Increased Viability of Sugar Transport-Deficient Mutant of the Periodontal Pathogen, Aggregatibacter actinomycetemcomitans.

The periodontal pathogen, Aggregatibacter actinomycetemcomitans is extremely sensitive to even a mildly acidic pH resulting from metabolic acids secreted during growth, losing viability rapidly as the pH goes below 6.0. Cells grown at high glucose concentration grow fast but rapidly lose viability. However, if the cells are grown at low glucose concentration, the pH of the growth medium first decreases slowly for about 24 h and then starts to increase. This increase of pH is indicative of cell death since the spontaneous rise of pH due to the presence of bicarbonate can no longer be opposed by secreted metabolic acids. By monitoring these pH changes on a petri dish, a method was developed to screen for sugar transport-deficient mutants from a library of transposon insertion mutants. Isolation of a mannose phosphotransferase mutant strain is described. The mutant cells were found to be more viable and for a longer period of time than wild-type cells both in high and low glucose concentrations due to slower metabolism and less acid secreted. This observation highlights the concern that spontaneous mutations in the sugar transport genes may be selected for in patients due to increased viability of the mutant cells especially in a biofilm.

Maillard reaction products inhibit the periodontal pathogen Aggregatibacter actinomycetemcomitans by chelating iron.

OBJECTIVE: To determine the mechanism of growth inhibition of Aggregatibacter actinomycetemcomitans by Maillard reaction products (MRP). DESIGN: Growth and cell viabilities in the presence or absence of MRP were measured for both the rough and smooth variants of the bacteria. Effects of addition of ferrous and ferric ions on the inhibition of the bacteria by MRP were determined. RESULTS: MRPs decreased the extent of complex formation of Chrome Azurol S with iron suggesting that MRPs can chelate iron effectively. The chelation causes growth inhibition of both the rough and smooth strains. At low concentrations of the inhibitor, lag time was extended by approximately 12 h while at high concentrations, cells were killed, decreasing cell viability by up to 8 orders of magnitude. Growth of both the rough and smooth strains could be restored to original level by addition of iron. For the rough strain, both ferrous and ferric ions could relieve the inhibition by MRP while for the smooth strain only ferrous ion was effective. CONCLUSION: MRPs inhibit the growth of A. actinomycetemcomitans by chelating iron and the inhibition can be relieved by addition of iron.

A Water-Soluble Antibiotic in Rhubarb Stalk Shows an Unusual Pattern of Multiple Zones of Inhibition and Preferentially Kills Slow-Growing Bacteria.

Organic extract of Rhubarb (Rheum officinale) roots is known to have several medicinal uses. However, not much research has been done with the rhubarb stalk. The aim of this research is to evaluate the anti-bacterial and anti-proliferative effects of the aqueous extract from rhubarb stalks. The crude aqueous extract was further purified using anion exchange and gel filtration. The purified compound demonstrated broad spectrum antibacterial activity against the Gram-negative bacteria, E. coli and Aggregatibacter actinomycetemcomitans, and Gram-positive bacteria, S. aureus. A time-kill assay demonstrated that the antibiotic has strong bactericidal activity. It also has anti-proliferative action against the breast cancer cell line MCF-7 with no cytotoxicity, although the crude extract had a significant cytotoxic effect. The antibiotic activity, as measured by the diameter of the zone of inhibition, increased by several fold in low nutrient and/or low salt agar, suggesting that the antibiotic preferentially kills slow-growing bacteria. The antibiotic also gives an unusual pattern of multiple zones of inhibition in which several zones of cell growth are seen within the zone of inhibition. In conclusion, the active component in the aqueous extract of rhubarb stalk has great potential as a strong bactericidal antibiotic and as an anti-proliferative drug.

Antibiotic in myrrh from Commiphora molmol preferentially kills nongrowing bacteria.

AIM: To demonstrate that myrrh oil preferentially kills nongrowing bacteria and causes no resistance development. METHOD: Growth inhibition was determined on regular plates or plates without nutrients, which were later overlaid with soft agar containing nutrients to continue growth. Killing experiments were done in broth and in buffer without nutrients. RESULTS: Bacterial cells were inhibited preferentially in the absence of nutrients or when growth was halted by a bacteriostatic antibiotic. After five passages in myrrh oil, surviving colonies showed no resistance to the antibiotic. CONCLUSION: Myrrh oil has the potential to be a commercially viable antibiotic that kills persister cells and causes no resistance development. This is a rare example of an antibiotic that can preferentially kill nongrowing bacteria.

Bacterial conjugation in the cytoplasm of mouse cells.

Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

tfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.

Does microwave sterilization of growth media involve any non-thermal effect?

Fast reactions mediated by microwaves are often attributed by many to non-thermal effect. We show here that rapid formation of Maillard reaction products during microwave sterilization of growth medium results from concentration effect and not any non-thermal effect. This leads to an improved method for microwave sterilization of growth media.

Sensitivity of the periodontal pathogen Aggregatibacter actinomycetemcomitans at mildly acidic pH.

BACKGROUND: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a capnophilic facultative anaerobe, is associated with localized aggressive periodontitis and endocarditis. When grown in broth, the cells begin to die rapidly after overnight growth. The cells also often lose viability on plates within a few days. The aim of this study is to identify the cause of the rapid loss of cell viability. METHODS: Cell viabilities, as measured by colony forming units, were determined for cells obtained from isolated colonies and from the dense part of a streak on plates. The effect of pH on cell viability was determined by growing cells in broth at various initial glucose concentrations and with or without added bicarbonate. RESULTS: A. actinomycetemcomitans cells were highly sensitive to even a mildly acidic pH of ≈6. Because the bacteria grew at a glucose concentration that is commonly used in many laboratories, there was a dramatic decrease in cell viability as the pH went <6, which happened long before the culture reached saturation. This was easily avoided by using a lower initial glucose concentration, and under these conditions, the addition of bicarbonate to the growth medium was not necessary. Cells resuspended in buffer without nutrients lost viability much faster at pH 6 than at a higher pH. On plates, the cell viability was much higher in isolated colonies than in the dense area of the streak. CONCLUSIONS: A. actinomycetemcomitans cells rapidly lost viability at even a mildly acidic pH. The problem was easily rectified by growing cells at a low glucose concentration.

Microwave sterilization of growth medium alleviates inhibition of Aggregatibacter actinomycetemcomitans by Maillard reaction products.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative agent of localized aggressive periodontitis and endocarditis. The bacteria grow slowly even in a rich medium and rapidly lose viability after about 19 h of growth. One of the reasons for the slow growth and for the decreased viability is the conventional method of making growth media by autoclaving. Faster growth and greater viability were observed in both broth and plates if the growth media were sterilized by microwave radiation rather than by autoclaving. One difference between autoclaved and microwaved media is that the autoclaved media are darker brown in color, which is known to be due to the Maillard reaction products, also known as Amadori products. The Maillard reaction products formed by autoclaving a mixture of lysine and glucose were shown to inhibit growth of A. actinomycetemcomitans.