Fred Breidt

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.

Protective Effects of Organic Acids on Survival of Escherichia coli O157:H7 in Acidic Environments

ABSTRACT Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation

ABSTRACT Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage.

Determination of 5-Log Reduction Times for Food Pathogens in Acidified Cucumbers during Storage at 10 and 25°C†‡

Outbreaks of acid-resistant foodborne pathogens in acid foods with pH values below 4.0, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. For acidified vegetable products with pH values between 3.3 and 4.6, previous research has demonstrated that thermal treatments are needed to achieve a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, or Salmonella enterica. For some acidified vegetable products with a pH of 3.3 or below, heat processing can result in unacceptable product quality. The purpose of this study was to determine the holding times needed to achieve a 5-log reduction in E. coli O157:H7, L. monocytogenes, and S. enterica strains in acidified vegetable products with acetic acid as the primary acidulant, a pH of 3.3 or below, and a minimum equilibrated temperature of 10 degrees C. We found E. coli O157:H7 to be the most acid-resistant microorganism for the conditions tested, with a predicted time to achieve a 5-log reduction in cell numbers at 10 degrees C of 5.7 days, compared with 2.1 days (51 h) for Salmonella or 0.5 days (11.2 h) for Listeria. At 25 degrees C, the E. coli O157:H7 population achieved a 5-log reduction in 1.4 days (34.3 h).

Characterization of Cucumber Fermentation Spoilage Bacteria by Enrichment Culture and 16S rDNA Cloning

UNLABELLEDnCommercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram-negative anaerobic bacteria supersede Gram-positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage.nnnPRACTICAL APPLICATIONnAn understanding of the microorganisms that cause commercial cucumber fermentation spoilage may aid in developing methods to prevent the spoilage from occurring.

Effects of pH, dissolved oxygen, and ionic strength on the survival of Escherichia coli O157:H7 in organic acid solutions.

The ability of Escherichia coli O157:H7 to survive in acidified vegetable products is of concern because of previously documented outbreaks associated with fruit juices. A study was conducted to determine the survival of E. coli O157:H7 in organic acids at pH values typical of acidified vegetable products (pH 3.2 and 3.7) under different dissolved oxygen conditions (< or = 0.05 and 5 mg/liter) and a range of ionic strengths (0.086 to 1.14). All solutions contained 20 mM gluconic acid, which was used as a noninhibitory low pH buffer to compare the individual acid effect to that of pH alone on the survival of E. coli O157:H7. E. coli O157:H7 cells challenged in buffered solution with ca. 5-mg/liter dissolved oxygen (present in tap water) over a range of ionic strengths at pH 3.2 exhibited a decrease in survival over 6 h at 30 degrees C as the ionic strength was increased. Cells challenged in 40 mM protonated L-lactic and acetic acid solutions with ionic strength of 0.684 achieved a > 4.7-log CFU/ml reduction at pH 3.2. However, under oxygen-limiting conditions in an anaerobic chamber, with < or = 0.05-mg/ liter oxygen, E. coli O157:H7 cells showed < or = 1.55-log CFU/ml reduction regardless of pH, acid type, concentration, or ionic strength. Many acid and acidified foods are sold in hermetically sealed containers with oxygen-limiting conditions. Our results demonstrate that E. coli O157:H7 may survive better than previously expected from studies with acid solutions containing dissolved oxygen.

Escherichia coli O157:H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity

A novel phage, Φ241, specific for Escherichia coli O157:H7 was isolated from an industrial cucumber fermentation where both acidity (pH ≤ 3.7) and salinity (≥5% NaCl) were high. The phage belongs to the Myoviridae family. Its latent period was 15 min and average burst size was 53 phage particles per infected cell. The phage was able to lyse 48 E. coli O157:H7 strains, but none of the 18 non-O157 strains (including E. coli O104:H7) or the 2 O antigen-negative mutants of O157:H7 strain, 43895Δper (also lacking H7 antigen) and F12 (still expressing H7 antigen). However, the phage was able to lyse a per-complemented strain (43895ΔperComp) which expresses O157 antigen. These results indicated that phage Φ241 is specific for O157 antigen, and E. coli strains lacking O157 antigen were resistant to the phage infection, regardless of the presence or absence of H7 antigen. SDS-PAGE profile revealed at least 13 structural proteins of the phage. The phage DNA was resistant to many commonly used restriction endonucleases, suggesting the presence of modified nucleotides in the phage genome. At the multiplicity of infection of 10, 3, or 0.3, the phage caused a rapid cell lysis within 1 or 2 h, resulting in 3.5- or 4.5-log-unit reduction in cell concentration. The high lytic activity, specificity and tolerance to low pH and high salinity make phage Φ241 a potentially ideal biocontrol agent of E. coli O157:H7 in various foods. To our knowledge, this is the first report on E. coli O157:H7 phage isolated from high acidity and salinity environment.

Redesign of an Undergraduate General Microbiology Lab to Include Authentic Discovery-Driven Research on Cucumber Fermentations

Many undergraduate introductory microbiology laboratory courses teach basic principles of bacteriology using classical protocol-based experiments, with limited critical thinking and inquiry-based learning practices. We initiated a comprehensive redesign in our General Microbiology Laboratory course to promote scientific critical and creative thinking, while strengthening core microbiology concepts and skills. As part of the redesign, a series of authentic discovery-driven labs, based on cucumber fermentations, were developed as an independent research module within the course curriculum. Integrating discovery-driven labs allowed students to be engaged problem solvers, applying the scientific process to develop hypotheses, design experiments, utilize quantitative reasoning, and effectively communicate results. The inquiry-guided research project was developed to evaluate the minimum concentration of salt (NaCl) required in fermentation brine to safely, and effectively, ferment cucumbers. Over 5 weeks, students assess different aspects of the fermentation process, including quantifying bacterial populations with differential and selective media, measuring pH and glucose concentration of brine solutions, and characterizing the microbial metabolic potential. Additionally, students isolate an unknown bacterium from their fermentations, identifying and characterizing the isolate using 16S rRNA gene sequencing and metabolic tests. Throughout the research project, students collect, graph, and analyze their observations, culminating in students creating and presenting a scientific research poster. With this lab redesign, students generate new knowledge contributing to our understanding of microbial ecology within food fermentations, learn core microbiology skills and techniques, and develop critical and creative thinking skills. The impact of their research is valuable to science educators, researchers, and industry partners.