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PubMed2006

Role of penicillin-binding protein 1b in competitive stationary-phase survival of Escherichia coli.

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.

Bacterial Proteins↗
PubMed2007

Survival of Campylobacter jejuni in different gas mixtures.

Campylobacter jejuni in fresh chilled chicken meat is known to be a major risk factor for human gastrointestinal disease. In the present study, the survival under chilled conditions of different C. jejuni strains exposed to different gas mixtures usually used for gas packaging of food was examined. Bolton broth and fresh, skinless chicken fillets were inoculated with six and four strains, respectively, and exposed to the gas mixtures 70/30% O(2)/CO(2), 70/30% N(2)/CO(2), and 100% N(2) (the latter only investigated in broth) at refrigeration temperature (4-5 degrees C). In broth culture, the strains survived significantly longer when exposed to 100% N(2) and 70/30% N(2)/CO(2) than in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001). For the two anaerobic gas mixtures, the reductions only reached 0.3-0.8 log(10) CFU mL(-1) within the same period. In the presence of oxygen, the numbers of C. jejuni were reduced by a minimum of 4.6 log(10) CFU mL(-1) over 21 days. When inoculated onto chicken fillets, the C. jejuni strains also died significantly faster in the oxygen-containing gas mixture, 70/30% O(2)/CO(2) (P<0.0001), reaching reductions of 2.0-2.6 log(10) CFU g(-1) after 8 days. In the gas mixture without oxygen (70/30% N(2)/CO(2)), no reductions were observed.

Animals↗
PubMed2006

Persistence of Helicobacter pylori in the oral cavity after systemic eradication therapy.

AIM: The present study aimed to evaluate if the oral cavity of chronic periodontitis patients can harbor Helicobacter pylori after systemic eradication therapy. MATERIALS AND METHODS: Samples of 30 patients (15 with gingivitis and 15 with chronic periodontitis) positive for H. pylori in the stomach were evaluated. Samples were collected 3 months after triple systemic antibiotic therapy from saliva, microbiota from the dorsum of the tongue, supra- and sub-gingival plaque as well as gastric biopsies. DNA of each sample was extracted by the boiling method and used as a template in polymerase chain reaction with the primers JW22/23. RESULTS: Eighteen patients (60%) harboured H. pylori in their mouths. Five patients (16.6%) were positive in saliva, two (6.6%) on the dorsum of the tongue, nine (30%) in supra-gingival plaque, 14 (46.6%) in sub-gingival plaque and three (10%) in the stomach. There was no statistically significant difference between study groups. CONCLUSION: Eradication of H. pylori after therapy was more effective for the stomach than for the mouth (p<0.001). Mouths of patients with gingivitis or with chronic periodontitis, who are positive for H. pylori in their stomachs, may be considered as reservoirs of these bacteria.

Adult↗
PubMed2006

Differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study.

BACKGROUND: The purpose of this clinical cross-over study was to examine the antibacterial and plaque-inhibiting properties of two chlorhexidine solutions compared with a negative control. MATERIAL AND METHODS: Twenty-one volunteers refrained from all oral hygiene measures, but rinsed instead twice daily with 10 ml of a conventional chlorhexidine solution (0.2%; CHX), a chlorhexidine solution with anti-discolouration system (ADS) (0.2%, alcohol-free chlorhexidine solution (CSP)) or a placebo solution (Pla). Plaque index (PI), plaque area (PA) and bacterial vitality were assessed after 24 h (PI1, vital flora (VF)1) and 96 h (PI2; VF2, PA). After a 10-day wash-out period, a new test cycle was started. RESULTS: Results for Pla were 0.94, 1.59, 27.4 (PI1, PI2, PA) and 79% and 72% (VF1 and VF2). CSP significantly reduced the parameter PI1, PI2 and PA to 0.67 (p=0.012), 1.0 and 15.7 (p<0.001). VF1 and VF2 (63% and 53%) were not significantly affected. The corresponding figures of CHX were 0.42, 0.43, 6.77, 33 and 16%, which were all significantly lower (all p<0.001). On comparing the two chlorhexidine solutions, CHX showed significantly higher reductions of all parameters. CONCLUSION: The results suggest that the 0.2% alcohol-containing solution showed superiority in inhibiting plaque re-growth and reducing bacterial vitality compared with the solution with ADS.

Adult↗
PubMed2005

Lethal photosensitization of oral pathogens via red-filtered halogen lamp.

UNLABELLED: OBJECTIVE OF INVESTIGATION: The ability of laser irradiation in the presence of photosensitizing agent to induce lethal effect on oral bacteria is well documented. We designed an in-vitro experiment to achieve phototoxic results on two common oral pathogens, using a high intensity, red filtered halogen lamp. Our goal was to determine the minimum duration of light exposure and drug dilution to achieve at least 50% reduction in bacteria counts. METHODS: Two common oral pathogens, Porphyromonas gingivalis and Prevotella intermedia were used in experiments. The source for light energy was a continuous working, high intensity, red filtered, halogen lamp (HL) with light transmitted through a flexible light guide over petri dishes containing live bacteria. Microorganisms were exposed to light for 5, 10 and 20 min. Methylene Blue (MB) in concentrations of 0.1, 0.075, 0.05, 0.025 and 0.01% was used as a photosensitizing agent. Light energy alone and MB alone was used as controls. RESULTS AND DISCUSSION: Optimum lethal photosensitization (50% or more bacteria killing) of oral pathogens was achieved using halogen light illumination for 5 min and longer with 0.05% MB or exposure to light for 20 min in the presence of 0.025 and 0.01% MB. Light exposure of 20 min in the absence of MB was not effective in killing bacteria. In the absence of light, MB at concentrations of 0.025 and 0.001% was not effective. Reduction of bacteria with the use of 0.05% MB alone was also insignificant. However, 0.075 and 0.1% MB, even in the absence of light was found to be bacteriocidal. CONCLUSIONS: Our in-vitro data indicate that we were able to achieve lethal photosensitization of two common oral pathogens with high intensity red filtered HL in the presence of diluted MB. In this era of increased incidence of antibiotic resistance, bacterial killing with laser or light energy in the presence of photosensitizing agent can prove to be a valuable treatment modality.

Filtration↗
PubMed2006

Fusion of fungicidal peptide dhvar4 to enterococcal peptide pheromone increases its bactericidal activity against Enterococcus faecalis.

Bacterial peptide pheromone has a high affinity to its membrane receptor. Fusion of these peptides to pore-forming antimicrobial peptide might enhance its bactericidal activity against pheromone-sensing bacteria. We constructed two chimeric peptides by fusing the pore-forming fungicidal peptide dhvar4 to the C-terminus of enterococcal peptide pheromones cCF10 and cOB1 individually. Comparison on the bactericidal activities against pheromone-sensing bacteria Enterococcus faecalis demonstrates that the chimeric peptides cCF10-dhvar4 and cOB1-dhvar4 are more potent than the parent peptide dhvar4. The LD(50)s of both chimeric peptides (1.0 microm) are 10 times lower than that of dhvar4 (10.8 microm). Free peptide pheromone could inhibit E. faecalis killing mediated by both chimeric peptides. As same as that of the parent peptide, both chimeric peptides kill bacteria by disrupting its cell membrane. These results indicate that fused enterococcal peptide pheromone increases the bactericidal activity of fungicidal peptide against E. faecalis by improving its ability to reach the cell membrane.

Anti-Bacterial Agents↗
PubMed2006

Human IRGM induces autophagy to eliminate intracellular mycobacteria.

Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.

Animals↗
PubMed2006

Antibacterial activity and mechanism of action of a novel anilinouracil-fluoroquinolone hybrid compound.

The anilinouracils (AUs) such as 6-(3-ethyl-4-methylanilino)uracil (EMAU) are a novel class of gram-positive, selective, bactericidal antibacterials which inhibit pol IIIC, the gram-positive-specific replicative DNA polymerase. We have linked various fluoroquinolones (FQs) to the N-3 position of EMAU to generate a variety of AU-FQ "hybrids" offering the potential for targeting two distinct steps in DNA replication. In this study, the properties of a hybrid, "251D," were compared with those of representative AUs and FQs in a variety of in vitro assays, including pol IIIC and topoisomerase/gyrase enzyme assays, antibacterial, bactericidal, and mammalian cytotoxicity assays. Compound 251D potently inhibited pol IIIC and topoisomerase/gyrase, displayed gram-positive antibacterial potency at least 15 times that of the corresponding AU compound, and as expected, acted selectively on bacterial DNA synthesis. Compound 251D was active against a broad panel of antibiotic-resistant gram-positive pathogens as well as several gram-negative organisms and was also active against both AU- and FQ-resistant gram-positive organisms, demonstrating its capacity for attacking both of its potential targets in the bacterium. 251D also was bactericidal for gram-positive organisms and lacked toxicity in vitro. Although we obtained strains of Staphylococcus aureus resistant to the individual parent compounds, spontaneous resistance to 251D was not observed. We obtained 251D resistance in multiple-passage experiments, but resistance developed at a pace comparable to those for the parent compounds. This class of AU-FQ hybrids provides a promising new pharmacophore with an unusual dual mechanism of action and potent activity against antibiotic-sensitive and -resistant gram-positive pathogens.

Aniline Compounds↗
PubMed2006

Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate.

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4 degrees C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

Animals↗
PubMed2006

Acanthamoeba castellanii promotion of in vitro survival and transmission of coxsackie b3 viruses.

This work was undertaken to determine whether Acanthamoeba could play a role in the survival and transmission of coxsackieviruses and focused on in vitro interactions between Acanthamoeba castellanii and coxsackie B3 viruses (CVB-3). Residual virus titer evaluations and immunofluorescence experiments revealed a remarkable CVB-3 adsorption on amoeba surfaces and accumulation inside cells. The survival of viruses was independent of the dynamics of amoeba replication and encystment. In addition, our results indicated that virus-infected amoebas can release infectious viruses during interaction with human macrophages. On the basis of these data, Acanthamoeba appears to be a potential promoter of the survival of coxsackieviruses and their transmission to human hosts.

Acanthamoeba castellanii↗
PubMed2006

Affinity-purified human immunoglobulin G that binds a lacto-N-neotetraose-dependent lipooligosaccharide structure is bactericidal for serogroup B Neisseria meningitidis.

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.

Antibodies, Bacterial↗
PubMed2006

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium.

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Acids↗
PubMed2007

Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo.

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.

Adaptation, Physiological↗
PubMed1998

Coiling phagocytosis discriminates between different spirochetes and is enhanced by phorbol myristate acetate and granulocyte-macrophage colony-stimulating factor.

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borrelia burgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides--microbes and phagocytes--in coiling phagocytosis. As seen with electron microscopy, morphologically similar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, but pseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B. burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.

Borrelia burgdorferi Group↗
PubMed2006

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus.

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Alphaproteobacteria↗
PubMed2006

Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease.

Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this phenomenon as endogenous MceA bactericidal activity to differentiate it from the action of extracellularly secreted MccE492. The toxicity of endogenous MceA is enhanced by an efficient targeting to the inner membrane. However, a periplasmic intermediate state is not required for MceA toxicity. Indeed, endogenous MceA remains fully active when it is fused to thioredoxin-1, a fast-folding protein that promotes retention of the C terminus of MceA in the cytoplasm. The C-terminal domain of MccE492 is required only for delivery from the extracellular environment to the periplasm, and it is not required for inner membrane damage. A common component is absolutely essential for the bactericidal activity of both endogenous MceA and extracellular MccE492. Indeed, toxicity is strictly dependent on the presence of ManYZ, an inner membrane protein complex involved in mannose uptake. Based on these findings, we propose a new model for cell entry, inner membrane insertion, and toxic activity of MccE492.

Anti-Bacterial Agents↗