Survey of raw milk cheeses for microbiological quality and prevalence of foodborne pathogens.

Cheese may be manufactured in the United States using raw milk, provided the cheese is aged for at least 60 days at temperatures not less than 35°F (1.7°C). There is now increased concern among regulators regarding the safety of raw milk cheese due to the potential ability of foodborne pathogens to survive the manufacturing and aging processes. In this study, 41 raw milk cheeses were obtained from retail specialty shops, farmers' markets, and on-line sources. The cheeses were then analyzed for the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, Staphylococcus aureus, and Campylobacter. Aerobic plate counts (APC), coliform and yeast/mold counts were also performed. The results revealed that none of the enteric pathogens were detected in any of the samples tested. Five samples contained coliforms; two of those contained E. coli at less than 10(2) cfu/g. Three other cheese samples contained S. aureus. The APC and yeast-mold counts were within expected ranges. Based on the results obtained from these 41 raw milk cheeses, the 60-day aging rule for unpasteurized milk cheeses appears adequate for producing microbiologically safe products.

A dose dependent impact of prebiotic galactooligosaccharides on the intestinal microbiota of healthy adults.

The goal of this research was to determine the effect of different doses of galactooligosaccharide (GOS) on the fecal microbiota of healthy adults, with a focus on bifidobacteria. The study was designed as a single-blinded study, with eighteen subjects consuming GOS-containing chocolate chews at four increasing dosage levels; 0, 2.5, 5.0, and 10.0g. Subjects consumed each dose for 3 weeks, with a two-week baseline period preceding the study and a two-week washout period at the end. Fecal samples were collected weekly and analyzed by cultural and molecular methods. Cultural methods were used for bifidobacteria, Bacteroides, enterobacteria, enterococci, lactobacilli, and total anaerobes; culture-independent methods included denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qRT-PCR) using Bifidobacterium-specific primers. All three methods revealed an increase in bifidobacteria populations, as the GOS dosage increased to 5 or 10g. Enumeration of bifidobacteria by qRT-PCR showed a high inter-subject variation in bifidogenic effect and indicated a subset of 9 GOS responders among the eighteen subjects. There were no differences, however, in the initial levels of bifidobacteria between the responding individuals and the non-responding individuals. Collectively, this study showed that a high purity GOS, administered in a confection product at doses of 5g or higher, was bifidogenic, while a dose of 2.5g showed no significant effect. However, the results also showed that even when GOS was administered for many weeks and at high doses, there were still some individuals for which a bifidogenic response did not occur.

Comparative genomics of the lactic acid bacteria.

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones.

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene. In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11. These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones.

Role of sigma(B) in adaptation of Listeria monocytogenes to growth at low temperature.

The activity of sigma(B) in Listeria monocytogenes is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, sigma(B) contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.

Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria.

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.

Mutational analysis of the role of HPr in Listeria monocytogenes.

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.

Bacteriocins inhibit glucose PEP:PTS activity in Listeria monocytogenes by induced efflux of intracellular metabolites.

Glucose transport by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) of listeria monocytogenes is inhibited by the bacteriocins, nisin, pediocin JD and leuconocin S. To investigate the mechanism of inhibition, PTS activity assays were performed with permeabilized, bacteriocin-treated L. monocytogens Scott A cells. In the presence of exogenous PEP, nisin stimulated the PTS while both pediocin JD and leuconocin S partially inhibited its activity. These results suggested that PTS enzymes were still active in bacteriocin-treated cells and the bacteriocin-induced PEP efflux may be a mechanism for inhibition of the PTS. To verify that PEP did efflux from bacteriocin-treated L. monocytogens Scott A cells, intracellular and extracellular PEP were measured by HPLC. All three bacteriocins induced efflux of PEP. Nisin, pediocin JD and leuconocin S also induced efflux of AMP, ADP and ATP. These studies indicate that bacteriocin inhibition of the glucose PEP:PTS in L. monocytogenes is due to efflux of intracellular metabolites, particularly

Cloning and expression of the Listeria monocytogenes scott A ptsH and ptsI genes, coding for HPr and enzyme I, respectively, of the phosphotransferase system.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of Listeria monocytogenes, we identified the ptsH and ptsI genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified L. monocytogenes HPr overexpressed in Escherichia coli was also capable of complementing an HPr defect in heterologous extracts of Staphylococcus aureus ptsH mutants. Additional studies of the transcriptional organization and control indicated that the ptsH and ptsI genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.

Identification of the gene encoding the alternative sigma factor sigmaB from Listeria monocytogenes and its role in osmotolerance.

Listeria monocytogenes is well known for its robust physiology, which permits growth at low temperatures under conditions of high osmolarity and low pH. Although studies have provided insight into the mechanisms used by L. monocytogenes to allay the physiological consequences of these adverse environments, little is known about how these responses are coordinated. In the studies presented here, we have cloned the sigB gene and several rsb genes from L. monocytogenes, encoding homologs of the alternative sigma factor sigmaB and the RsbUVWX proteins, which govern transcription of a general stress regulon in the related bacterium Bacillus subtilis. The L. monocytogenes and B. subtilis sigB and rsb genes are similar in sequence and physical organization; however, we observed that the activity of sigmaB in L. monocytogenes was uniquely responsive to osmotic upshifting, temperature downshifting, and the presence of EDTA in the growth medium. The magnitude of the response was greatest after an osmotic upshift, suggesting a role for sigmaB in coordinating osmotic responses in L. monocytogenes. A null mutation in the sigB gene led to substantial defects in the ability of L. monocytogenes to use betaine and carnitine as osmoprotectants. Subsequent measurements of betaine transport confirmed that the absence of sigmaB reduced the ability of the cells to accumulate betaine. Thus, sigmaB coordinates responses to a variety of physical and chemical signals, and its function facilitates the growth of L. monocytogenes under conditions of high osmotic strength.

Bacteriocin inhibition of two glucose transport systems in Listeria monocytogenes.

Listeria monocytogenes transports glucose by proton motive force-mediated and phosphoenolpyruvate-dependent phosphotransferase systems (PEP-dependent PTS). Inhibition of both systems by nisin, pediocin JD and leuconosin S is reported here for four strains of L. monocytogenes. Intracellular and extracellular adenosine triphosphate (ATP) and extracellular inorganic phosphate were measured in energized L. monocytogenes Scott A cells to determine whether inhibition of the PEP-dependent PTS might occur as a result of bacteriocin-induced leakage of intracellular components. Addition of nisin resulted in a decrease in intracellular ATP with an increase in extracellular ATP. Leuconosin S and pediocin JD induced a depletion of intracellular ATP. ATP efflux was low for the leuconosin S-treated cells and barely detectable for pediocin JD-treated cells. Addition of nisin, leuconosin S and pediocin JD induced efflux of inorganic phosphate. It appears that bacteriocin-mediated inhibition of the glucose PEP-dependent PTS occurs as a result of hydrolysis or efflux of ATP, PEP and other essential molecules from L. monocytogenes cells.

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.

Cloning and expression of the Zymomonas mobilis "production of ethanol" genes in Lactobacillus casei.

This study describes the expression of the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) in Lactobacillus casei 686. To promote transcription, the promoter and ribosome binding site (RBS) from the Lactococcus lactis subsp. lactis-derived vector, pMGE36e, were inserted upstream of the pdc gene. The former sequences were positioned such that translation of pdc was coupled to translation of an 81-base pair open reading frame terminating within the pdc initiation site. The recombinant plasmid (pRSG02) was electroporated into L. casei, and transformants were obtained. Northern analysis confirmed the production of a 3. 1-kb transcript corresponding to the predicted size of the PET operon. Western blot analyses revealed that the recombinant strain expressed both enzymes. The recombinant produced more than twice the ethanol produced by the parental L. casei strain.

Cloning and characterization of the galactokinase gene from Streptococcus thermophilus.

The objective of this research was to clone and characterize the galactokinase gene (galK) from Streptococcus thermophilus F410. Partially digested genomic DNA was cloned into pBR322 and transformed into galK Escherichia coli, and a galactose-fermenting transformant was isolated. Restriction analysis revealed that the transformant resulted from a Sau3A-HindIII 4.0-kb fragment. Galactokinase activity in the recombinant was 10 times that of the parent strain. Analysis of the DNA sequence showed the presence of a 1.3-kb open reading frame that had high homology with the galK gene from other organisms. A putative ribosome-binding site, start and stop codons, and -10 and -35 sequences were identified. The predicted protein had a molecular mass of 49 kDa, which corresponded to the estimated size of a band apparent by SDS-PAGE. Amino acid sequence homologies with other galactokinases ranged from 50 to 62% similarity. Northern blots were performed between the galK gene and mRNA from S. thermophilus. No hybridization signals were observed for cells grown in glucose, but cells grown in lactose or galactose gave moderate and strong signals. The results suggest that repression of the galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.

Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD.

Glucose uptake by Listeria monocytogenes Scott A was inhibited by the bacteriocin pediocin JD and by the protonophore carbonyl cyanide m-chlorophenyhydrazone. Experiments with monensin, nigericin, chlorhexidine diacetate, dinitrophenol, and gramicidin, however, showed that glucose uptake could occur in the absence of a proton motive force. L. monocytogenes cell extracts phosphorylated glucose when phosphoenolpyruvate (PEP) was present in the assay mixture, and whole cells incubated with 2-deoxyglucose accumulated 2-deoxyglucose-6-phosphate, indicating the presence of a PEP-dependent phosphotransferase system in this organism. Glucose phosphorylation also occurred when ATP was present, suggesting that a proton motive force-mediated glucose transport system may also be present. We conclude that L. monocytogenes Scott A accumulates glucose by phosphotransferase and proton motive force-mediated systems, both of which are sensitive to pediocin JD.

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.

Lactose Uptake Driven by Galactose Efflux in Streptococcus thermophilus: Evidence for a Galactose-Lactose Antiporter.

Galactose-nonfermenting (Gal) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated [C]lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force (Deltap) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a Deltap of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in GalS. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system.

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus.

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.

Carbohydrate Metabolism by Streptococcus thermophilus : A Review.

Despite the widespread use of Streptococcus thermophilus as a starter culture in the manufacture of many fermented dairy products, only recently has an understanding of the basic processes regarding carbohydrate metabolism been developed. Although S. thermophilus is related to other lactic streptococci by virtue of their common use in dairy fermentations, available information indicates that S. thermophilus is serologically, genetically, and physiologically distinct from the Group N, mesophilic streptococci. Carbohydrate metabolism, in particular, occurs by different processes in S. thermophilus than in the Group N streptococci ( Streptococcus lactis and Streptococcus cremoris ). The latter organisms utilize lactose by a specific phosphoenolpyruvate-dependent phosphotransferase system in which the lactose hydrolysis products, glucose and galactyose-6-phosphate, are concurrently metabolized to lactic acid. In contrast, S. thermophilus lacks phosphotransferase activity and instead possesses a lactose permease. After hydrolysis by ß-galactosidase, only glucose is further metabolized and galactose is released into the extracellular medium. Most strains are unable to ferment galactose and are phenotypically galactose-negative. The rapid growth rates of S. thermophilus on lactose and slow growth rates on glucose and galactose are likely due to the differences between the lactose and monosaccharide transport activities. Galactose transport by S. thermophilus requires an exogenous energy source and is mediated by a galactose permease. Galactose is further metabolized in galactose-positive cells by the enzymes of the Leloir pathway, specifically, galactokinase, galactose-1-phosphate uridyl transferase, and uridine-5-diphospho-glucose-4-epimerase. The latter two enzymes are eonstituitively expressed; however, in galactose-positive cells galactokinase and the galactose permease are induced by galactose in the absence of lactose. The phenotypic differences between galactose-positive and galactose-negative S. thermophilus are, in part, due to differences in the galactokinase and galactose permease activities. Galactose released into the medium by lactose-grown, galactose-positive cells can be subsequently metabolized, homofermentatively, to lactic acid. However, the important practical implications of released galactose has produced the need for isolation and development of S. thermophilus strains which ferment the lactose components, glucose and galactose, completely and simultaneously.

Phosphotransferase Activity in Clostridium acetobutylicum from Acidogenic and Solventogenic Phases of Growth.

Clostridium acetobutylicum cells, when energized with fructose, transported and phosphorylated the glucose analog 2-deoxyglucose by a phosphoenolpyruvate-dependent phosphotransferase (PT) system. Butanol up to 2% did not inhibit PT activity, although its chaotropic effect on the cell membrane caused cellular phosphoenolpyruvate and the 2-deoxyglucose-6-phosphate to leak out. Cells harvested from the solventogenic phase of batch growth had a significantly lower PT activity than did cells from the acidogenic phase.