Effects of Acid Adaptation of Escherichia coli O157:H7 on Efficacy of Acetic Acid Spray Washes To Decontaminate Beef Carcass Tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4°C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

Quantification of the Relative Effects of Temperature,pH, and Water Activity on Inactivation of Escherichia coli in Fermented Meat by Meta-Analysis

Outbreaks of Escherichia coli infections linked to fermented meats have prompted much research into the kinetics of E. coli inactivation during fermented meat manufacture. A meta-analysis of data from 44 independent studies was undertaken that allowed the relative influences of pH, water activity (a_w), and temperature on E. coli survival during fermented meat processing to be investigated. Data were reevaluated to determine rates of inactivation, providing 484 rate data points with various pH (2.8 to 6.14), a_w (0.75 to 0.986), and temperature (−20 to 66°C) values, product formulations, and E. coli strains and serotypes. When the data were presented as an Arrhenius model, temperature (0 to 47°C) accounted for 61% of the variance in the ln(inactivation rate) data. In contrast, the pH or a_w measured accounted for less than 8% of variability in the data, and the effects of other pH- and a_w-based variables (i.e., total decrease and rates of reduction of those factors) were largely dependent on the temperature of the process. These findings indicate that although temperatures typically used in fermented meat manufacture are not lethal to E. coli per se, when other factors prevent E. coli growth (e.g., low pH and a_w), the rate of inactivation of E. coli is dominated by temperature. In contrast, inactivation rates at temperatures above ∼50°C were characterized by smaller z values than those at 0 to 47°C, suggesting that the mechanisms of inactivation are different in these temperature ranges. The Arrhenius model developed can be used to improve product safety by quantifying the effects of changes in temperature and/or time on E. coli inactivation during fermented meat manufacture.Fermented meats encompass a diverse range of product styles in which raw, ground meat is preserved by the processes of fermentation and drying (or maturation). These products are typically manufactured without a bactericidal heat treatment, and instead, inhibition of growth and inactivation of contaminating pathogens rely upon the collective effects of acid pH, reduced water activity (a_w), and the presence of lactic acid and, potentially, curing salts (nitrate and/or nitrite) and spices. However, pathogenic Escherichia coli can contaminate and survive in fermented meat products at levels sufficient to cause serious illness in consumers, as evidenced by numerous outbreaks of E. coli infections epidemiologically linked to uncooked fermented meat products (8, 9, 54, 64). Knowledge of the kinetics of nonthermal inactivation of E. coli, and the factors affecting it, is important to be able to optimize the safety of fermented meat processes.Several research groups have conducted studies on the survival of pathogenic E. coli during the processing of specific fermented meat products (4, 6, 14, 16, 23, 25). In some cases, the effects of alternative ingredients or processing parameters have also been determined (12, 21, 24, 30). Such investigations have allowed the lethality of a specific fermented meat process to be determined and the microbiological safety of that product, in regards to E. coli, to be described. However, most of these studies have been essentially empirical and product/process specific, i.e., they have not sought to discern key variables or their interactions that influence the extent of inactivation of E. coli or other pathogens during meat fermentation and maturation. Thus, while they are very important and useful, it has been difficult to extrapolate the results of those studies to different fermented meat processes, confounding efforts to assess product safety without the requirement for challenge tests or to give manufacturers the confidence to develop new or modified processes that remain safe.In an attempt to identify the main factors that influence the inactivation of E. coli in fermented meat products, we utilized data that already existed in the scientific literature and reassessed that information via a process similar to meta-analysis. Meta-analysis is a statistical technique that involves amalgamating, summarizing, and reviewing previous quantitative research to identify trends. It is used, albeit rarely in the area of microbiology, as a means to address a wide variety of questions where a reasonable body of primary research studies exists. In a preliminary investigation, based on an analysis of limited published and other data, Ross and Shadbolt (51) observed that inactivation of E. coli in fermented meat processes was dominated by temperature and that pH and a_w levels appeared to be less influential, except insofar as creating conditions inimical to E. coli growth. The objective of the present investigation was to rigorously test that observation by collation and analysis of a large data set describing the inactivation of E. coli during manufacture of fermented meats and to attempt to identify underlying patterns in the responses of E. coli to conditions encountered during fermented meat processes. From the observations of Ross and Shadbolt (51), and because the inactivation of E. coli in fermented meat is not instantaneous, we based our analyses on the rate of E. coli inactivation, calculated from viable count data and processing times reported by a variety of published and unpublished sources. We sought to relate the inactivation rate to reported environmental conditions, including pH, a_w, and temperature, and to summarize the observations in a predictive mathematical model.     

Salt, Alone or in Combination with Sucrose, Can Improve the Survival of Escherichia coli O157 (SERL 2) in Model Acidic Sauces

The commercial production of microbiologically safe and stable sauces containing acetic acid is guided by the Comité des Industries des Mayonnaises et Sauces Condimentaires de la Communauté Économique Européenne's (CIMSCEE) code. The CIMSCEE safety value is calculated using a linear regression equation combining weighted contributions of pH and aqueous-phase concentrations of undissociated acetic acid, NaCl, and sugars. By implication, the CIMSCEE safety equation predicts that increasing concentrations of hurdles will always increase inactivation of the target pathogen. In this study, the time to achieve a 3-log₁₀ reduction of an acid-resistant, acid-adapted, Shiga toxin-producing Escherichia coli (STEC) O157 isolate was determined experimentally for 81 formulations at various pHs and acetic acid, NaCl, and sucrose concentrations in a broth model. The combinations were intended to simulate the aqueous phase of acidic sauces and dressings. Experimental data were fitted to the log logistic model to estimate the time to 3-log₁₀ reduction (t_3D). Comparison of fitted t_3D estimates with CIMSCEE values showed agreement in predicting safety (as defined by CIMSCEE) for the majority of formulations. However, CIMSCEE safety predictions were “fail dangerous” for 13 of 81 formulations. Among these formulations and others, the observed E. coli t_3D initially increased and then decreased with increasing osmolalities (NaCl and sucrose). Relative protection increased with exposure time where the protective effect of NaCl predominated. While commercial acidic sauces are not considered high-risk vehicles for STEC, interactions among hurdles that decrease their combined effectiveness are deserving of further investigation because they may reveal mechanisms of broader relevance in the inactivation of pathogens in foods.     

Effect of Three Factors in Cheese Production (pH, Salt, and Heat) on Mycobacterium avium subsp. paratuberculosis Viability

Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20°C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log₁₀ concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10³ cells of M. avium subsp. paratuberculosis per ml.     

Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.The use of selected starter cultures (SSC) in fermented food production is widely applied throughout the food industry, in particular for wine, dairy products, sausages, and a variety of vegetables (3, 11). The advantages of their use are related to the improvement of the process control, hygiene, and quality with respect to fermented foods obtained through indigenous fermentation. Vinegar is one of the fermented beverages produced without SSC inoculation, in both small- and large-scale production, mainly for the following reasons: (i) the majority of vinegars have low commercial value, and often technological innovation is not considered profitable, and (ii) there is limited knowledge of the ecophysiology of acetic acid bacteria (AAB) due to the difficulty in accessing, sampling, isolating, and preserving strains (2, 12, 15, 16, 17). Among vinegars, traditional balsamic vinegar (TBV) is an Italian aged condiment produced by “seed vinegar,” the so-called “mother of vinegar” that is an indigenous starter culture withdrawn from acetifying vinegar through back-slopping procedures. The raw material is a fermented and cooked grape must (here indicated as must) at a soluble solids content ranging from 20 to 60°Bx (10). TBV production is regulated by denomination of protected origin guidelines that specify procedures and final product features. In particular, the raw material characteristics, the production process (e.g., must cooking, alcoholic fermentation, acetic oxidation, and ageing), features of the production area (no environmental condition management is permitted), and analytical and sensorial parameters are stated as follows: acidity (not less than 4.5% [wt/wt], expressed as grams of acetic acid per 100 g of product), density at 20°C (not less than 1.240 g per liter), color, aroma, and taste. The production is performed in wood barrels, and the process is carried out by sequential refilling to acetify the must and replace the volume lost by evaporation. AAB grow on the surface of liquid by biofilm formation. No addition of any substance can be made except for the acetifying must as a starter (7). Microbial studies of TBV reported culture-dependent and -independent approaches to evaluating AAB occurrence in TBV musts (5, 10). These studies highlighted the occurrence of Gluconacetobacter europaeus as a widespread indigenous species, as well as Acetobacter pasteurianus, Acetobacter aceti, and Acetobacter malorum. However, no comprehensive studies of AAB diversity and the correlation between species occurrence and technological steps of TBV production have been published, due mainly to the difficulty of easy access to AAB microflora in vinegar matrix by both culture-dependent and -independent approaches.Regarding production technology, at least one drawback of current production procedures has been acknowledged. It concerns the difficulty of start-up acetification, which affects the minimum acidity value required for the final product. In fact, some studies showed that many variables regulate AAB growth and activity. Above all is the sugar concentration among substrates and the temperature among physical parameters. To efficiently control the acetification start-up, it is necessary to understand the function of AAB responsible for the initial colonization of musts and to investigate the microbial succession suitable to complete the acetification. Our previous researches on TBV showed that AAB strains exhibit different growing abilities. In particular, strains of Acetobacter pasteurianus grow quickly on laboratory synthetic media, wine, and cooked must. In contrast, strains belonging to G. europaeus do not grow or grow very slowly on cooked and fermented must (9, 10).The goal of this study was to implement a laboratory SSC to test it on a factory scale for TBV production purposes. In particular, we focused our attention on the effect of A. pasteurianus strain AB0220 on the acetification and dynamics of species at the end of the process. The SSC effectiveness was assessed by monitoring analytical parameters (acetic acid, ethanol, and pH), species succession, and strain persistence during three stages by the following molecular analyses: PCR/denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus (ERIC)/PCR sequencing using genomic DNA extracted from biofilms recovered on GYC (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) plates.     

Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC_h7) and other key virulence genes (eae, stx₁, and stx₂). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.Escherichia coli serotype O157:H7 was first linked to human illness in the early 1980s, when it was determined to cause severe abdominal pain with initially watery diarrhea that progressed to grossly bloody diarrhea accompanied by little or no fever (42). Initially, E. coli O157:H7 can cause nonbloody diarrhea through attachment to, and subsequent destruction of, intestinal microvilli (24). In addition to microvillus damage, serious health complications can arise due to the ability of E. coli O157:H7 to produce Shiga toxins (Stx1 and Stx2). Shiga toxins are very potent cytotoxins that are absorbed into the intestinal microvasculature and initiate apoptosis of vascular epithelium, resulting in hemorrhagic colitis (41). Persistent uptake of these toxins may lead to more severe manifestations of disease, such as hemolytic-uremic syndrome, which may ultimately result in kidney failure (24). Most recent estimates have identified E. coli O157:H7 as the cause of at least 70,000 cases of food-borne illness annually in the United States, and in 4% of cases life-threatening hemolytic-uremic syndrome develops (37). Epidemiological studies have implicated the consumption of meat, dairy products, produce, and water contaminated by animal feces, as well as person-to-person contact and direct contact with farm animals or their environment, as routes of E. coli O157:H7 transmission leading to human illness (36).It is generally accepted that cattle and other animals are the major reservoir of E. coli O157:H7, but it is still not clear if animals are colonized for prolonged periods with E. coli O157:H7 or if they transiently shed this organism following repeated exposure to it through ingestion of contaminated feedstuffs or water or through exposure to other contaminated environmental sources. Based on results of numerous epidemiological studies (4, 6, 21, 30, 32), the prevalence of E. coli O157:H7 in feedlot cattle is highly variable and can range from less than 1% to 80%. Several other studies (7, 8, 23) have found evidence of persistent E. coli O157:H7 colonization in individual cattle, supporting the hypothesis that at least some animals are susceptible to persistent E. coli O157:H7 colonization. Multiple experimental inoculation studies (15, 23, 39, 46) showed that E. coli O157:H7 persists in the bovine gastrointestinal (GI) tract for at least 14 days up to 140 days postinfection. Studies have implicated the lower GI tract and specifically the recto-anal junction (RAJ) as the major location of E. coli O157:H7 colonization and proliferation (9, 12, 23, 39); however, this organism also can be found throughout the bovine GI tract (7, 8, 31, 40, 54).It stands to reason that if the E. coli O157:H7 prevalence in cattle presented for harvest were reduced, there would be a decrease in the probability of beef product contamination, if good manufacturing procedures were used. Although there is consensus concerning the importance of preharvest pathogen mitigation and its role in minimizing entry of E. coli O157:H7 into harvest facilities, there is disagreement about the significance of “supershedders” (animals that excrete large quantities of a pathogen for various amounts of time) for E. coli O157:H7 transmission dynamics at the preharvest level (12, 34, 35, 39). Utilizing statistical modeling, researchers have estimated that, on average, the prevalence of “supershedders” in a population is 4% and that these animals excrete 50 times more E. coli O157:H7 than other animals colonized by this organism (34). Additionally, the same researchers suggested that approximately 80% of E. coli O157:H7 transmission is generated by a few “supershedders” (35).Research by our group discovered a unique association between E. coli O157:H7 prevalence in pen floor fecal pats and carcass contamination by this pathogen (57). When the prevalence in fecal pats from a pen floor exceeded 20%, carcasses of animals from the pen had E. coli O157:H7 prevalence values of 14.3, 2.9, and 0.7% before evisceration, after evisceration, and after final intervention, respectively. However, when the prevalence in pen floor fecal pats was less than 20%, the preeviscerated carcass prevalence value was 6.3%, and there was no detectable E. coli O157:H7 contamination of carcass samples after evisceration and after final intervention (57). Thus, we hypothesize that animals which persistently excrete normal levels of E. coli O157:H7 over prolonged periods (persistent shedders [PS]) rather than animals that periodically shed abnormally high levels (supershedders) are the most significant source of E. coli O157:H7 contamination in the food continuum. Although previous studies suggested that cattle may be persistently colonized by E. coli O157:H7 and shed this organism in their feces for prolonged periods, molecular subtyping data are required to further investigate whether cattle are persistently colonized by the same strain (i.e., molecular subtype) or if they are repeatedly exposed to different strains through contaminated feedstuffs, water, or other environmental sources. Thus, the objectives of this study were to determine if naturally colonized feedlot cattle persistently shed E. coli O157:H7, using combined cultural microbiological analyses, molecular subtyping approaches, and in vitro virulence phenotype assays to probe the factors (agent, host, environment, or a combination of these factors) that contribute to the complex ecology of E. coli O157:H7 persistence at the preharvest level.     

Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination

The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10⁴ CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (≥40 CFU/100 cm²) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (≥200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.It is now well established that at the time of harvest, hides are the major source of Escherichia coli O157:H7 contamination on beef carcasses (1, 4, 22). Thus, reducing the levels of food-borne pathogens on cattle hides has been the focus of many pre- and postharvest research efforts. For postharvest applications, hide interventions (i.e., washing of hide-on carcasses with various antimicrobial agents) are direct approaches and have been shown to be efficacious for reducing hide and carcass contamination rates (2, 4, 5, 22).In the area of preharvest research, several approaches have been taken to reduce the prevalence of E. coli O157:H7 in feces of cattle presented for slaughter. These approaches include, among others, feeding cattle probiotics (dietary administration of beneficial bacteria to compete with E. coli O157:H7), vaccination, and bacteriophage treatment (8, 24, 30). These intervention approaches are indirect. By reducing the fecal pathogen load, the pathogen prevalence and the level on hides are reduced through lower cross-contamination at the feedlot, and subsequently, carcass contamination rates decrease. While the effectiveness of preharvest interventions varies, no preharvest intervention is 100% effective in reducing the fecal prevalence of E. coli O157:H7. It is not known what level of pathogen reduction in feces would be necessary to significantly reduce hide and carcass contamination during processing. Key pieces of information needed to address this question are the number of shedding cattle in a pen needed to contaminate the hides of most of the cattle in the same pen and at what level the shedding cattle are contaminated.Aside from the number of cattle shedding a pathogen, the concentration of the pathogen in feces plays a pivotal role in spreading the pathogen between animals. Recently, cattle shedding E. coli O157:H7 at levels of >10⁴ CFU/g (“supershedders”) have been associated with high rates of transmission of the pathogen between cohort animals (18, 23). Matthews et al. reported that 20% of the E. coli O157:H7 infections in cattle on Scottish farms were responsible for 80% of the transmission of the organism between animals (18). Another study reported similar findings; 9% of the animals shedding E. coli O157:H7 produced over 96% of the total E. coli O157:H7 fecal load for the group (23). While a number of studies have indicated the importance of supershedders in fecal transmission dynamics, there is a general lack of information concerning the effects of high shedding rates on hide prevalence and load. Accordingly, the objectives of this study were (i) to investigate the dynamics of E. coli O157:H7 prevalence and levels in feces and on hides of feedlot cattle over time and (ii) to determine how pathogen prevalence and levels on hides in a pen are affected by individuals shedding E. coli O157:H7 at high levels.In the analysis presented here, fecal shedding was analyzed using the following three categories based on the level of E. coli O157:H7 being shed: shedding positive (presumed concentration, ≥1 CFU/g), high-density shedder (≥200 CFU/g), and supershedder (≥10⁴ CFU/g). Several definitions of E. coli O157:H7 supershedders have been offered previously. One-time shedding levels of >10³ or >10⁴ CFU/g have been used in multiple studies (17, 23, 24), while other groups have required persistent colonization of the rectoanal junction, as well as high cell counts, for an animal to qualify as a supershedder (10). Recently, Chase-Topping et al. (9) reviewed the requirements for supershedder status and provided a working definition: an animal that excretes >10⁴ CFU/g. In doing this, Chase-Topping et al. noted the high stringency of this definition and acknowledged that with such a definition some supershedders will be missed if they are sampled at times other than peak shedding times (9). In the current study, this was a concern. In an attempt to investigate the link between high-shedding-level animals and hide contamination, greater leeway was needed in the classification. When it is sampled on a monthly basis, an animal shedding at high levels can have a large impact on the hide status of pen cohorts between sampling intervals but not be shedding at peak levels on the day of sample collection. Hence, the categories described above were selected to analyze the relationship between fecal shedding and hide contamination.     

Modulation of Responses of Vibrio parahaemolyticus O3:K6 to pH and Temperature Stresses by Growth at Different Salt Concentrations

Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at a 1% or 3% NaCl concentration, and the growth and survival of the organism were examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus that in 1% NaCl increased survival under both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42°C and −20°C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (encoded by cadA), the organism''s main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of the stress response, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two-component regulator common to many Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl was examined using a cytotoxicity assay, and we found that V. parahaemolyticus grown in 1% NaCl was significantly more toxic than that grown in 3% NaCl.Vibrio parahaemolyticus is a Gram-negative bacterium that inhabits coastal waters worldwide. Vibrio parahaemolyticus grows optimally in warmer waters and is most commonly isolated during the summer months, often in association with plankton, crustaceans, mollusks, and fish (16, 17). During the winter months, the organism is typically scarce and usually is isolated from sediment samples (16). While V. parahaemolyticus has been shown to be the etiological agent of disease in several kinds of crustaceans and shellfish, it is most notably a pathogen of humans (17). Vibrio parahaemolyticus was first discovered in Japan during an outbreak of gastroenteritis in 1950 (12). It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia (6, 39). Infection is most frequently associated with the consumption of oysters harvested from warm waters, particularly along the U.S. Gulf Coast, where vibrios grow to high levels during the summer months (6, 7, 42). Newly released data from the CDC comparing the incidence rates of laboratory-confirmed infections by gastrointestinal pathogens in 1996 to 2008 revealed an increase of 47% for Vibrio infections, of which V. parahaemolyticus accounted for 55%, while rates for all other enteric pathogens decreased or remained the same (5). An outbreak of V. parahaemolyticus infections which caused rapid hospitalization of those infected occurred in India in 1995 (28). These infections were caused by a single serogroup, a new, highly virulent O3:K6 strain, which has now disseminated globally (1, 6, 20, 26, 34, 38). Recent studies report the recovery of O3:K6 isolates from the water in southern Chile, a region that previously was considered too cold to support the growth of this organism (4, 11, 13).All V. parahaemolyticus strains inhabit marine, brackish, and estuarine waters, where fluctuations in salinity pose a constant challenge to the adaptive response of the organism. Vibrio parahaemolyticus is moderately halophilic in nature and requires a minimum of 0.086 M (0.5%) NaCl for growth (29). It has also been demonstrated that this organism has the ability to grow in medium containing NaCl concentrations upwards of 1.5 M, making V. parahaemolyticus more osmotolerant than many other Vibrio species, such as V. cholerae, V. vulnificus, and V. fischeri, which occupy similar niches (27). In a recent study, we examined the genome of V. parahaemolyticus O3:K6 (designated RIMD2210633) and identified homologues of ectoine and betaine synthesis genes, as well as homologues of four single-component compatible solute transporters and two multicomponent compatible solute transporters (27). The large compendium of compatible solute systems in V. parahaemolyticus suggests that they might play an additional role(s) in survival.Within offshore waters, V. parahaemolyticus is generally faced with NaCl concentrations of 3.5% salinity (35 ppt), but in estuarine systems and within oysters (which are osmoconformers), it must adapt to changes in salinity. In addition, as a human pathogen, once inside the human host, like most enteric pathogens, V. parahaemolyticus must overcome the inorganic-pH challenge presented by gastric acid from the stomach and organic acids found within the intestine, as well as decreasing salinity (salinity in the intestine is approximately 300 mM NaCl). Organic acids have the ability to cross the cell membrane and enter the cytoplasm of the cell, whereas inorganic acids remain in the extracellular environment. Once in the cells, the organic acids can disassociate, decreasing the cytoplasmic pH and increasing the turgor pressure within the cell due to increases in anions from the acids (9). Thus, inorganic and organic acids can affect cells very differently.We suggest that the ability to grow at different NaCl concentrations, such as those vibrios would encounter in estuarine environments, allows V. parahaemolyticus to adapt more effectively to other environmental stresses (temperature fluctuations) and to the challenges that occur upon invasion of the human host (low pH). In this study, we show that V. parahaemolyticus RIMD2210633 cells grown at 3% NaCl are more resistant to acid and temperature stresses than cells grown at 1% NaCl. We demonstrate that V. parahaemolyticus grown in 3% NaCl is better able to survive sublethal and lethal acid shock conditions, as well as persistent high- and low-temperature conditions. We determined possible regulatory mechanisms involved in stress responses by examining the global regulator genes toxRS and rpoS. Last, we examined how changing environmental conditions, such as high and low NaCl and low pH, might affect the virulence of V. parahaemolyticus by determining its cytotoxicity toward human intestinal (Caco-2) cells.     

Inclusion of Dried or Wet Distillers' Grains at Different Levels in Diets of Feedlot Cattle Affects Fecal Shedding of Escherichia coli O157:H7

Our objectives were to evaluate the prevalence of Escherichia coli O157:H7 in cattle fed diets supplemented with 20 or 40% dried distillers'' grains (DG) (DDG) or wet DG (WDG) and assess whether removing DG from diets before slaughter affected fecal shedding of E. coli O157:H7. Eight hundred forty steers were allocated to 70 pens (12 steers/pen). Treatments were no DG (control), 20% DDG or WDG, and 40% DDG or WDG, and each was replicated in 14 pens. In phase 1, eight floor fecal samples were collected from each pen every 2 weeks for 12 weeks for isolation of E. coli O157:H7 and detection of high shedders. In phase 2, half of the pens with DG were transitioned to the no-DG control diet, and pen floor fecal samples were collected weekly from all pens for 4 weeks. During phase 1, prevalence of E. coli O157:H7 was 20.8% and 3.2% for high shedders. The form of DG had no significant effect on fecal E. coli O157:H7 shedding. The prevalence levels of E. coli O157:H7 and the numbers of high shedders were not different between diets with 0 or 20% DG; however, cattle fed 40% DG had a higher prevalence and more high shedders than cattle fed 0 or 20% DG (P ≤ 0.05). During phase 2, overall and high-shedder prevalence estimates were 3.3% and <0.1%, respectively, and there were no differences between those for different DG forms and inclusion levels or when DG was removed from diets. The form of DG had no impact on E. coli O157:H7; however, fecal shedding was associated with the DG inclusion level.Cattle are asymptomatic reservoirs for Escherichia coli O157:H7, a food-borne pathogen associated with gastrointestinal disease in thousands of Americans each year. The organism colonizes the hindgut of cattle (18, 27) and is shed in cattle feces. Once shed, E. coli O157:H7 can contaminate food and water, creating a food safety risk (20). Contamination of beef products occurs during slaughter and is associated with the prevalence of E. coli O157:H7 in feces and on the hides of cattle at harvest (5, 8, 12).The prevalence of E. coli O157:H7 in cattle is associated with many factors, including season, geographic location, and diet. Previous work has shown that cattle fed diets containing distillers'' grains (DG), an ethanol fermentation coproduct, have a higher prevalence of E. coli O157:H7 than cattle fed diets without DG (10, 28). Distillers'' grains are a valuable feed commodity for cattle producers, and use of these coproducts has increased with the expansion of the ethanol industry (14, 17). Distillers'' grains for use in cattle diets are available in wet (WDG) or dry (DDG) form. The association between feeding DG and E. coli O157:H7 prevalence has been shown with both forms (10, 28), but no study has directly compared the two forms. The levels of DG supplementation in cattle diets generally range from 10 to 50% (dry matter basis) depending on whether the coproduct is used as a protein or energy source. As a protein supplement, DG is included at 10 to 15%; as an energy source, the DG level is generally dictated by coproduct availability and grain price (14). There is some indication that E. coli O157:H7 prevalence is different for cattle fed different levels of DG (19). However, no study has specifically evaluated the relationship between E. coli O157:H7 prevalence and DG inclusion level. Evaluation of these two factors (form and inclusion level) is important for furthering our understanding of the association between DG and E. coli O157:H7 in cattle.We also were interested in determining whether removing the DG component of the diet would lower fecal prevalence of E. coli O157:H7. Such a strategy may lead to potential mitigation options and would provide further evidence of a positive association between feeding DG and E. coli O157:H7 prevalence in cattle. In this two-phase study, our objectives were to (i) concurrently evaluate the effect of DG inclusion level and form on E. coli O157:H7 prevalence in feedlot cattle and (ii) determine if removing DG from cattle diets subsequently reduces the fecal prevalence of E. coli O157:H7.     

Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

The application of whole cells containing cytochrome P-450_BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450_BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450_BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450_BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.Cytochrome P-450_BM-3 monooxygenase (CytP450_BM-3) is a soluble NADPH-dependent monooxygenase from Bacillus megaterium ATCC 14581 (13). It is a class II P-450 enzyme that contains flavin adenine dinucleotide, flavin mononucleotide, and a heme moiety (17). Unlike most CytP450 monooxygenases, which consist of a distinct monooxygenase and a reductase, CytP450_BM-3 contains these functionalities in a single polypeptide (3, 15, 18).The enzyme hydroxylates a variety of long-chain aliphatic substrates, such as fatty acids, alkanols, and alkylamides at the ω-1, ω-2, and ω-3 positions (4, 17), and oxidizes unsaturated fatty acids to epoxides in vitro (17, 23) with high enantioselectivity. Oxidation of eicosapentenoic acid (C_20:5) and arachidonic acid (C_20:4) yielded 17(S),18(R)-epoxyeicosatetraenoic acid (94% enantiomeric excess [e.e.]) for the former and a mixture of 18-(R)-hydroxyarachidonic acid (92% e.e.) and 14(S),15(R)-epoxyeicosatrienoic acid at 98% e.e. for the latter substrate (8). Recently, it has been demonstrated that the enzyme also produces α,ω diacids from ω-oxo fatty acids by oxidation of the terminal aldehyde functionality (9). The catalytic constant (k_cat) of CytP450_BM-3 is among the highest found for P-450 monooxygenases, ranging from 15 s⁻¹ for laureate to 75 s⁻¹ for pentadecanoic acid (11). For comparison, a typical microsomal P-450 monooxygenase from human liver (CYP2J2) had a k_cat of 10⁻³ s⁻¹ for arachidonic acid (32), compared to a k_cat of 55 s⁻¹ for CytP450_BM-3 for the same substrate (8).This high catalytic efficiency prompted us to investigate the applicability of CytP450_BM-3 as a biocatalyst for the subterminal hydroxylation of long-chain fatty acids (LCFAs). Since these subterminal hydroxy LCFAs are chiral molecules, their application in the production of enantiopure synthetic building blocks, especially for pharmaceutical agents, could be envisioned. Further, long-chain hydroxy acids find applications as precursors for polymers or cyclic lactones, which are used as components of fragrances and as antibiotics. Although chemical syntheses have been developed for ω-1 hydroxy fatty acids (from C₁₂ to C₁₈) (26, 28, 29) and for ω-2 and ω-3 hydroxyoctadecanoic acids (2), they require expensive functionalized substrates and are in general complicated, multistep processes (26, 28, 29) which cannot be carried out with unmodified fatty acids as inexpensive starting material. In principle, such inexpensive substrates can be oxidized to hydroxy fatty acids by biocatalysts, either in vitro or in vivo. The latter is preferred, since whole cells actively regenerate the NADPH required for fatty acid oxidation with monooxygenases such as CytP450_BM-3. In designing a suitable whole-cell biocatalyst, several additional points had to be considered.First, uptake must be efficient. Second, degradation of substrate or product must be avoided. In fact, biotransformations of fatty acids with whole cells are usually inefficient due to limited uptake of these compounds at neutral pH, and when taken up, they are degraded via β-oxidation. The transport of LCFAs in Escherichia coli is mediated via the fadL and fadD gene products. FadL is the transporter that carries LCFAs across the outer membrane and is absolutely required for LCFA transport (20). FadD, the acyl coenzyme A (CoA) synthetase, is located at the inner side of the cytoplasmic membrane and is required for formation of the acyl coenzyme A thioester, after which the activated fatty acids are channeled into the β-oxidation cycle for fatty acid degradation (21, 22). Thus, we used a FadD mutant, E. coli K27, as a suitable host for the production of subterminal hydroxyalkanoic acids (20). E. coli K27 cannot couple free fatty acids to coenzyme A, thus preventing substrate or product degradation by the host. Such fadD mutants are, however, also impaired in efficient uptake of fatty acids (20). We circumvented this by introducing a fatty acid uptake system from Pseudomonas oleovorans encoded on pGEc47. Finally, we introduced the P-450_BM-3 monooxygenase on pCYP102 into the fadD mutant E. coli. The resulting recombinant, E. coli K27(pCYP102, pGEc47), is a promising tailored biocatalyst for the oxidation of saturated LCFAs to ω-1, ω-2, and ω-3 hydroxy fatty acids.     

Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Comparison of the Thermostability Properties of Three Acid Phosphatases from Molds: Aspergillus fumigatus Phytase, A. niger Phytase, and A. niger pH 2.5 Acid Phosphatase

Enzymes that are used as animal feed supplements should be able to withstand temperatures of 60 to 90°C, which may be reached during the feed pelleting process. The thermostability properties of three histidine acid phosphatases, Aspergillus fumigatus phytase, Aspergillus niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were investigated by measuring circular dichroism, fluorescence, and enzymatic activity. The phytases of A. fumigatus and A. niger were both denatured at temperatures between 50 and 70°C. After heat denaturation at temperatures up to 90°C, A. fumigatus phytase refolded completely into a nativelike, fully active conformation, while in the case of A. niger phytase exposure to 55 to 90°C was associated with an irreversible conformational change and with losses in enzymatic activity of 70 to 80%. In contrast to these two phytases, A. niger pH 2.5 acid phosphatase displayed considerably higher thermostability; denaturation, conformational changes, and irreversible inactivation were observed only at temperatures of ≥80°C. In feed pelleting experiments performed at 75°C, the recoveries of the enzymatic activities of the three acid phosphatases were similar (63 to 73%). At 85°C, however, the recovery of enzymatic activity was considerably higher for A. fumigatus phytase (51%) than for A. niger phytase (31%) or pH 2.5 acid phosphatase (14%). These findings confirm that A. niger pH 2.5 acid phosphatase is irreversibly inactivated at temperatures above 80°C and that the capacity of A. fumigatus phytase to refold properly after heat denaturation may favorably affect its pelleting stability.     

Survival or Growth of Escherichia coli O157:H7 in a Model System of Fresh Meat Decontamination Runoff Waste Fluids and Its Resistance to Subsequent Lactic Acid Stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 ± 0.1) or in water (pH 7.2 ± 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25°C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4°C and lowest at 25°C. The pathogen survived without growth in water washings at 4 and 10°C, while it grew by 0.8 to 2.7 log cycles at 15 and 25°C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10°C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25°C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15°C > 10°C > 4°C, while at 25°C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15°C may maintain a higher acid resistance than when acid habituated at 4°C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (10¹⁰, 10⁹, and 10⁸ PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 10⁹ PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.     

Substrate Selectivity and Biochemical Properties of 4-Hydroxy-2-Keto-Pentanoic Acid Aldolase from Escherichia coli

4-Hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli was identified as a class I aldolase. The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept α-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.Aldolase-catalyzed reactions are involved in the latter stages of many bacterial catabolic pathways responsible for the degradation of aromatic compounds (3). In particular, many of the bacterial meta cleavage pathways for aromatic degradation proceed via the common intermediate 4-hydroxy-2-keto-pentanoic acid (HKP) (3), whose cleavage to acetaldehyde and pyruvic acid (Fig. ​(Fig.1)1) is catalyzed by an aldolase enzyme. Previous biochemical work on HKP aldolase is limited to the enzyme activity from Pseudomonas strains. HKP aldolase from Pseudomonas sp. strain CF600 was shown to be activated by Mn²⁺ ions (9), whereas 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas putida (11) requires Mg²⁺ ions for catalytic activity, and these enzymes appear to fall into the class II family of aldolases. Open in a separate windowFIG. 1Reaction catalyzed by HKP aldolase. Also illustrated are the previous reaction on the phenylpropionate catabolic pathway, the lactone derivative of HKP, and the method used for the coupled enzyme assay.It has been reported previously that the HKP aldolase activity in extracts of Escherichia coli would process both enantiomers of the substrate, unlike the corresponding activities from Pseudomonas and Acinetobacter spp., which were enantioselective (2). We have previously established that the preceding enzyme on the phenylpropionic acid catabolic pathway, namely, 2-hydroxypentadienoic acid hydratase (MhpD), catalyzes a stereospecific hydration reaction (8). We therefore wished to examine the stereoselectivity and substrate selectivity of HKP aldolase from E. coli.HKP aldolase activity was detectable in extracts of E. coli W3110 in a stopped assay involving treatment of HKP with extract for 30 min, followed by heat treatment and then addition of lactate dehydrogenase and NADH. Levels of enzyme activity were low (7.0 mU/mg of protein) and were not enhanced by inclusion of phenylpropionic acid in the growth media (1). The enzyme was purified by precipitation with 50 to 80% ammonium sulfate, phenyl-agarose hydrophobic-interaction chromatography, Q Sepharose anion-exchange fast protein liquid chromatography (FPLC), and Mono Q anion-exchange FPLC. The purified enzyme had a specific activity of 184 mU/mg of protein, a 26-fold purification overall. Although not purified to homogeneity, the purified enzyme was entirely free of background NADH oxidase activity and could be examined in a continuous assay by incubation with lactate dehydrogenase and NADH. Maximum activity was obtained in the pH range 6.25 to 6.75, with sharp inflections of activity at pH 6.0 and 8.0.The purified HKP aldolase showed no observable dependence on divalent metal ions, unlike the purified Pseudomonas aldolases (9, 11). Furthermore, treatment of the enzyme with the metal chelator EDTA at 10 mM resulted in no loss of enzyme activity. Moreover, treatment of enzyme with sodium borohydride in the presence of substrate HKP resulted in 100% loss of activity, indicative of an imine linkage. Treatment with sodium borohydride in the absence of substrate resulted in only a slight (<10%) loss of activity; thus, the imine linkage is formed only upon addition of the substrate. These data imply that the E. coli enzyme is a class I aldolase utilizing an imine linkage between the C-2 carbonyl of HKP and the ɛ-amino group of a lysine residue at the active site. Since 80% amino acid sequence identity has been determined between the Pseudomonas strain CF600 DmpG and E. coli MhpE gene products corresponding to the respective HKP aldolases (4), the difference in behavior between the E. coli and Pseudomonas enzymes is most surprising. We note that although the Pseudomonas HKP aldolase was reported to show a six- to eightfold activation by Mn²⁺, the enzyme retained residual activity after EDTA treatment and the presence of an imine linkage was not investigated (9).The stereoselectivity of the enzymatic reaction was examined by treatment of racemic HKP with the enzyme for various reaction times, followed by acid-catalyzed lactonization of the remaining HKP substrate to give 2-keto-4-methyl-γ-butyrolactone, followed by organic acids HPLC analysis. Time-dependent consumption of HKP was observed, but after long reaction times approximately 60% of the substrate remained (Fig. ​(Fig.2A).2A). Kinetic studies subsequently showed that the equilibrium position lies strongly in favor of the forward reaction; therefore, these data imply that the enzyme utilizes only one enantiomer of the substrate. Since the earlier study of the E. coli enzyme stereospecificity was carried out with crude extract (2), it is possible that there is a second aldolase with the opposite stereospecificity in E. coli, although we observed only one peak of activity upon enzyme purification. Open in a separate windowFIG. 2Analysis of HKP aldolase-catalyzed reaction via HPLC analysis of the lactone derivative α-methyl-γ-methyl-γ-butyrolactone. (A) Forward reaction using racemic HKP as the substrate (percentage of initial peak area). (B) Reverse reaction using pyruvic acid and acetaldehyde as substrates (concentrations determined by peak area, versus authentic standards).The reverse reaction was assayed by incubation of enzyme with acetaldehyde and pyruvate, followed by lactonization of the HKP product under acidic conditions, and HPLC analysis. Time-dependent formation of product was observed (Fig. ​(Fig.2B),2B), indicating that HKP aldolase also catalyzes the reverse reaction. Concentrations of product were deduced by calibration with known amounts of synthetic 2-keto-4-methyl-γ-butyrolactone (10); thus, after 24 h the reaction mixture contained 3.7 mM HKP. Initial concentrations of acetaldehyde and pyruvic acid were 360 mM and 180 mM, respectively; thus, a K_eq of 17 M in favor of the forward reaction can be deduced. From the rate of reaction over the first hour, it was calculated that the reverse reaction proceeds at 13% of the rate of the forward reaction.The substrate selectivity for the reverse reaction catalyzed by HKP aldolase was examined with respect to the carbonyl donor pyruvic acid and the carbonyl acceptor acetaldehyde. No product formation was observed when propionaldehyde was used in place of acetaldehyde; thus, the enzyme is highly selective for the carbonyl acceptor. However, lactone products with similar retention times and λ_maxs were observed by HPLC using either α-ketobutyric acid or phenylpyruvic acid as the substrate for the reverse reaction. The apparent rates of formation of the new lactone derivatives are similar to those observed with pyruvic acid as the substrate (Table ​(Table1);1); thus, α-ketobutyric acid and phenylpyruvic acid appear to be converted efficiently by the enzyme.

TABLE 1

Substrate selectivity for the MhpE-catalyzed forward and reverse reactions^a

Cytoplasmic Membrane Lipoprotein LppC of Streptococcus equisimilis Functions as an Acid Phosphatase

The function of the streptococcal cytoplasmic membrane lipoprotein, LppC, was identified with isogenic Streptococcus equisimilis H46A and Escherichia coli JM109 strain pairs differing in whether they contained [H46A and JM109(pLPP2)] or lacked (H46A lppC::pLPP10 and JM109) the functional lppC gene for comparative phosphatase determinations under acidic conditions. lppC-directed acid phosphatase activity was demonstrated zymographically and by specific enzymatic activity assays, with whole cells or cell membrane preparations as enzyme sources. LppC acid phosphatase showed optimum activity at pH 5, and the enzyme activity was unaffected by Triton X-100, l-(+)-tartaric acid, or EDTA. Database searches revealed significant structural homology of LppC to the Streptococcus pyogenes LppA, Flavobacterium meningosepticum OplA, Helicobacter pylori HP1285, and Haemophilus influenzae Hel [e (P4)] proteins. These results suggest a possible function for these proteins and establish a novel function of streptococcal cell membrane lipoproteins.In a previous study from this laboratory, we reported the cloning and nucleotide sequence of a novel Streptococcus equisimilis chromosomal gene, designated lppC, which encodes a 32.4-kDa lipoprotein associated with the streptococcal cytoplasmic membrane or the outer membrane of Escherichia coli when expressed in this organism (5). The lppC gene is located immediately 3′ to and is transcribed independently of the unrelated gapC gene that codes for glyceraldehyde-3-phosphate dehydrogenase (4). As revealed by Southern, Northern, and Western analyses, homologs of lppC (and gapC) are conserved and also expressed in Streptococcus pyogenes (5). Database searches performed at that time found homology of LppC only to the hel gene-encoded outer-membrane antigen e (P4) from Haemophilus influenzae (6), to which it exhibits 58% sequence similarity. The biological function of e (P4) has remained elusive until very recently, when it was reported to be involved in the uptake of hemin as a source of porphyrin, an essential growth factor for H. influenzae when grown aerobically (9). Our attempts to provide evidence for a role of lppC in hemin uptake failed as, unlike the hel gene, lppC was unable to complement hemA mutants of E. coli for growth on hemin as the sole porphyrin source in aerobic conditions. Furthermore, S. equisimilis H46A, the source of lppC, was incapable of hemin binding or of growing on this compound in iron-limited medium (5).Sequence database searching was continued at regular intervals for additional homologs of LppC and revealed weak structural similarity at low quality (quality score, 92.3) to the aphA gene product of E. coli MG1655 (sequence identity and similarity between LppC and AphA, 20.3 and 46.7%, respectively). Thaller et al. (16) had cloned and sequenced the aphA gene in the meantime and functionally characterized its product as an acid phosphatase. Sequence similarity between LppC and AphA prompted me to explore the possibility that the streptococcal protein has similar enzymatic activity. Here I provide biochemical, serological, and genetic evidence that the LppC protein does function as an acid phosphatase.     

Induction of Acid Resistance of Salmonella typhimurium by Exposure to Short-Chain Fatty Acids

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05). The results suggest that the virulence of S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.     

Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation

We investigated the effect of the ecological factors pH, temperature, ionic strength, and lactate, acetate, and ethanol levels on Candida milleri and two strains of Lactobacillus sanfranciscensis, organisms representative of the microflora of sourdough. A mathematical model describing the single and combined effects of these factors on the growth of these organisms was established in accordance with the following criteria: quality of fit, biological significance of the parameters, and applicability of the in vitro data to in situ processes. The growth rates of L. sanfranciscensis LTH1729 and LTH2581 were virtually identical under all conditions tested. These organisms tolerated >160 mmol of undissociated acetic acid per liter. Growth occurred in the pH range of 3.9 to 6.7 and was completely inhibited by 4% NaCl. C. milleri had a lower optimum temperature for growth (27°C) than the lactobacilli. The growth of the yeast was not affected by pH in the range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete inhibition of growth occurred at 150 mmol of undissociated acetic acid per liter, but acetate at concentrations of up to 250 mmol/liter exerted virtually no effect. The model provides insight into factors contributing to the stability of the sourdough microflora and can facilitate the design of novel sourdough processes.