Avelino Alvarez-Ordóñez

Modifications in membrane fatty acid composition of Salmonella typhimurium in response to growth conditions and their effect on heat resistance.

The effects of growth temperature (in the range 10-45 degrees C) and acidification up to pH 4.5 of the culture medium (Brain Heart Infusion, BHI) with different organic acids (acetic, citric and lactic) and hydrochloric acid on membrane fatty acid composition and heat resistance of Salmonella typhimurium CECT 443 were studied. The heat resistance was maximal in cells grown at 45 degrees C (cells grown in non-acidified BHI showed a D58-value of 0.90 min) and decreased with decreasing growth temperature up to 10 degrees C (D58-value of 0.09 min). The growth of cells in acidified media caused an increase in their heat resistance. In general, acid adapted cells showed D-values of between 1.5 and 2 times higher than the corresponding for non-acid adapted control cells. This cross-protection response, which has important implications in food processing, was not dependent on the pH value and the acid used to acidify the growth medium. A membrane adaptation corresponding to an increase in the unsaturated to saturated fatty acids ratio (UFA/SFA) and membrane fluidity was observed at low growth temperature. Moreover, the acidification of the growth medium caused a decrease in UFA/SFA ratio and in the C18:1 relative concentration, and an increase in cyclopropane fatty acids (CFA) content mainly due to the increase in cyc19 relative concentration. Thus, acid adapted cells showed CFA levels 1.5 times higher than non-acid adapted control cells. A significant proportion of unsaturated fatty acids were converted to their cyclopropane derivatives during acid adaptation. These changes in membrane fatty acid composition result in cells with decreased membrane fluidity. A clear relation between membrane fatty acid composition and heat resistance was observed. In general, D-values were maximum for cells with low UFA/SFA ratio, and, consequently, with low membrane fluidity. Moreover, CFA formation played a major role in protecting acid adapted cells from heat inactivation. However, changes observed in membrane fatty acid composition are not enough to explain the great thermotolerance of cells grown at 45 degrees C. Thus, other mechanisms, such as the synthesis of Heat Shock Proteins, could be responsible for this increase in the bacterial heat resistance.

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria.

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.

Comparison of acids on the induction of an Acid Tolerance Response in Salmonella typhimurium, consequences for food safety

Salmonellatyphimurium inactivation at pH 3.0 in Brain Heart Infusion (BHI) and Meat Extract (ME) was studied using stationary-phase cells grown in non-acidified BHI (pH 7.4) and ME (pH 6.6) and acidified BHI and ME at pH values of 6.4, 5.4 and 4.5 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids. Cells grown in buffered BHI (pH 7.0) were used as non-acid adapted control cells. Acid adapted S. typhimurium cells obtained in both media (BHI and ME) were more resistant to extremely acidic conditions when ME was used as challenge medium, although the ability of S. typhimurium to survive extreme pH conditions also depended on growth medium and type of acidulant used. Acid adapted cells grown in BHI developed a higher Acid Tolerance Response (ATR) than those grown in ME. When cells were grown in acidified BHI, no bacterial inactivation was observed after three hours of acid challenge in ME. Furthermore, when cells were grown in acidified ME at pH values of 6.4 and 5.4, D-values obtained using ME as challenge medium were, respectively, 6-9 and 10-15 fold higher than those found when BHI was used as challenge medium. In all cases, the order of acids in inducing the ATR was citric>acetic>lactic>malic⩾hydrochloric>ascorbic. These findings represent a concern for food safety as the increase in the acid resistance of acid adapted cells could allow for S. typhimurium survival in the strong acidic environment of the gastrointestinal tract.

Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

Relationship between membrane fatty acid composition and heat resistance of acid and cold stressed Salmonella senftenberg CECT 4384.

This study evaluates the adaptative response to heat (63 degrees C) and the modifications in membrane fatty acid composition of Salmonella senftenberg after its growth in an acidified medium and after its exposure to combinations of acid and cold stresses. Cells were grown in Brain Heart Infusion (BHI) buffered at pH 7.0 and acidified up to pH 4.5 (fresh cultures) and kept at refrigeration temperature (4 degrees C) for 7 days (refrigerated cultures). The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, but combinations of sublethal stresses reduced S. senftenberg heat tolerance, specially when the growth medium pH was decreased. Acid-adapted cells showed D(63)-values ranging from 3.10 to 6.27 min, while non-acid-adapted cells showed D(63)-values of 1.07 min. As pH decreased, over the pH range studied (7.4-4.5), D(63)-values of the resulting cells increased. However, refrigerated acid-adapted cells showed lower D(63)-values, which ranged from 0.95 to 0.49 min. A linear relationship between the thermotolerance of S. senftenberg cells and the previous growth medium pH was found in both fresh and refrigerated cultures, which allowed us to predict changes in heat resistance of S. senftenberg that occur at any pH value within the range used in the present study in which most foodstuffs are included. Both acidification of the growth medium and refrigeration storage of cells induced modifications in membrane fatty acid composition, which were clearly linked to their heat resistance. Acid-adapted cells, regardless of the pH value of the growth medium, showed the lowest UFA/SFA ratio and a CFA content 1.5-2-fold higher than that observed for non-acid-adapted cells. On the other hand, the UFA/SFA ratio found for S. senftenberg cells exposed to a cold stress was 1.2-1.8-fold higher than that observed for non-refrigerated cultures. This increase in the UFA/SFA ratio was specially high for acid-adapted cells. The highest thermotolerance was observed for cells with low UFA/SFA ratio, and, consequently, having a low membrane fluidity. However, changes observed in CFA content did not explain the great heat sensitivity of refrigerated acid-adapted cells.

Salmonella spp. survival strategies within the host gastrointestinal tract

Human salmonellosis infections are usually acquired via the food chain as a result of the ability of Salmonella serovars to colonize and persist within the gastrointestinal tract of their hosts. In addition, after food ingestion and in order to cause foodborne disease in humans, Salmonella must be able to resist several deleterious stress conditions which are part of the host defence against infections. This review gives an overview of the main defensive mechanisms involved in the Salmonella response to the extreme acid conditions of the stomach, and the elevated concentrations of bile salts, osmolytes and commensal bacterial metabolites, and the low oxygen tension conditions of the mammalian and avian gastrointestinal tracts.

Monitoring occurrence and persistence of Listeria monocytogenes in foods and food processing environments in the Republic of Ireland.

Although rates of listeriosis are low in comparison to other foodborne pathogenic illness, listeriosis poses a significant risk to human health as the invasive form can have a mortality rate as high as 30%. Food processors, especially those who produce ready-to-eat (RTE) products, need to be vigilant against Listeria monocytogenes, the causative pathogen of listeriosis, and as such, the occurrence of L. monocytogenes in food and in the food processing environment needs to be carefully monitored. To examine the prevalence and patterns of contamination in food processing facilities in Ireland, 48 food processors submitted 8 samples every 2 months from March 2013 to March 2014 to be analyzed for L. monocytogenes. No positive samples were detected at 38% of the processing facilities tested. Isolates found at the remaining 62% of facilities were characterized by serotyping and Pulsed Field Gel Electrophoresis (PFGE). A general L. monocytogenes prevalence of 4.6% was seen in all samples analyzed with similar rates seen in food and environmental samples. Differences in prevalence were seen across different food processors, food sectors, sampling months etc. and PFGE analysis allowed for the examination of contamination patterns and for the identification of several persistent strains. Seven of the food processing facilities tested showed contamination with persistent strains and evidence of bacterial transfer from the processing environment to food (the same pulsotype found in both) was seen in four of the food processing facilities tested.

Acid tolerance in Salmonella typhimurium induced by culturing in the presence of organic acids at different growth temperatures

The influence of growth temperature and acidification of the culture medium up to pH 4.25 with acetic, citric, lactic and hydrochloric acids on the growth and subsequent acid resistance at pH 3.0 of Salmonella typhimurium CECT 443 was studied. The minimum pH value which allowed for S. typhimurium growth within the temperature range of 25-37 degrees C was 4.5 when the pH was reduced using citric and hydrochloric acids, and 5.4 and 6.4 when lactic acid and acetic acid were used, respectively. At high (45 degrees C) or low (10 degrees C) temperatures, the growth pH boundary was increased about 1 pH unit. The growth temperature markedly modified the acid resistance of the resulting cells. In all cases, D-values were lower for cells grown at 10 degrees C and significantly increased with increasing growth temperature up to 37 degrees C, at which D-values obtained were up to 10 times higher. Cells grown at 45 degrees C showed D-values similar to those found for cells grown at 25 degrees C. The growth of cells in acidified media, regardless of the pH value, caused an increase in their acid resistance at the four incubation temperatures, although the magnitude of the Acid Tolerance Response (ATR) observed depended on the growth temperature. Acid adapted cultures at 10 degrees C showed D-values ranging from 5.75 to 6.91 min, which turned out to be about 2 times higher than those corresponding to non-acid adapted cultures, while higher temperatures induced an increase in D-values of at least 3.5 times. Another finding was that, while at 10 and 45 degrees C no significant differences among the effect of the different acids tested in inducing an ATR were observed, when cells were grown at 25 and 37 degrees C citric acid generally turned out to be the acid which induced the strongest ATR. Results obtained in this study show that growth temperature is an important factor affecting S. typhimurium acid resistance and could contribute to find new strategies based on intelligent combinations of hurdles, which could prevent the development or survival of Salmonella spp. in foods. The fact that moderately low temperatures (10 degrees C) markedly decrease the acid resistance and increase the growth pH boundary of S. typhimurium suggests the convenience to control the temperature during food processing as a strategy to prevent the growth and survival of this pathogenic microorganism.

Changes in Fourier transform infrared spectra of Salmonella enterica serovars Typhimurium and Enteritidis after adaptation to stressful growth conditions.

The effects of growth conditions (temperature in the range 10-45 degrees C, sodium chloride concentration in the range 0-4%, aerobic vs. anaerobic growth and acidification of the growth medium, up to pH 4.5) on the Fourier transform infrared (FT-IR) spectra of Salmonella enterica serovars Typhimurium and Enteritidis were studied using multivariate statistical methods (Hierarchical Cluster Analysis and Factor Analysis). Although all environmental factors tested affected S. Typhimurium and S. Enteritidis FT-IR spectra to some extent, growth temperature was the most influential factor within the five spectral regions. The w(4) spectral region (1200 to 900 cm(-1)) was the most variable region, suggesting that S. Typhimurium and S. Enteritidis modulate their cell wall and cell membrane composition in response to shifts in growth temperature. Changes in membrane fluidity were determined by monitoring the vibrational modes of the acyl chain v(s)CH(2) symmetric stretching band by FT-IR spectroscopy. For cells grown in unsupplemented media an increase in growth temperature was linked to a decrease in membrane fluidity. Even though the effect of NaCl concentration, pH and atmosphere was considered of less importance, cells grown in acidified media also showed a reduction in their membrane fluidity, and the addition of sodium chloride to the culture medium was associated with an increase in the bacterial membrane fluidity. These findings can help interpret how important adaptive mechanisms for the survival of pathogenic bacteria in foods are, and show that FT-IR spectroscopy is a useful tool to understand how environmental conditions mimicking those in certain food products affect the cell. Also, FT-IR can be used to perform a rapid discrimination between bacterial phenotypes with different adaptive tolerance responses to environmental stress.

Swine dysentery: aetiology, pathogenicity, determinants of transmission and the fight against the disease.

Swine Dysentery (SD) is a severe mucohaemorhagic enteric disease of pigs caused by Brachyspira hyodysenteriae, which has a large impact on pig production and causes important losses due to mortality and sub-optimal performance. Although B. hyodysenteriae has been traditionally considered a pathogen mainly transmitted by direct contact, through the introduction of subclinically infected animals into a previously uninfected herd, recent findings position B. hyodysenteriae as a potential threat for indirect transmission between farms. This article summarizes the knowledge available on the etiological agent of SD and its virulence traits, and reviews the determinants of SD transmission. The between-herds and within-herd transmission routes are addressed. The factors affecting disease transmission are thoroughly discussed, i.e., environmental survival of the pathogen, husbandry factors (production system, production stage, farm management), role of vectors, diet influence and interaction of the microorganism with gut microbiota. Finally, prophylactic and therapeutic approaches to fight against the disease are briefly described.