Bacteria In Exponential Phase Research Articles

Bioleaching of the α-alumina layer of spent three-way catalysts as a pretreatment for the recovery of platinum group metals

Acid bioleaching of Al by Acidithiobacillus thiooxidans has been explored as an environmentally friendly pretreatment to facilitate the extraction of platinum group metals from spent three-way catalysts (TWC). Biogenic sulfur obtained from desulfurization bioreactors improved the production of acid by A. thiooxidans compared to commercially available elemental sulfur. The lixiviation abilities of bacteria-free biogenic acid and biogenic acid with exponential or stationary phase bacteria were compared against a control batch produced by commercial H2SO4. The maximum Al leaching percentage (54.5%) was achieved using biogenic acids with stationary-phase bacteria at a TWC pulp density of 5% w/v whereas bacteria-free biogenic acid (23.4%), biogenic acid with exponential phase bacteria (21.7%) and commercial H2SO4 (24.7%) showed lower leaching abilities. The effect of different pulp densities of ground TWC (5, 30, and 60% w/v) on Al leaching and bacterial growth was determined. While greater Al leaching yields were obtained at lower TWC pulp density solutions (54.5% at 5% w/v and 2.5% at 60% w/v), higher pulp densities enhanced microbial growth (2.3 × 109 cells/mL at 5% w/v and 9.5 × 1010 cells/mL at 60% w/v). The dissolution of the metal from the solid into the liquid phase triggered the production of biological polymeric substances that were able to absorb traces of both Al (up to 24.80% at 5% w/v) and Pt (up to 0.40% at 60% w/v).

Comparison of bacteria in different metabolic states by micro-Raman spectroscopy

It was shown that several metabolic states of bacteria with various characteristics such as chemical composition participate in the formation of biofilms. To study the connections and differences among different bacterial metabolic states, five species of bacteria in exponential phase, stationary phase and biofilm have been compared and investigated by micro-Raman spectroscopy. The spectral differences between different metabolic states showed that the chemical composition varied among those metabolic states. Moreover, as can be shown by the spectral differences and principal components (PCs), different species and strains of bacteria behave differently. Furthermore, a principal component analysis (PCA) combined with support vector machines (SVM) was applied to distinguish species of bacteria within the same metabolic states. Our study provides valuable data for the comparison of bacteria between different metabolic states utilizing micro-Raman spectroscopy in combination with chemometrics models.

Cysteine Potentiates Bactericidal Antibiotics Activity Against Gram-Negative Bacterial Persisters.

PurposeBacterial metabolism regulators offer a novel productive strategy in the eradication of antibiotic refractory bacteria, particularly bacterial persisters. However, the potential of amino acids in the fight against Gram-negative bacterial persisters has not been fully explored. The aim of this study is to investigate the potentiation of amino acids to antibiotics in combating Gram-negative bacterial persisters and to reveal the underlying mechanisms of action.MethodsBactericidal activity of antibiotics in the absence or presence of amino acids was evaluated through detecting the reduction of bacterial CFUs. The ratio of NAD+/NADH in E. coli B2 persisters was determined using assay kit with WST-8. Bacterial respiration and ROS production were measured by the reduction of iodonitrotetrazolium chloride and fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate, respectively.ResultsIn this study, we found that cysteine possesses excellent synergistic bactericidal activity with ciprofloxacin against multiple Gram-negative bacterial persisters. Furthermore, the potentiation of cysteine was evaluated in exponential and stationary-phase E. coli ATCC 25922 and E. coli B2. Interestingly, cysteine significantly improves three bactericidal antibiotics killing against stationary-phase bacteria, but not exponential-phase bacteria, implying that the effect of cysteine correlates with the metabolic state of bacteria. Mechanistic studies revealed that cysteine accelerates the bacterial TCA cycle and promotes bacterial respiration and ROS production. These metabolic regulation effects of cysteine re-sensitive bacterial persisters to antibiotic killing.ConclusionCollectively, our study highlights the synergistic bactericidal activity of bacterial metabolism regulators such as cysteine with commonly used antibiotics against Gram-negative bacterial persisters.

Cell wall hydrolase as a surface-associated protein target for the specific detection of Lactobacillus rhamnosus using flow cytometry

Abstract Flow cytometry (FC) is a very popular technique for to the study of different eukaryotic cell phenotypes. In addition, FC has many applications in the field of food microbiology, although the few number of specific molecules that can be potentially targeted with fluorescence-conjugated antibodies limits its potential applications. The present work represents a step forward in the application of FC for detection of specific foodborne or probiotic bacteria by using fluorescent antibodies binding specific surface-associated protein. To illustrate this novel approach, we have used a monoclonal and polyclonal antibodies targeting cell-wall hydrolase (CWH), one of the major surface-associated proteins of the probiotic Lactobacillus rhamnosus GG. In order to verify the specific union of the antibodies to this surface protein, ten different bacterial strains belonging to different bacteria species taxonomically related and no related to L. rhamnosus, were tested. The optimal conditions for bacteria detection corresponded to a final suspension of 5E107 bacteria in exponential phase of growth using FC buffer, without freezing, without adding extra BSA to the FC buffer and without fixing bacteria. Our results showed that GG strain was detected using the polyclonal anti-CWH serum in a higher extent (91.62%) than the rest of strains (L. casei 393: 64.49%; L. amylovorus: 46.13%, E. coli: 45.13%; L. acidophilus DSM20079T: 35.83%; B. longum NCIMB8809: 33.68%; L. gasseri BM7/10: 32.41%; L. reuteri DSM21016: 29.26%; L. plantarum NCIMB8826: 26.33% and L. delbruekii: 22.52%). The percentage of L. rhamnosus labeled with the monoclonal antibody was 53.06, which contrasted with the 91.62% labeled with the polyclonal antibody. The low affinity of the monoclonal antibody was perhaps due to the lack of immunogenicity of the single epitope chosen for antibody generation (CWHp). The high efficiency observed for the polyclonal anti-CWH serum is maybe a consequence of being constituted by a mixture of antibodies that recognizes different epitopes in the L. rhamnosus GG protein. With this proof of concept, we have shown for the first time that foodborne or probiotic bacteria can be specifically detected using antibodies targeting adequate molecules present on their surface.

Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 μM and 200 μM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 μM, and a respective minimum bactericidal concentration (MBC) of 12.5 μM and 25 μM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 μM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 μM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 μM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 μM EPA. For exponential-phase bacteria, 100 μM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 μM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention and treatment of periodontal diseases.

Antimicrobial Effect of Photosensitized Rose Bengal on Bacteria and Viruses in Model Wash Water

Peroxides and chlorite-based sanitizers are commonly used for washing procedures. The efficacy of these sanitizers is significantly reduced in the presence of organic content and may result in the formation of harmful secondary products. Therefore, new food-grade sanitizers with enhanced antimicrobial efficacy are needed. In this work, we tested a visible light-activated photosensitizer, Rose Bengal (RB), for the reduction of microbial load in simulated wash water. Escherichia coli BL21 and bacteriophage T7 were selected as model bacterial and viral targets, respectively. Effects of duration of illumination, growth phase of the bacterium, and the presence of organic matter on efficacy of inactivation were evaluated. Photosensitized RB was able to achieve 6 log colony-forming units (CFU)/mL reduction in the exponential-phase bacteria in the presence of high organic content (2000 ppm LB broth) within 45 min of treatment. The results also demonstrated that the stationary-phase microbes were significantly more resistant to photoinactivation as compared to the exponential-phase microbes at both low and high organic loads (200 and 2000 ppm). The results indicate that RB dye can be internalized in bacteria and induces damage to the cell membrane upon photoactivation. Viral inactivation studies demonstrated that photoactivation of RB can achieve 5 log PFU/mL reduction in viral load in the presence of 2000 ppm LB after 30 min of treatment. Overall, these results highlight the efficacy of RB as an antimicrobial against bacteria and viruses in simulated wash water and demonstrate its potential as an alternative sanitizer for the food industry.

Sigma S-dependent antioxidant defense protects stationary-phase Escherichia coli against the bactericidal antibiotic gentamicin.

Stationary-phase bacteria are important in disease. The σ(s)-regulated general stress response helps them become resistant to disinfectants, but the role of σ(s) in bacterial antibiotic resistance has not been elucidated. Loss of σ(s) rendered stationary-phase Escherichia coli more sensitive to the bactericidal antibiotic gentamicin (Gm), and proteomic analysis suggested involvement of a weakened antioxidant defense. Use of the psfiA genetic reporter, 3'-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that Gm generated more reactive oxygen species (ROS) in the mutant. HPF measurements can be distorted by cell elongation, but Gm did not affect stationary-phase cell dimensions. Coadministration of the antioxidant N-acetyl cysteine (NAC) decreased drug lethality particularly in the mutant, as did Gm treatment under anaerobic conditions that prevent ROS formation. Greater oxidative stress, due to insufficient quenching of endogenous ROS and/or respiration-linked electron leakage, therefore contributed to the greater sensitivity of the mutant; infection by a uropathogenic strain in mice showed this to be the case also in vivo. Disruption of antioxidant defense by eliminating the quencher proteins, SodA/SodB and KatE/SodA, or the pentose phosphate pathway proteins, Zwf/Gnd and TalA, which provide NADPH for ROS decomposition, also generated greater oxidative stress and killing by Gm. Thus, besides its established mode of action, Gm also kills stationary-phase bacteria by generating oxidative stress, and targeting the antioxidant defense of E. coli can enhance its efficacy. Relevant aspects of the current controversy on the role of ROS in killing by bactericidal drugs of exponential-phase bacteria, which represent a different physiological state, are discussed.

Regulated Shift from Helical to Polar Localization of Listeria monocytogenesCell Wall-Anchored Proteins

Many virulence factors of Gram-positive bacterial pathogens are covalently anchored to the peptidoglycan (PG) by sortase enzymes. However, for rod-shaped bacteria little is known about the spatiotemporal organization of these surface proteins in the cell wall. Here we report the three-dimensional (3D) localization of the PG-bound virulence factors InlA, InlH, InlJ, and SvpA in the envelope of Listeria monocytogenes under different growth conditions. We found that all PG-anchored proteins are positioned along the lateral cell wall in nonoverlapping helices. However, these surface proteins can also become localized at the pole and asymmetrically distributed when specific regulatory pathways are activated. InlA and InlJ are enriched at poles when expressed at high levels in exponential-phase bacteria. InlA and InlH, which are σ(B)dependent, specifically relocalize to the septal cell wall and subsequently to the new pole in cells entering stationary phase. The accumulation of InlA and InlH in the septal region also occurs when oxidative stress impairs bacterial growth. In contrast, the iron-dependent protein SvpA is present at the old pole and is excluded from the septum and new pole of bacteria grown under low-iron conditions. We conclude that L. monocytogenes rapidly reorganizes the spatial localization of its PG proteins in response to changes in environmental conditions such as nutrient deprivation or other stresses. This dynamic control would distribute virulence factors at specific sites during the infectious process.

Effect of Heat Exposure on Invasiveness of Listeria monocytogenes Strains

The effect of a 54°C heat exposure lasting 20 and 60 minutes on the invasiveness of 10 Listeria monocytogenes strains in the exponential and stationary phases was investigated. It was shown that heat exposure significantly reduced the L. monocytogenes count. A maximal reduction of 5 log took place after 60 minutes of heating. No significant differences in survival between exponential and stationary phases were observed for most strains. Twenty minutes of heat exposure resulted in a reduction of the invasiveness of L. monocytogenes, which was more pronounced in the exponential-phase bacteria. Prolongation of heating to 60 minutes was shown to have a significant impact on the invasiveness of five stationary-phase strains. It was demonstrated that heat exposure influences the survival of L. monocytogenes strains as expected. Also, the invasiveness was significantly changed in a time- and growth phase-dependent manner. Low reductions of bacterial counts in milder conditions, that is, 20 minutes of heat, were accompanied by a decrease of the invasiveness of all L. monocytogenes strains. Prolongation of heating time to 60 minutes resulted in significant reduction of bacterial numbers. However, bacteria, especially those in the stationary phase, which survived this treatment can become more invasive.

Antimicrobial mechanism of pore-forming protegrin peptides: 100 pores to kill E. coli

Antimicrobial peptides (AMPs), important effector molecules of the innate immune system, also provide templates for designing novel antibiotics. Protegrin, an especially potent AMP found in porcine leukocytes, was recently shown to form octameric transmembrane pores. We have employed a combination of experiments and models spanning length scales from the atomistic to the cellular level in order to elucidate the microbicidal mechanism of protegrin. Comparison of the modeling and experimental data suggests that approximately 10–100 protegrin pores are necessary to explain the observed rates of potassium leakage and Escherichia coli death in exponential-phase bacteria. The kinetics of viability loss suggest that bacterial death results largely from uncontrolled ion exchange processes and decay of transmembrane potential. However, ion exchange processes alone cannot account for the experimentally observed cell swelling and osmotic lysis—a redundant “overkill” mechanism most likely to occur in locales with high protegrin concentrations. Although our study is limited to protegrin and E. coli, the timeline of events described herein is likely shared by other AMPs that act primarily by permeabilizing microbial membranes. This work provides many of the missing links in describing antimicrobial action, as well as providing a quantitative connection between several previous experimental and simulation studies of protegrin.

Factors Affecting Preference Responses of the Freshwater Ciliate Uronema nigricans to Bacterial Prey

To enhance our understanding of the factors affecting feeding selectivity of bacterivorous protists in aquatic systems, we examined the preference responses of the freshwater ciliate Uronema nigricans towards three bacterial prey taxa, Pseudomonas luteola, Serratia rubidaea, and Aeromonas hydrophila. Potential factors influencing the predator-prey contact rate included the previous feeding history of the ciliate and physiological state of bacteria. Preference indexes were obtained from multiple-choice mazes in which ciliates moved preferentially towards alternative bacteria or the prey species on which they had been feeding. Uronema nigricans showed differential attraction towards the offered prey types, and these preferences varied as a function of the ciliate feeding history: U. nigricans growing on P. luteola showed lower preference responses towards the offered bacteria than U. nigricans growing on S. rubidaea. The bacteria in stationary phase elicited a higher degree of attraction than bacteria in exponential phase, probably due to a higher concentration of carbohydrates in the former. Therefore, this protist will preferentially swim towards bacteria in stationary growth phase, although the degree of this response will be affected by the recent feeding history of the ciliate.

Inactivation of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages during maturation/storage

The purpose of this study was to evaluate maturation and storage conditions as a way to increase the safety of non-heat treated fermented sausages. The specific objectives were to investigate the effects of storage time and temperature on the levels of Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica in fermented sausages and in broth, and to validate how well the broth experiments and some published models can predict inactivation in sausage. One strain each of E. coli, L. monocytogenes and Y. enterocolitica with induced acid tolerance was inoculated into sausage batters representing a typical Swedish recipe for cold-smoked sausages. The sausages were fermented at 27 °C for 39 or 48 h and then stored at different temperatures (8, 15, or 20–22 °C) for up to 44 days. The levels of the experimental strains, lactic acid bacteria, and pH, a w, and lactic acid was measured during the maturation/storage period. Inactivation in BHI broths adjusted to pH 4.4 or 4.6, water activity of 0.93, and with 1, 1.3 or 2% lactic acid added was also studied. For all strains inactivation rates increased with temperature in both broths and sausages. At 8 °C the storage time required for a one-log reduction in sausage ranged from 21 days for E. coli, > 16 days for L. monocytogenes, to 18 days for Y. enterocolitica. At temperatures of 20 °C or more, the storage time needed for a one log reduction was shorter: between 7 to 11 days for E. coli, 4 to 7 days for L. monocytogenes, and 1 to 4 days for Y. enterocolitica. A published model based on temperature only yielded a good prediction of E. coli inactivation in sausage. A linear model based on the rate estimated in broth yielded a fair prediction of L. monocytogenes inactivation. The performance of other inactivation models validated was unsatisfactory. Significant E. coli growth which occurred in batters without salt during initial phases of fermentation resulted in a subsequent increased inactivation rate, possibly due to increased susceptibility to stress of exponential phase bacteria. The results indicate that the practice of utilising a short maturation period and storage at refrigeration temperatures may result in unsatisfactory reductions of pathogens if present. Thus, inclusion of a maturation period above refrigeration temperatures before distribution may increase the safety of these products.

Contributions of Listeria monocytogenes σ B and PrfA to expression of virulence and stress response genes during extra- and intracellular growth

Listeria monocytogenes sigmaB and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of sigmaB- and PrfA-dependent genes in exponential-phase L. monocytogenes wild-type and DeltasigB strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed L. monocytogenes showed sigmaB-dependent increases in opuCA (10- and 17-fold higher, respectively) and gadA transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by plcA transcript levels, was up to 95-fold higher in intracellular L. monocytogenes as compared to non-stressed bacteria, sigmaB activity was only slightly higher in intracellular than in non-stressed bacteria. Increased plcA transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both prfA expression and PrfA activity. qRT-PCR assays were designed to measure expression of prfA from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream plcA promoter was very low. The relative contribution to total prfA transcription from the sigmaA-dependent P1prfA promoter ranged from approximately 17 % to 30 %, while the contribution of the P2prfA region, which appears to be transcribed by both sigmaA and sigmaB, ranged from approximately 70 % to 82 % of total prfA transcript levels. In summary (i) sigmaB is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular L. monocytogenes and (ii) the partially sigmaB-dependent P2prfA promoter region contributes the majority of prfA transcripts in both intra- and extracellular bacteria.

Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.

Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied. To investigate the effect of viable and nonviable cells of Propionibacterium acnes in both exponential and stationary growth phases, and of P. acnes GroEL on cytokine production by human primary keratinocytes. Actively proliferating or contact-inhibited keratinocytes were cocultured with viable or formaldehyde-killed P. acnes cells in either the exponential or stationary phase of growth. Culture supernatants were assayed by enzyme-linked immunosorbent assay for the cytokines interleukin (IL)-1alpha, tumour necrosis factor (TNF)-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF). Keratinocytes were also stimulated with different concentrations of P. acnes GroEL and supernatants assayed for cytokines. Viable P. acnes in the stationary phase of growth stimulated keratinocyte monolayers to produce significantly higher amounts of IL-1alpha, TNF-alpha and GM-CSF than unstimulated keratinocytes. Viable exponential-phase bacteria stimulated production of significantly higher amounts of TNF-alpha and GM-CSF but these levels were significantly lower than those for stimulation with stationary-phase bacteria. Nonviable P. acnes from either growth phase was not able to stimulate cytokine production. P. acnes GroEL at concentrations in the range 0.05-1.0 micro g mL(-1) was able to induce increased production of cytokines by keratinocytes in a dose-dependent manner. This was analogous to stimulation with Escherichia coli GroEL. Stimulation of cytokine production by P. acnes and P. acnes GroEL may be important in the pathogenesis of inflammatory acne vulgaris and may have wider implications for the immunomodulation of the human immune system by commensal skin microorganisms.

Temperature and growth phase regulate the transcription of the O-antigen gene cluster of Yersinia enterocolitica O:3.

Temperature seems to regulate both O-antigen gene cluster operons of Y. enterocolitica O:3 mainly during the stationary phase of growth. In exponential phase bacteria both operons are actively transcribed independent of temperature since temperature shift during exponential phase did not have signifigant effect on the luciferase activity. Dilution of stationary phase bacteria grown at RT decreases the light production very rapidly and dramatically. Our results suggest that in stationary phase Y. enterocolitica O:3 bacteria the O-antigen expression is controlled by a regulatory circuit which may include an activator at RT and/or a represser at 37°C. Further work is needed to elucidate regulatory mechanisms.

Mycobacterium avium resists exposure to the acidic conditions of the stomach.

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistance.

Mycobacterium avium resists exposure to the acidic conditions of the stomach

Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS. There is evidence that in AIDS patients, the main route for M. avium infection is the gastrointestinal tract. The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens. Incubation of three clinical isolates of M. avium under acidic pH revealed resistance of M. avium grown both to the exponential and stationary phase at pH 2.2 for 2 h. Inhibition of protein synthesis had no effect on the acid tolerance. When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria. M. avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M. avium in the presence of buffer. Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2. Isoosmolarity of Hank’s balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation. These findings indicate that M. avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M. avium is found in the environment results in increased acid resistance.

Thermoprotection by glycine betaine and choline.

Glycine betaine is mostly known as an osmoprotectant. It is involved in the osmotic adaptation of eukaryotic and bacterial cells, and accumulates up to 1 M inside cells subjected to an osmotic upshock. Since, like other osmolytes, it can act as a protein stabilizer, its thermoprotectant properties were investigated. In vitro, like protein chaperones such as DnaK, glycine betaine and choline protect citrate synthase against thermodenaturation, and stimulate its renaturation after urea denaturation. In vivo, the internal concentration of glycine betaine is neither increased nor decreased after heat shock (this contrasts with a massive increase after osmotic upshock). However, even in exponential-phase bacteria grown in usual minimal salts media, the internal glycine betaine concentration attains levels (around 50 mM) which can protect proteins against thermodenaturation in vitro. Furthermore, glycine betaine and choline restore the viability of a dnaK deletion mutant at 42 degrees C, suggesting that glycine betaine not only acts as a thermoprotectant in vitro, but also acts as a thermoprotectant for Escherichia coli cells in vivo.