Functional amyloid formation by Streptococcus mutans.

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe.

BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans.

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.

The Biology of Streptococcus mutans.

As a major etiological agent of human dental caries, Streptococcus mutans resides primarily in biofilms that form on the tooth surfaces, also known as dental plaque. In addition to caries, S. mutans is responsible for cases of infective endocarditis with a subset of strains being indirectly implicated with the onset of additional extraoral pathologies. During the past 4 decades, functional studies of S. mutans have focused on understanding the molecular mechanisms the organism employs to form robust biofilms on tooth surfaces, to rapidly metabolize a wide variety of carbohydrates obtained from the host diet, and to survive numerous (and frequent) environmental challenges encountered in oral biofilms. In these areas of research, S. mutans has served as a model organism for ground-breaking new discoveries that have, at times, challenged long-standing dogmas based on bacterial paradigms such as Escherichia coli and Bacillus subtilis. In addition to sections dedicated to carbohydrate metabolism, biofilm formation, and stress responses, this article discusses newer developments in S. mutans biology research, namely, how S. mutans interspecies and cross-kingdom interactions dictate the development and pathogenic potential of oral biofilms and how next-generation sequencing technologies have led to a much better understanding of the physiology and diversity of S. mutans as a species.

Biology of Oral Streptococci.

Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity, which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this article, we discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus, because this genus is now divided into eight distinct groups, and oral species are found in six of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation, thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali, displaying an important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans. Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence.

IVIAT: a novel method to identify microbial genes expressed specifically during human infections.

In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in human infections, without the use of animal models. This technology is expected to facilitate the discovery of new targets for vaccines, antimicrobials and diagnostic strategies in a wide range of microbial pathogens.

Regulation of carnitine palmitoyltransferase synthesis in spontaneously diabetic BB Wistar rats.

The long-term regulation of hepatic mitochondrial carnitine palmitoyltransferase (CPT) was studied in control, insulin-treated, and untreated spontaneously diabetic BB Wistar rats. The activity of CPT was elevated approximately twofold in the untreated diabetic rats. This corresponded to an approximately equivalent elevation in the immunoreactive CPT activity. mRNACPT was assayed by reticulocyte lysate translation and by dot blot to a CPT oligonucleotide probe. The level of mRNACPT was approximately proportional to the observed CPT activity. A cDNA probe to CPT was developed, and transcriptional activity for CPT was assessed in isolated hepatic nuclei. Again, transcription of CPT mRNA was approximately proportional to the observed activity. We therefore conclude that at least part of the long-term regulation of hepatic CPT in spontaneously diabetic BB Wistar rats is the product of increased de novo synthesis of CPT protein brought about by regulation at the transcriptional level. Additional control of the amount of CPT may be via the regulation of RNA processing and turnover and enzyme insertion into the mitochondrial membrane.

Breaking the bacterial protein targeting and translocation model: oral organisms as a case in point.

Insights into the membrane biogenesis of oral and throat bacteria have highlighted key differences in protein localization by the general secretion pathway compared with the well-studied Escherichia coli model system. These intriguing novelties have advanced our understanding of both how these microorganisms have adapted to survive and cause disease in the oral cavity, and the field of protein translocation as a whole. This review focuses on findings that highlight where oral bacteria differ from the E. coli paradigm, why these differences are biologically important, and what questions remain about the differences in pathway function. The majority of insight into protein translocation in microbes of the oral cavity has come from streptococcal species, which will be the main topic of this review. However, other bacteria will be discussed when relevant. An overview of the E. coli model of protein targeting and translocation is provided for comparison.

Hepatic and cardiac carnitine palmitoyltransferase activity. Effects of adriamycin and galactosamine.

Carnitine palmitoyltransferase (CPT) activity is located on both the outer and inner sides of the mitochondrial inner membrane and is influenced by the surrounding lipids of the inner mitochondrial membrane. Both adriamycin and galactosamine interact with mitochondrial lipids as a part of their mechanism of toxicity, and thus these agents might be expected to affect CPT activity. Addition of adriamycin to both intact rat liver and heart mitochondria (CPT-A, outer CPT) and inverted submitochondrial vesicles (CPT-B, inner CPT) depressed CPT in the forward direction of reaction (palmitoyl-l-carnitine formation), but the CPT-B activity was more sensitive to the inhibitor. Adriamycin depressed the CPT-A reverse reaction (palmitoyl-CoA formation) to 40% of control, but it had no effect on the CPT-B reverse reaction. In vivo galactosamine administration depressed CPT-A and CPT-B 20-30% and did not affect subsequent action of in vitro adriamycin. Addition of cardiolipin (0.25 to 1.0 mg/assay) increased activity of the CPT-A forward reaction of both control and galactosamine-treated rats, but it did not affect CPT-B activity. The results suggest that CPT-A and CPT-B may be influenced differently by perturbants that affect lipids of the membrane.

Effects of clofibrate and acetylsalicylic acid on hepatic carnitine palmitoyltransferase synthesis.

Clofibrate and acetylsalicylic and have both been reported to increase carnitine palmitoyltransferase (CPT) activity when administered to rats. The purpose of the present study was to determine the mechanism of the increase in CPT activity. Rats (150-200 g) were fed one of the following: chow, chow with 0.5% clofibrate, or chow with 1% acetylsalicylic acid for 2 weeks. At the end of this time, hepatic CPT activity was increased 4-fold over control in the clofibrate group and 3.6-fold over control in the acetylsalicylic acid group. Immunoreactive protein increased 4.0- and 3.6-fold, respectively, over control. Transcription rates of hepatic nuclei were increased 2.8- and 1.9-fold over control in the clofibrate and acetylsalicylic acid groups, and hepatic mRNA levels increased 2.8- and 2.0-fold respectively. These data indicate that increases in CPT activity caused by clofibrate and acetylsalicylic acid administration are due, at least in part, to increased CPT protein, resulting from increased transcription rate and levels of mRNA specific for CPT.

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochondrial carnitine palmitoyltransferase and beta-oxidation of fatty acids.

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and a structural polyamine analogue which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces profound mitochondrial structural damage and inhibition of fatty acid oxidation. Carnitine palmitoyltransferase-A (CPT-A) is located on the outer surface of the inner mitochondrial membrane and is the putative rate-controlling enzyme for mitochondrial long-chain fatty acid oxidation. The present experiments were designed to determine if MGBG inhibits CPT-A. Liver, heart and skeletal muscle mitochondria were isolated from rats following 24 hr of starvation. Measuring the reaction in the direction of palmitoylcarnitine plus CoA formation from palmitoyl-CoA plus carnitine ("forward reaction"), MGBG was competitive with l-carnitine. The MGBG CPT-A Ki values were (mM): liver, 5.0 +/- 0.6 (N = 15); heart 3.2 +/- 1.2 (N = 3); and skeletal muscle, 2.8 +/- 1.0 (N = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0, which was not significantly different from intact mitochondria or inverted vesicles (4.9 mM). Purified hepatic CPT had a Ki of 4.2 mM. MGBG did not inhibit purified CPT in the "reverse reaction" (palmitoyl-CoA plus carnitine formation from palmitoylcarnitine plus CoA). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit either CPT activity or acid-soluble product formation from 1-[14C]palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by l-carnitine. Fluorescence polarization, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe, indicated that MGBG increased membrane rigidity in a dose-dependent manner. This effect was not altered by l-carnitine. MGBG also inhibited purified pigeon breast carnitine acetyltransferase (CAT; Ki = 1.6 mM). While MGBG appeared to be competitive with l-carnitine for both CPT and CAT, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not reversed by carnitine.

The Ca2+ second messenger system and interleukin-1-alpha modulation of hepatic gene transcription and mitochondrial fat oxidation.

Cytokines have been implicated in the modulation of fat metabolism after sepsis. Carnitine palmitoyltransferase (CPT), the regulatory enzyme of hepatic mitochondrial long-chain fatty-acid oxidation, is involved in the control of hepatic fat oxidation in sepsis. Using either H4IIe rat hepatoma cells or rat hepatocytes in primary culture, we tested the hypothesis that interleukin-1-alpha (IL-1 alpha) would modulate CPT transcription (CPT mRNA), CPT translation (35S-methionine CPT protein incorporation), and hepatic mitochondrial oxidation of 1-Carbon 14-labeled (14C) palmitate to ketone bodies (acid soluble products). We showed that IL-1 alpha significantly increased CPT mRNA, 35S-methionine incorporation CPT protein, and hepatic mitochondrial oxidation of 1-14C-palmitate to acid soluble products. We further hypothesized that the Ca2+ second messenger system may play a role in the IL-1 alpha induction of hepatic CPT gene transcription. We showed that either calcium ionophore (A23187) or phorbol myristate acetate increased CPT gene transcription and that either calcium chelation, protein kinase C inhibition (acridine orange), or chronic exposure to phorbol myristate acetate significantly inhibited IL-1 alpha induction of CPT mRNA. We conclude that the IL-1 alpha increases in hepatic mitochondrial fatty-acid oxidation may be, in part, secondary to increased CPT gene transcription and translation and that the Ca2+ second messenger system may play an important role in IL-1 alpha induction of CPT gene transcription.

The possible inhibitory role of the leucine-zipper DNA binding protein c-fos in the regulation of hepatic gene expression after sepsis.

BACKGROUND: The leucine-zipper c-fos has been implicated in the regulation of gene expression. We investigated the possible role of c-fos in the regulation of hepatic gene expression after sepsis. Based on previous data demonstrating that sepsis inhibits hepatic gene expression of carnitine palmitoyltransferase (CPT), we hypothesized that c-fos may play a role in the inhibition of CPT gene expression after sepsis. METHODS: We studied c-fos gene expression after peritoneal sepsis induced by cecal ligation and puncture (CLP) or sham-CLP. To investigate the possible inhibitory role of c-fos on CPT gene transcription, we investigated the effect of c-fos on c-jun-driven CPT promoter-chloramphenicol acyltransferase reporter gene expression in a HepG2 hepatoma cell cotransfection model. To investigate the possible role of cyclic adenosine monophosphate (cAMP) in the regulation of c-fos in vivo, we treated either the sham-CLP group or the CLP group with either vehicle or cAMP. RESULTS: Peritoneal sepsis in the rat model resulted in a four-fold increase in hepatic c-fos mRNA and c-fos protein. In the cotransfection model, c-fos significantly inhibited c-jun-induced chloramphenicol acyltransferase activity. Treatment with cAMP resulted in a 50% decrease in c-fos protein in either the sham-CLP or CLP group. CONCLUSIONS: We conclude that (1) sepsis increases hepatic c-fos transcription and translation, (2) c-fos inhibits c-jun-induced CPT gene expression, and (3) cAMP probably does not directly mediate the increase in c-fos after sepsis.

Sepsis-induced release of interleukin-6 may activate the immediate-early gene program through a hypothalamic-hypophyseal mechanism.

BACKGROUND: The immediate-early gene c-fos has been implicated in transcriptional regulation after sepsis. We test the hypothesis that sepsis-induced central nervous system release of interleukin (IL)-6 regulates hepatic c-fos gene expression. METHODS: Using a stereotaxically placed intracerebral-ventricular (ICV) catheter in rats with and without hypophysectomy, we measured hepatic c-fos protein accumulation after treatment with either IL-6 or vehicle control. Using a rat cecal ligation and puncture (CLP) model, we studied the following groups: (1) sham-CLP, (2) CLP, (3) hypophysectomized sham-CLP, and (4) hypophysectomized CLP and measured hepatic c-fos mRNA. RESULTS: ICV IL-6 treatment increased hepatic c-fos protein in the IL-6-treated group compared with the vehicle-treated group, and hypophysectomy inhibited the ICV IL-6-mediated increase in c-fos protein. After peritoneal sepsis, CLP increased hepatic c-fos messenger RNA compared to either the sham-CLP or the hypophysectomized sham-CLP group, and hypophysectomy before CLP inhibited hepatic c-fos mRNA compared with the CLP group. CONCLUSIONS: ICV IL-6 results in an increase in hepatic fos protein that is mediated through a hypothalamic-hypophyseal mechanism. Peritoneal sepsis results in an increase in hepatic c-fos gene expression that may be, in part, mediated by central nervous system release of IL-6 through a hypothalamic-hypophyseal mechanism.

Pharmacologic action of L-carnitine on hypertriglyceridemia in obese Zucker rats.

Administration of pharmacologic amounts of L-carnitine was studied in the hypertriglyceridemic Zucker rat. When administered subcutaneously, doses from 250 to 2,000 mg/kg/d significantly decreased plasma triglycerides in obese rats over eight to 12 weeks, with no effect on plasma triglycerides in lean rats. Oral doses at the same high levels were not effective in decreasing plasma triglycerides. Triglyceride secretion rate was reduced from 367 micrograms/min to 168 micrograms/min in treated obese rats. Concurrently, liver lipid was increased twofold in obese treated rats, and the livers of these rats showed significant fatty infiltration. The mechanism of action of carnitine in decreasing plasma triglycerides appeared to be via decreased secretion of triglycerides by the liver of obese rats. There was no effect of L-carnitine in lean or obese rats on the following variables: carnitine palmitoyltransferase-A kinetics or malonyl CoA inhibition, mitochondrial or peroxisomal oxidative capacity, lipoprotein lipase in heart, muscle, and adipose, or fecal lipids. The effect of pharmacologic L-carnitine thus appears to be an inhibition of triglyceride synthesis and/or secretion by the liver.

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.

Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Cloning and inactivation of the gene responsible for a major surface antigen on Streptococcus mutans.

To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequencing and characterization of the 185 kDa cell surface antigen of Streptococcus mutans.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen I/II of Streptococcus mutans, serotype c (strain NG5) has been sequenced. The deduced amino acid sequence of antigen I/II (1561 residues) includes a putative signal peptide (residues 1-38), as well as a transmembrane region (residues 1537-1556). The N-terminal part of the protein (residues 39-550) is particularly rich in alanine and includes three tandem repeats of a sequence of 82 residues. This region is predicted to be alpha-helical, adopting a coiled-coil formation, and may account for the cell surface hydrophobicity associated with expression of antigen I/II. In contrast the C-terminal region (residues 800-1549) is proline-rich, favouring an extended conformation. Comparison with the sequence determined from Strep. mutans strain MT8148 showed that antigen I/II is highly conserved with the exception of a short central region (residues 750-805). N-terminal sequencing of purified antigens I and II components indicated that antigen I extends from the amino-terminus of the intact Mr 185,000 surface antigen while antigen II extends from residue 996.

Sublethal sanitizer stress and adaptive response of Escherichia coli O157:H7.

The effect of sublethal exposure to peroxyacetic acid (PAA) sanitizer on adaptation to peroxidative stress and development of thermal cross-resistance was investigated in Escherichia coli O157:H7. Acute sublethal PAA sanitizer exposure was used to represent a contact scenario. Cultures were grown in Trypticase soy-yeast extract broth. Acute treatment cultures were pretreated with 0.1% PAA, then all cultures were challenged at either 80 mM H202 or 54 degrees C. Acute and peroxide control cultures showed substantially increased peroxidative tolerance (D80mM > 2 h) versus negative control cultures not exposed to sanitizer (D80mM = 0.19+/-0.03 h). The inactivation rate of the acetic acid control (D80mM = 0.21+/-0.05 h) was similar to the negative control rate. Acute (D54 degrees C = 0.55+/-0.07 h) cultures did not exhibit increased thermal resistance versus the control (D54 degrees C = 0.54+/-0.07 h). Thermal injury was determined as difference in D54 degrees C value (deltaD54 degrees c) obtained on pyruvate and deoxycholate media. Thermal-induced injury was not observed in either control (deltaD54 degrees C = 0.04 h) or acute (deltaD54 degrees C = 0.05 h) cultures.