Acetoacetate and ethyl acetoacetate as novel inhibitors of bacterial biofilm.

Acetoacetate (AAA) was identified as a biofilm inhibitor in a previous study, where the effect of 190 carbon and nitrogen sources on biofilm amounts by Escherichia coli O157:H7 was determined. With this study, we tested the effect of AAA on growth and biofilm amounts of Cronobacter sakazakii, Serratia marcescens and Yersinia enterocolitica. AAA reduced growth and biofilm amounts of the three pathogens, albeit at rather high concentrations of 10 to 35 mg ml-1 . Acetoacetate at a concentration of 5 mg ml-1 reduced Y. enterocolitica mRNA transcripts of the flagellar master regulator operon flhD, the invasion gene inv, and the adhesion gene yadA. Transcription of the regulator of plasmid-encoded virulence genes virF, the plasmid-encoded virulence gene yopQ, and ymoA were largely unaffected by AAA. Importantly, AAA did not cause an increase in transcription of any of the tested virulence genes. As a more cost efficient homologue of AAA, the effect of ethyl acetoacetate (EAA) was tested. EAA reduced growth, biofilm amounts and live bacterial cell counts up to 3 logs. IC50 values ranged from 0·31 mg ml-1 to 5·6 mg ml-1 . In summary, both AAA and EAA inhibit biofilm, but EAA appears to be more effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial biofilms are communities of bacteria that form on surfaces and are extremely difficult to remove by conventional physical or chemical techniques, antibiotics or the human immune system. Despite advanced technologies, biofilm still contributes to 60 to 80% of human bacterial infections (NIH and CDC) and cause problems in many natural, environmental, bioindustrial or food processing settings. The discovery of novel substances that inhibit biofilm without increasing the virulence of the bacteria opens doors for countless applications where a reduction of biofilm is desired.

ß-Phenylethylamine as a novel nutrient treatment to reduce bacterial contamination due to Escherichia coli O157:H7 on beef meat.

Bacterial infection by Escherichia coli O157:H7 through the consumption of beef meat or meat products is an ongoing problem, in part because bacteria develop resistances towards chemicals aimed at killing them. In an approach that uses bacterial nutrients to manipulate bacteria into behaviors or cellular phenotypes less harmful to humans, we screened a library of 95 carbon and 95 nitrogen sources for their effect on E. coli growth, cell division, and biofilm formation. In the initial screening experiment using the Phenotype MicroArray(TM) technology from BioLog (Hayward, CA), we narrowed the 190 starting nutrients down to eight which were consecutively tested as supplements in liquid beef broth medium. Acetoacetic acid (AAA) and ß-phenylethylamine (PEA) performed best in this experiment. On beef meat pieces, PEA reduced the bacterial cell count by 90% after incubation of the PEA treated and E. coli contaminated meat pieces at 10°C for one week.

A combination of assays reveals biomass differences in biofilms formed by Escherichia coli mutants.

AIMS: The aim of this study was to develop an assay system that can quantify the amount of biomass in biofilms formed by different isogenic mutants of an Escherichia coli K-12 strain. METHODS AND RESULTS: The reported assay, which is based on the BacTiter-Glo assay from Promega, uses bioluminescence to detect the intracellular concentration of ATP, which correlates with viable bacterial cell numbers. The quantitative data obtained with this ATP assay were compared to those obtained with the conventional crystal violet assay. As a qualitative control, scanning electron microscopy was performed. CONCLUSIONS: The ATP assay, the crystal violet assay and scanning electron microscopy yielded similar results for six of the eight strains tested. For the remaining two strains, the images from the scanning electron microscopy confirmed the results from the ATP assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The ATP assay, in combination with other quantitative and qualitative assays, will allow us to perform genetic studies on the regulatory network that underlies the early steps in E. coli biofilm formation.

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli.

Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K-12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC'-'lacZ translational fusion was expressed at three- to fourfold higher levels in csrA wild-type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription-translation of flhDC'-' lacZ in S-30 extracts and bound to the 5' segment of flhDC mRNA in RNA mobility shift assays. The steady-state level of flhDC mRNA was higher and its half-life was approximately threefold greater in a csrA wild-type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post-transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.

The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group.

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group.

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.

Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group.

The Bacillus cereus group comprises the four valid species Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis and bacillus anthracis. Some isolates of B. cereus are known to be psychrotolerant (growth at 7 degrees C or below). Here, specific sequence differences are described between the 16S rDNA, the 23S rDNA, the 16S-23S rDNA spacer region and the genes of the major cold-shock protein homologue cspA in a variety of psychrotolerant and mesophilic B. cereus and B. mycoides strains. Randomly amplified polymorphic DNA analysis using three different primers clearly separated psychrotolerant strains of both species from the rest of the B. cereus group, as did inverse PCR patterns of the rDNA operons. These data strongly support a hitherto unrecognized fifth sub-group within the B. cereus species group comprising psychrotolerant, but not mesophilic, B. cereus strains. Despite the latter finding, the DNA sequences investigated exhibited a high degree of sequence similarity indicating a close relationship between the species of the B. cereus group. Considering the unusual importance of B. cereus in both food poisoning and food spoilage and to avoid merging all species of the group, a new species, Bacillus weihenstephanensis sp. nov., comprising psychrotolerant 'cereus' strains, is proposed. Isolates of the new species grow at 4-7 degrees C but not at 43 degrees C and can be identified rapidly using rDNA or cspA targeted PCR. The type strain is B. weihenstephanensis WSBC 10204T (= DSM 11821T).

Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli.

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Cell cycle regulation of flagellar genes.

The expression of the flagellar master operon, flhDC, peaked in the middle of three consecutive cell cycles. The level of expression was lowest at the time of cell division. The expression of the second-level operon, flhB, peaked at cell division. The swimming speed of individual cells was also highest at the time of cell division.

The Escherichia coli flagellar transcriptional activator flhD regulates cell division through induction of the acid response gene cadA.

FlhD is a positive regulator of cadA. A mutant with a transposon-mediated lacZ fusion to cadA exhibited a cell division phenotype similar to that of the flhD mutant and had FlhD-dependent beta-galactosidase activity. Under different growth conditions, the cell division rate correlated with the level of expression of cadA.

A regulator of the flagellar regulon of Escherichia coli, flhD, also affects cell division.

The role of an activator of flagellar transcription in Escherichia coli, flhD, was investigated in the regulation of cell division. When grown in tryptone broth, flhD mutant cells divided exponentially until they reached a cell density of 2.5 x 10(9) cells per ml. Wild-type cells and flhC mutant cells divided exponentially until they reached a cell density of 4 x 10(7) cells per ml. flhD mutant cells divided 5 times more than wild-type cells before they reduced their cell division rate and reached a cell density 37 times higher than that of wild-type or flhC mutant cultures. In stationary phase, the biomasses of all cultures were similar; however, flhD mutant cells were significantly smaller. Additional tryptone, Casamino Acids, and individual amino acids, added at the beginning of growth, allowed wild-type cells to grow to higher cell densities. Serine was determined to have the greatest effect. In contrast, the addition of Casamino Acids did not exhibit an effect upon flhD mutant cells. flhD mutant cells exhibited normal rates of uptake of serine and other amino acids. In both wild-type and flhD mutant cultures, the concentrations of serine in the media dropped from 140 to 20 microM within the first 2 h of growth. Serine concentrations and cell division rates were highly correlated. Wild-type cells reduced their cell division rate at a medium concentration of 50 microM serine, and the addition of serine at this time caused cells to resume a higher rate of division. We conclude that the reduction of the cell division rate in wild-type cells is caused by the depletion of serine from the medium and that flhD mutant cells seem to be unable to sense this depletion.

Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli.

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids.

We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1. Cells carrying any of these three mutations exhibited identical phenotypes. Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium. As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate. The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate. We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth. During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate. As they began the transition to stationary phase, both cell types consumed L-tryptophan. Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.