Number Of Gram-positive Bacteria Research Articles

Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes

BackgroundIn a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis.Methodology/Principal FindingsBy qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there.Conclusions/SignificanceOur data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat.

Listeria monocytogenesPrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol

In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection.

Antagonistic activity against nosocomial clinical isolates withBacillus subtilis MZ-7

Antibacterial activities were detected in thirty-six bacteria from six soil samples. One strain designated as Mz-7 produced an inhibitory substance which was active against number of Gram-positive bacteria as well as clinical isolates obtained from a local hospital. Strain Mz-7 was identified asBacillus subtilis by 16S rRNA sequence analysis and standard biochemical tests. Maximum production was observed at mid of stationary phase slightly before sporulation took place. Optimum conditions for growth were standardised. The antibiotic product was purified by chloroform precipitation and detected by polyacrylamide gel electrophoresis and mass spectrometry. It has lipopeptide nature and did not lose its activity on treatment at different pH values, temperatures and enzymes. The pronounce activity against multidrug resistant clinical isolates and the favourable biochemical properties of this antibiotic from the newly isolate strain suggest a new and effective agent against resistance in pathogenic microbes.

Complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-( N-methyl)isatin. : A study of their antimicrobial activity

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-( N-methyl)isatin (HL 1 and HL 2, respectively) were synthesized and characterized, being the crystal structures of HL 1, HL 2 and [Ni(L 1) 2] · 2CHCl 3 elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL 1and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15–1.50 μg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3–25 μg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50 μg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.

A double-blind, placebo-controlled, parallel group study of oral trovafloxacin on bowel microflora in healthy male volunteers

Background: Treatment with oral antibiotic drugs generally influences normal fecal flora. These changes can be both beneficial (eg, elimination of aerobic, gram-negative bacilli) and detrimental (eg, the appearance of resistant pathogenic micro-organisms). Trovafloxacin, a new fluoroquinolone with in vitro activity against anaerobes, and gram-negative, gram-positive, and atypical pathogens, is a potentially beneficial antimicrobial for bowel sterilization. This double-blind trial investigated the effect of trovafloxacin on the normal microbial bowel flora of healthy male subjects. Methods: Subjects were randomized (in a 2:1 ratio) to receive either 200 mg trovafloxacin once daily for 10 days or a matching placebo. Fecal samples were collected at two baseline occasions, on visit days 4, 7, 10, and 17, and at follow-up. Bacterial species were identified and quantified in the fecal samples. Results: Twelve subjects received the active drug and seven received placebo. No Enterobacteriaceae were found in samples from days 4 to 10 in subjects receiving trovafloxacin. No changes in Enterobacteriaceae were found throughout the study in subjects receiving placebo. Incidental Enterobacteriaceae were isolated from subjects in the trovafloxacin group at the end of the study. No clinically significant differences were found in either group with respect to prevalence, appearance, or disappearance of aerobic gram-positive cocci, anaerobic bacteria, or yeasts. All tested Enterobacteriaceae were highly susceptible to trovafloxacin. No increase in minimum inhibitory concentration values was seen in day 17 and follow-up samples for isolated Escherichia coli strains. No Clostridium difficile was found in day 17 or follow-up samples from subjects in the trovafloxacin group. All tests for clostridium toxin were negative. Conclusions: During the treatment period, E. coli could not be cultured from the feces of the 12 healthy subjects receiving 200 mg trovafloxacin daily during days 4 to 10. All isolated Enterobacteriaceae were susceptible to trovafloxacin and no changes in susceptibility were found after the treatment period. In subjects treated with trovafloxacin, the prevalence and number of gram-positive bacteria were rapidly reduced. Trovafloxacin is able to selectively and reversibly suppress bowel flora.

Bacterial numbers and biomass in leachate and in subsoil of a submerged paddy field

By a leaching experiment with glass columns packed with submerged paddy soils, the relationships among numbers of total bacteria, total Gram-positive bacteria, culturare aerobic bacteria and a amount of bacterial biomass both in the leachate, and in the subsoil after leaching incubation were studied. The leachate from soil columns was collected every 3 d during the 30-d incubation period. The soil columns were packed with plow layer soil samples with and without rice straw (RS) amendment, and the subsoil column was connected to the plow layer soil column without RS. Numbers of total bacteria, culturable aerobic bacteria, and a amount of bacterial biomass in the leachate decreased with the incubation time. There was no correlation between the number of total bacteria in the leachate and the concentration of total organic carbon in the leachate. Bacteria less than 0.1 µsm3 in size predominated in the leachate, especially in that from the plow layer soil column with RS. Percentages of the number of Gram-positive bacteria in the leachate were very low (less than 7% of the total bacterial number), while the percentage in the subsoil after the leaching experiment was in the range of 21–82%. The sum of the number of bacteria percolated from the plow layer soil column with RS during the 30-d period of incubation and the sum of the amount of biomass C were 39 and 77% less than the corresponding values for the bacteria percolated from the plow layer soil column without RS. Percentages of culturable aerobic bacteria among the total bacteria ranged between 2.8 and 37% in the leachate, while less than 0.6% in the subsoil after the leaching experiment.

Mannich Reaction Product of Quinolin-8-ol (Oxine) and its Antibacterial Activity

Quinolin-8-ol (1) on reaction with 2,6-dichloroaniline and m-nitrobenzaldehyde afforded 7-(α-hydroxy- m-nitrobenzyl)quinolin-8-ol (2), which is an unusual Mannich reaction product. The structure of (2) was determined by spectroscopic and single-crystal X-ray diffraction studies. Preliminary antimicrobial screening showed that (1) and (2) have activity against a large number of Gram-positive and Gram-negative bacteria.

Improved cloning vectors and transformation procedure for Lactococcus lactis

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MG1363 of L. lactis which routinely yielded 1 x 10(7) to 5 x 10(7) transformants micrograms-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2.5% w/v. Although growth of L. lactis strain MG1363 was inhibited by the use of 0.5 mol l-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.

Bacteriological conditions of a Jamaican chicken hatchery

The bacteriological profile of a chicken hatchery in Jamaica was examined. The bacterial numbers in each room of the hatchery and the effect of washing with disinfectant on the bacterial population were determined. A representative number of the bacterial isolates before and after washing the hatchery was identified. The results indicate that, while washing with quaternary ammonium compounds did not affect the Gram-negative bacteria, the number of Gram-positive bacteria was significantly decreased. Bacteria of the genera Pseudomonas, Plesiomonas and Enterobacter were predominant in the post-washing flora. The water used to wash the hatchery was contaminated and therefore a possible source of contamination.

Levels of Vancomycin in Aqueous Humor After Topical Eye Drops Administration

Vancomycin is an antibiotic which is especially active against Gram positive bacteria. At present, numerous infections of the anterior segment of the eye are caused by the Staphilococcus aureus and epidermis. Strains which are resistant to methicilin are more and more frequent and for this reason Vancomycin is becoming the antibiotic of choice to combat these infections since no resistance of the Staphilococcus to it has been demonstrated. The authors evaluated the levels of Vancomycin in aqueous humor after the administration of topical eye-drops. For the first two hours after the last administration, levels of 0.52 micrograms/ml were detected. These inhibit the growth of the majority of bacteria sensitive to Vancomycin. Between two and four hours, the levels decreased to 0.15 micrograms/ml; these are therapeutic levels for a large number of Gram positive bacteria. From four hours after the last administration, these levels are undetectable. Due to the success of its penetration, topical administration of Vancomycin should be considered as a therapeutic modality against infections by gram positive bacteria of the anterior segment. The ideal guideline for administration would be one drop every two hours, especially if the infection is severe. In this way, subconjunctival injection could be avoided.

Production of a monoclonal antibody specific for aFlavobacteriumspecies isolated from soil

Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods (Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g−1 soil can be enumerated.

Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods ( Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g −1 soil can be enumerated.

Secretion of recombinant proteins into the culture medium by Escherichia coli and Staphylococcus aureus.

The ability of staphylococcal protein A (SPA) to bind to the Fc part of IgG has been used for the purification of a number of heterologous gene products as fusion proteins. Both the SPA promoter and signal sequence function in Escherichia coli, as well as in a number of Gram-positive bacteria, which facilitates comparisons of the expressed specific products in different hosts. The expression system developed for E. coli yields excretion of the fusion protein to the growth medium, which makes E. coli a competitive alternative to Gram-positive bacteria for the expression of secreted products. The human peptide hormones insulin-like growth factors (IGF) I and II were expressed using the protein A system in E. coli and Staphylococcus aureus. Despite a high degree of structural homology, large differences in the yields were observed in the two hosts. This underlines the importance of investigating different bacterial hosts for a particular protein product.

Lipopolymers, isoprenoids, and the assembly of the gram-positive cell wall.

Several lines of evidence suggest that Gram-positive bacterial cell surface polymers are synthesized by stepwise addition of polymer subunits to an amphipathic acceptor. In the case of membrane-bound lipopolymers such as mannan and lipoteichoic acid, the finished product may be covalently linked to a lipid anchor. In the case of polymers that are transferred into preexisting cell wall, such as teichoic acid and peptidoglycan, two alternative fates might be possible: (1) transfer into wall with concomitant or later cleavage of the lipid anchor, with recycling of the lipid anchor or secretion of the lipid anchor into the growth medium, and (2) transfer into wall without cleavage of the lipid anchor, resulting in maintenance of the covalent relationship between lipid anchor and polymer chain. In the latter case, a close relationship should be established between the cell wall and the plasma membrane. A number of Gram-positive bacteria have been shown to be resistant to plasmolysis. Therefore, a model for the assembly of the Gram-positive cell wall is proposed which takes into account a role for lipopolymeric intermediates and which views the establishment of resistance to plasmolysis as the natural consequence of such a mechanism.

ChemInform Abstract: SYNTHESIS, SPECTRAL BEHAVIOR AND MICROBIOLOGICAL PROPERTIES OF SOME DI(NITROTHIENYL)SULFIDES

AbstractBromnitrothjophene (I) reagieren mit Natriumsulfid zu symmetrischen Sulfiden (II)‐(IV).

The effect of dietary xylitol on the ability of rat caecal flora to metabolise xylitol.

The effect of dietary xylitol on the ability of the rat caecal flora to metabolise xylitol was investigated. Xylitol metabolism in micro-organisms has generally been assessed in terms of pH change and acid production which are often insensitive in demonstrating low rates of substrate utilisation. Using a rapid and sensitive radioisotopic assay, in which 14CO2 production from [U-14C] xylitol was measured, it was possible to show that the caecal microflora obtained from rats can metabolise xylitol. This activity was increased 10, 15, 30 and 40-fold in the caecal flora taken from rats fed diets containing 2.5,5,10 and 20% xylitol, respectively. Using the caecal microflora of normally fed rats, the order of 14CO2 production from 14C-labelled sugars and sugar alcohols was glucose greater than fructose greater than xylose greater than sorbitol greater than or equal to xylitol. The feeding of glucose and fructose did not alter the 14CO2 producing activities, whereas xylose feeding increased xylose metabolism, sorbitol feeding increased sorbitol and xylitol metabolism, xylitol feeding increased sorbitol, xylose and its own metabolism and arabitol feeding increased xylose and sorbitol metabolism. Marked changes were also observed in the population of the caecal flora of xylitol-fed rats, with increases in the number of gram-positive bacteria, compared to rats on a normal diet. Possible mechanisms for these effects involve mutation, selection of micro-organisms capable of metabolising sugar alcohols, and the induction of enzymes involved in sugar alcohol metabolism.

Significance of bacterial and white cell counts in midstream urines.

The results of cultures carried out on urine collected by a midstream technique (MSU) have been compared with the results of cultures of urine collected by suprapubic bladder aspiration (SPA) in the same 903 patients. Comparisons indicate that when there are more than 100 x 10(6)/l (100 000/ml) Gram-negative bacteria in the midstream urine this finding is confirmed by suprapubic aspiration in 92% of patients, and in 70% when the urine contained this number of Gram-positive bacteria. When the culture contained more than one species (mixed organisms), the presence of infection was confirmed in only 11%. When there were 10-100 x 10(6)/l bacteria in the MSU the finding of Gram-negative bacilli still indicated urinary infection in 74% of patients, but the presence of Gram-positive organisms was confirmed in only 30% and mixed organisms in 2%. In asymptomatic patients, the presence of white cells in the urine was not helpful in confirming the diagnosis of infection. The nature of the organism found in the MSU is almost as important as the number of bacteria in assessing the validity of the result.

Chemical studies on actinoxanthin.

The antitumor protein actinoxanthin exhibits high inhibitory activity against a number of gram-positive bacteria and some strains of transplantable leucoses and related tumors. Actinoxanthin was shown to consist of a single polypeptide chain crosslinked by two disulfide bonds and to contain 107 amino acid residues. Reduced and alkylated actinoxanthin was digested with chymotrypsin, thermolysin and trypsin. Based on the sequence analysis of fragments so obtained the complete amino acid sequence and the location of disulfide bonds of actinoxanthin has been proposed. The high degree homology of some regions of actinoxanthin and the antitumor protein neocarzinostatin have been revealed.

A new antibiotic X-5108 OF Streptomyces origin. I. Production, isolation and properties.

A new antibiotic, designated X-5108, produced by a new species of microorganism named Streptomyces goldiniensis var. goldiniensis, has been isolated in essentially pure but amorphous form. Assay methods, and cultivation and isolation procedures for the antibiotic are described. The antibiotic is active in vitro primarily against a number of gram-positive bacteria, whereas in vivo it is especially active against Streptococcus pyogenes infections of mice and as a poultry growth promotant. It shows a very low toxicity in mice with an LD50 of >lg/kg sbc, and >4 g/kg p.o. Its empirical formula is C44H62N2O12. Structural studies reveal one methoxyl, one N-methyl and six C-methyl groups. UV, IR and NMR spectra and optical rotation of the pure antibiotic are given. The antibiotic appears to belong to a new chemical class.