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PubMed2002

Microbiological assay for determination of ofloxacin injection.

A simple, sensitive and specific agar diffusion bioassay for the antibacterial ofloxacin was developed using a strain of Micrococcus luteus ATCC 9341 as the test organism, ofloxacin at concentration ranging 12-27 microg ml(-1) could be measured in injection. A prospective validation of the method showed that method was linear (r=0.9994) and precise (CV=1.14). UV spectrophotometric and high performance liquid chromatographic techniques were chosen as a comparison methods for the determination of ofloxacin. The results obtained by three methods were statistically analyzed by analysis of variance (ANOVA) and the results obtained indicate that there is no significant difference among these methods.

Anti-Infective Agents↗
PubMed2001

Adesmia aegiceras: antimicrobial activity and chemical study.

The antimicrobial activity of the ethanolic extract of Adesmia aegiceras was studied by the agar-well diffusion method. Antibacterial activity against Micrococcus luteus and eight pathogenic bacterial strains as well as antifungal activity against Candida albicans, was detected. Bacterial and fungal strains exhibited similar concentration-response curves (EC50 and Rmax values) and similar MIC. The MBC/MIC was about 8. These data would indicate the potential usefulness of the A. aegiceras extract as a microbiostatic, antiseptic or disinfectant agent. Furthermore, chemical study of the bioactive alcoholic extract was performed, which revealed quercetin, isorhamnetin-3-rutinoside, isovitexin, pinitol and chlorogenic acid as its main components.

Anti-Bacterial Agents↗
PubMed1999

In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution.

The aim of the present study was to evaluate the in vivo antimicrobial activity of 2% chlorhexidine gluconate (FCFRP-USP) used as a root canal irrigating solution in teeth with pulp necrosis and radiographically visible chronic periapical reactions. Culture techniques and measurement of the inhibition zone were used. Twenty-two root canals of incisors and molars of 12 patients were used. After accessing the canal, the first root canal sample was collected with two sterile paper points that were transferred to a tube containing reduced transport fluid. The root canal was instrumented using chlorhexidine solution. A small sterile cotton pellet was placed at the root canal entrance, and the cavity was sealed with zinc oxide-eugenol cement. The canals were maintained empty for 48 h. Three sterile paper points were then introduced to absorb the root canal fluid (second sample). One paper point was placed on an agar plate inoculated with Micrococcus luteus ATCC 9341 and incubated for 24 h at 37 degrees C, and the other two were submitted to microbiological evaluation. Present in 10 cases at baseline, mutans streptococci was reduced by 100% at the second assessment. Treatment showed an efficiency of 77.78% for anaerobic microorganisms at the second assessment. These data suggest that chlorhexidine prevents microbial activity in vivo with residual effects in the root canal system up to 48 h.

Adolescent↗
PubMed2005

Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses.

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.

Aedes↗
PubMed2002

A family of autocrine growth factors in Mycobacterium tuberculosis.

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

Bacterial Proteins↗
PubMedDate not supplied

Microbiological assay for the determination of telithromycin in tablets.

This study describes the development and validation of a microbiological assay, applying the cylinder-plate method, for the determination of the antibiotic telithromycin. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism. The response graphs for standard and sample solutions were linear (r = 0.9987), and no parallelism deviations were detected in the tested concentrations (0.25, 0.5, and 1.0 microg/mL). The interday precision was 2.67%. Recovery values were between 96.75 and 100.91%. A preliminary stability study of telithromycin showed that the microbiological assay is specific for the determination of telithromycin in the presence of its degradation products. The proposed method allows the quantitation of telithromycin in pharmaceutical dosage form and can be used for drug analysis in routine quality control.

Anti-Bacterial Agents↗
PubMed2006

Structure-activity relationships in nitrothiophenes.

The structure and electronic properties of a series of biologically active 2-nitrothiophenes (1) have been calculated using both semi-empirical and ab initio molecular orbital methods. Multi-linear regression analysis suggests that there is a reasonable correlation between the experimental activity of the derivatives against either Escherichia coli or Micrococcus luteus and calculated properties such as the HOMO energies, the total atomic charges and ring angles at the heterocyclic sulfur atom, but there is no correlation with the calculated solvation energies or dipole moments. The presence or absence of an additional nitro group at the 3-position of the ring also has a significant effect on the activity. From the derived QSAR equations, the 2-chloro- or 2-bromo-3,5-dinitrothiophenes (1a and 1c) are predicted to show the highest activity against both bacteria, while 2-nitrothiophene (1n) is predicted to be the least active, in line with the experimental results.

Anti-Bacterial Agents↗
PubMed2006

Bacterial contamination in the anterior chamber after povidone-iodine application and the effect of the lens implantation device.

PURPOSE: To assess the incidence of anterior chamber bacterial contamination during cataract surgery, and compare results of injector implantation and forceps implantation of foldable intraocular lenses (IOLs). SETTING: Department of Ophthalmology and Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary. METHODS: This prospective randomized controlled clinical study comprised 97 eyes of 96 patients. Antibiotic eyedrops were not used; however, povidone-iodine 10% solution was used to prepare the eyebrow and eyelids and povidone-iodine 5% to disinfect the ocular surface. A Steri-Drape (3M) was used to surround the eye. Aqueous fluid samples were aspirated from the anterior chamber at the beginning and the end of surgery. The samples were cultured for 14 days under aerobic and anaerobic conditions simultaneously. Cataract surgery was performed using a sutureless, superotemporal, clear corneal phacoemulsification technique. The IOL was implanted with an injector (n = 47) or a forceps (n = 50), with the instrument randomly selected. The frequency of positive bacterial cultures with each implantation method was compared using the Fisher exact test. RESULTS: Bacteria were found in the conjunctival samples in 21 eyes (21.65%) before povidone-iodine application and in 4 eyes (4.12%) after disinfection. The anterior chamber sample before surgery was culture positive for Staphylococcus epidermidis in 2 eyes and for Micrococcus luteus in 1 eye. After surgery, the culture was positive for S epidermidis in 1 eye (2.15%) in the injector group and 1 eye (2.00%) in the forceps group (P = .74). Neither sample came from an eye that had a positive culture preoperatively. There were no intraoperative complications. CONCLUSIONS: In uneventful clear corneal phacoemulsification, meticulous technique can prevent antibiotic use during surgery. No difference in anterior chamber bacterial contamination was found between IOL implantation using an injector or a forceps.

Adult↗
PubMed1993

A new approach to use of bacteriolytic enzymes as a tool for species identification: selection of species-specific indicator strains with bacteriolytic activity towards Enterococcus strains.

We describe the bacteriolytic activity of 377 group D Enterococcus isolates expressed towards 25 Enterococcus strains belonging to different species and Micrococcus luteus ATCC 4698. Of the 26 indicator strains used to reveal bacteriolytic activity, 5 were lysed by all of the strains of some species and were not lysed by all of the strains of other species. The use of these indicator strains allowed us to devise a new method to differentiate group D Enterococcus strains, based on qualitative analysis (lysis or no lysis of the indicator strains) of bacteriolytic activity. The bacteriolytic patterns obtained fell into six bacteriolytic groups corresponding (98% agreement) to species or groups of enterococci as determined by a comparison with data from a phenetic similarity study.

Bacterial Typing Techniques↗
PubMed2006

Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D-nanoLC MS/MS.

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.

Amino Acid Sequence↗
PubMed1998

Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics.

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [14C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein-loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol- or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5-10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.

Anti-Bacterial Agents↗
PubMed2002

Comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage.

AIMS: To determine the transfer efficiency of micro-organisms from fomites to hands and the subsequent transfer from the fingertip to the lip. METHODS AND RESULTS: Volunteers hands were sampled after the normal usage of fomites seeded with a pooled culture of a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium (Serratia rubidea) and phage PRD-1 (Period A). Activities included wringing out a dishcloth/sponge, turning on/off a kitchen faucet, cutting up a carrot, making hamburger patties, holding a phone receiver, and removing laundry from the washing machine. Transfer efficiencies were 38.47% to 65.80% and 27.59% to 40.03% for the phone receiver and faucet, respectively. Transfer efficiencies from porous fomites were <0.01%. In most cases, M.luteus was transferred most efficiently, followed by phage PRD-1 and S. rubidea. When the volunteers' fingertips were inoculated with the pooled organisms and held to the lip area (Period B), transfer rates of 40.99%, 33.97%, and 33.90% occurred with M. luteus, S. rubidea, and PRD-1, respectively. CONCLUSIONS: The highest bacteral transfer rates from fomites to the hands were seen with the hard, non-porous surfaces. Even with low transfer rates, the numbers of bacteria transferred to the hands were still high (up to 10(6) cells). Transfer of bacteria from the fingertip to the lip is similar to that observed from hard surfaces to hands. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious doses of pathogens may be transferred to the mouth after handling an everyday contaminated household object.

Adolescent↗
PubMed2006

Characterization of a novel C-type lectin, Bombyx mori multibinding protein, from the B. mori hemolymph: mechanism of wide-range microorganism recognition and role in immunity.

To investigate the system used by insects to recognize invading microorganisms, we examined proteins from the larval hemolymph of Bombyx mori that bind to the cell surface of microorganisms. Two hemolymph proteins that bound to the cell surfaces of Micrococcus luteus and Saccharomyces cerevisiae were shown to be identical. This protein bound to all 11 microorganisms examined-5 Gram-negative bacteria, 3 Gram-positive bacteria, and 3 yeasts-and was consequently designated B. mori multibinding protein (BmMBP). The sequence of the cDNA encoding BmMBP revealed that it was a C-type lectin with two dissimilar carbohydrate-recognition domains (CRD1 and CRD2) distantly related to known insect C-type lectins. CRD1 and CRD2 were prepared as recombinant proteins and their binding properties were investigated using inhibition assays. Each domain had wide, dissimilar binding spectra to sugars. These properties enable BmMBP to bind to two sites on a microorganism, facilitating high-affinity binding to many types of microorganisms. The dissociation constants of BmMBP with M. luteus cells and S. cerevisiae were 1.23 x 10(-8) and 1.00 x 10(-11) M, respectively. rBmMBP triggered the aggregation of hemocytes from B. mori larvae in vitro and microorganisms recognized by BmMBP were surrounded by aggregated hemocytes in vivo, forming a nodule, which is the typical cellular reaction in insect immune responses. These observations suggest that BmMBP functions as a trigger for the nodule reaction and that the multirecognition characteristic of BmMBP plays an important role in the early stages of infection by a variety of microorganisms.

Amino Acid Sequence↗
PubMed1998

Which conditions promote a remanent (persistent) bactericidal activity of chlorine covers?

The interaction of disinfectants containing active chlorine compounds (hypochlorite, chloramine T) with skin surfaces produces a so called chlorine cover which is a true chemical transformation of the protein matrix of the outermost horny skin layer by forming covalent N-Cl bonds. Though its intensity can easily be measured, the results on its remanent (persistent) bactericidal action until now have not been described consistently. While Gottardi and Karl observed a well-defined remanent action which correlated with the cover strength in the artificially with E. coli contaminated arm, Koller, Rotter, and Gottardi were not able to prove it, neither in artificially contaminated hands nor in the case of the resident bacterial colonization. To clarify these not consistent results, the bactericidal activity of chlorine covers on transient (dry contamination of the finger tips with Micrococcus luteus) as well as on resident colonization (after wearing gloves for 3 h) has been re-investigated using contact plates for recovering the bacteria. The results are showing that the remanent bactericidal action of chlorine covers strongly depends on the skin milieu, with moist conditions caused by sweat (surgical gloves) favoring the bactericidal activity. Elution experiments prove that protein constituents (detected by UV-spectrometry) continuously are transported to the skin-surface. It is obvious that the latter equilibrate with the chlorine cover by forming bactericidal N-Cl compounds with the effect that oxidation capacity fixed at the protein matrix is mobilized enabling the remanent bactericidal action.

Chloramines↗
PubMed2005

Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa.

We have cloned and characterized a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the superfamily Cerambyocidea. This peptide is active against Micrococcus luteus and Escherichia coli, and the amino acid sequence deduced by cloning of the cDNA identifies it as a coleopteran cecropin. Sequence comparisons and phylogenetic analyses performed using Clustal X suggest that this cecropin is evolutionarily intermediate between dipteran and lepidopteran cecropins. The results of MALDI-TOF mass spectrometry indicate that the mature form of this antibacterial peptide is 35 amino acid residues in length and has an amidated C-terminal isoleucine. This report is the first description of a cecropin from a coleopteran insect.

Amino Acid Sequence↗
PubMed1997

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge.

The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E. coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M.luteus infection since anti-E.coli activity was similar in haemolymph from both E.coli or M.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4 kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.

Animals↗
PubMed1995

A promoter associated with the neisserial repeat can be used to transcribe the uvrB gene from Neisseria gonorrhoeae.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrB mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrB proteins of E. coli, Micrococcus luteus, and Streptococcus pneumoniae. A gonococcal uvrB mutant was constructed and found to be extremely sensitive to UV radiation. Transcriptional fusions between portions of the gonococcal uvrB upstream region and a reporter gene were used to localize promoter activity, and the transcriptional start point of the gonococcal uvrB gene was mapped in E. coli by primer extension. A corresponding sigma 70 promoter was identified within a copy of the 26-bp neisserial repeat, and this identification provided the first evidence of a promoter associated with this repetitive element in N. gonorrhoeae.

Amino Acid Sequence↗
PubMed2001

Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats.

The effect of an intestinal microflora consisting of selected microbial species on myoelectric activity of small intestine was studied using germ-free rat models, with recording before and after specific intestinal colonization, in the unanesthetized state. Intestinal transit, neuropeptides in blood (RIA), and neuromessengers in the intestinal wall were determined. Clostridium tabificum vp 04 promoted regular spike burst activity, shown by a reduction of the migrating myoelectric complex (MMC) period from 30.5 +/- 3.9 min in the germ-free state to 21.2 +/- 0.14 min (P < 0.01). Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11 reduced the MMC period from 27.9 +/- 4.5 to 21.5 +/- 2.1 min (P < 0.02) and accelerated small intestinal transit (P < 0.05). Micrococcus luteus showed an inhibitory effect, with an MMC period of 35.9 +/- 9.3 min compared with 27.7 +/- 6.3 min in germ-free rats (P < 0.01). Inhibition was indicated also for Escherichia coli X7 gnotobiotic rats. No consistent changes in slow wave frequency were observed. The concentration of neuropeptide Y in blood decreased after introduction of conventional intestinal microflora, suggesting reduced inhibitory control. Intestinal bacteria promote or suppress the initiation and aboral migration of the MMC depending on the species involved. Bacteria with primitive fermenting metabolism (anaerobes) emerge as important promoters of regular spike burst activity in small intestine.

Action Potentials↗