantified by dilution plating on potato dextrose agar (PDA) and modified Rose Bengal media (MDRB) for viability and qPCR for gene presence. AFB1 was quantified by HPLC and ELISA. A. flavus viability, but not gene copies, significantly decreased with increasing doses of radiation (PDA: p < 0.001; MDRB: p < 0.001; qPCR: p = 0.026). AFB1 concentration did not significantly change with increasing doses of radiation (HPLC: p = 0.153; ELISA: p = 0.567). Our results imply that X-ray irradiation is an effective means of reducing viable A. flavus without affecting AFB1 concentrations. Reducing the hazard of fungal spores and halting AFB1 production at the targeted dose are important steps to safely and reproducibly move forward research on the global mycotoxin challenge.