Erwin M. Berendsen

Bacterial Spores in Food: Survival, Emergence, and Outgrowth

Spore-forming bacteria are ubiquitous in nature. The resistance properties of bacterial spores lie at the heart of their widespread occurrence in food ingredients and foods. The efficacy of inactivation by food-processing conditions is largely determined by the characteristics of the different types of spores, whereas food composition and storage conditions determine the eventual germination and outgrowth of surviving spores. Here, we review the current knowledge on variation in spore resistance, in germination, and in the outgrowth capacity of spores relevant to foods. This includes novel findings on key parameters in spore survival and outgrowth obtained by gene-trait matching approaches using genome-sequenced Bacillus spp. food isolates, which represent notorious food spoilage and pathogenic species. Additionally, the impact of strain diversity on heat inactivation of spores and the variability therein is discussed. Knowledge and quantification of factors that influence variability can be applied to improve predictive models, ultimately supporting effective control of spore-forming bacteria in foods.

Two distinct groups within the Bacillus subtilis group display significantly different spore heat resistance properties.

The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups (p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z-values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply.

A mobile genetic element profoundly increases heat resistance of bacterial spores

Bacterial endospores are among the most resilient forms of life on earth and are intrinsically resistant to extreme environments and antimicrobial treatments. Their resilience is explained by unique cellular structures formed by a complex developmental process often initiated in response to nutrient deprivation. Although the macromolecular structures of spores from different bacterial species are similar, their resistance to environmental insults differs widely. It is not known which of the factors attributed to spore resistance confer very high-level heat resistance. Here, we provide conclusive evidence that in Bacillus subtilis, this is due to the presence of a mobile genetic element (Tn1546-like) carrying five predicted operons, one of which contains genes that encode homologs of SpoVAC, SpoVAD and SpoVAEb and four other genes encoding proteins with unknown functions. This operon, named spoVA2mob, confers high-level heat resistance to spores. Deletion of spoVA2mob in a B. subtilis strain carrying Tn1546 renders heat-sensitive spores while transfer of spoVA2mob into B. subtilis 168 yields highly heat-resistant spores. On the basis of the genetic conservation of different spoVA operons among spore-forming species of Bacillaceae, we propose an evolutionary scenario for the emergence of extremely heat-resistant spores in B. subtilis, B. licheniformis and B. amyloliquefaciens. This discovery opens up avenues for improved detection and control of spore-forming bacteria able to produce highly heat-resistant spores.

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

ABSTRACT High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D 120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.

High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain.

Effects of altered TatC proteins on protein secretion efficiency via the twin-arginine translocation pathway of Bacillus subtilis

Protein translocation via the Tat machinery in thylakoids and bacteria occurs through a cooperation between the TatA, TatB and TatC subunits, of which the TatC protein forms the initial Tat substrate-binding site. The Bacillus subtilis Tat machinery lacks TatB and comprises two separate TatAC complexes with distinct substrate specificities: PhoD is secreted by the TatAdCd complex, whereas YwbN is secreted by the TatAyCy complex. To study the role of the Gram-positive TatC proteins in Tat-dependent protein secretion efficiency, we applied several genetic engineering approaches to modify and analyse the B. subtilis TatCd and TatCy proteins. Cytoplasmic and transmembrane domain exchange between TatCd and TatCy resulted in stable chimeric proteins that were unable to secrete both known substrates of the B. subtilis Tat system. Site-directed mutagenesis of conserved residues in the N-terminal part of both TatC proteins revealed significant differences in the degree of importance of these residues between TatCd, TatCy and Escherichia coli TatC. In addition, two small C-terminal deletions in TatCy completely abolished YwbN translocation, indicating that this terminus is essential for Tat translocation activity. Important differences from previous observations for E. coli TatC and implications for substrate binding and translocation are discussed.

A transposon present in specific strains of Bacillus subtilis negatively affects nutrient- and dodecylamine-induced spore germination

Spore germination shows a large inter-strain variability. Spores of certain Bacillus subtilis strains, including isolates from spoiled food products, exhibit different germination behavior from spores of the well-studied model organism Bacillus subtilis 168, often for unknown reasons. In this study, we analyzed spore germination efficiencies and kinetics of seventeen B. subtilis strains with previously sequenced genomes. A subsequent gene-trait matching analysis revealed a correlation between a slow germination phenotype and the presence of a mobile genetic element, i.e., a Tn1546-like transposon. A detailed investigation of the transposon elements showed an essential role of a specific operon (spoVA2mob ) in inhibiting spore germination with nutrients and with the cationic surfactant dodecylamine. Our results indicate that this operon negatively influences release of Ca-DPA by the SpoVA channel and may additionally alter earlier germination events, potentially by affecting proteins in the spore inner membrane. The spoVA2mob operon is an important factor that contributes to inter-strain differences in spore germination. Screening for its genomic presence can be applied for identification of spores that exhibit specific properties that impede spore eradication by industrial processes.

Draft Genome Sequences of Four Bacillus thermoamylovorans Strains Isolated from Milk and Acacia Gum, a Food Ingredient

ABSTRACT The thermophilic bacterium Bacillus thermoamylovorans produces highly heat-resistant spores that can contaminate food products, leading to their spoilage. Here, we present the whole-genome sequences of four B. thermoamylovorans strains, isolated from milk and acacia gum.

Next-Generation Whole-Genome Sequencing of Eight Strains of Bacillus cereus, Isolated from Food

ABSTRACT Bacillus cereus can contaminate food and cause emetic and diarrheal foodborne illness. Here, we report whole-genome sequences of eight strains of B. cereus, isolated from different food sources.

Two complementary approaches to quantify variability in heat resistance of spores of Bacillus subtilis

Realistic prediction of microbial inactivation in food requires quantitative information on variability introduced by the microorganisms. Bacillus subtilis forms heat resistant spores and in this study the impact of strain variability on spore heat resistance was quantified using 20 strains. In addition, experimental variability was quantified by using technical replicates per heat treatment experiment, and reproduction variability was quantified by using two biologically independent spore crops for each strain that were heat treated on different days. The fourth-decimal reduction times and z-values were estimated by a one-step and two-step model fitting procedure. Grouping of the 20 B. subtilis strains into two statistically distinguishable groups could be confirmed based on their spore heat resistance. The reproduction variability was higher than experimental variability, but both variabilities were much lower than strain variability. The model fitting approach did not significantly affect the quantification of variability. Remarkably, when strain variability in spore heat resistance was quantified using only the strains producing low-level heat resistant spores, then this strain variability was comparable with the previously reported strain variability in heat resistance of vegetative cells of Listeria monocytogenes, although in a totally other temperature range. Strains that produced spores with high-level heat resistance showed similar temperature range for growth as strains that produced low-level heat resistance. Strain variability affected heat resistance of spores most, and therefore integration of this variability factor in modelling of spore heat resistance will make predictions more realistic.