Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein

Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.     

Clavulanic acid: a review

Natural antibiotics are almost universal secondary metabolites, not essential for the growth of the producing organisms generally produced at low growth rates or after growth has ceased. Clavulanic acid (CA), a naturally occurring powerful inhibitor of bacterial beta-lactamases is a major beta-lactam antibiotic produced by organism Streptomyces clavuligerus and is active against a wide spectrum of Gram-positive and Gram-negative bacteria. The review discusses the biosynthetic pathway, fermentative production, downstream processing and applications of CA.     

Ability of bacteria isolated from the hospital environment to proliferate in infusion fluids

The ability of some bacteria isolated from environmental sources to grow in commonly administered infusion fluids was investigated. Infusion fluids may become contaminated with bacteria present in the environment in which they are being administered and the ability of such bacteria to proliferate within commonly used infusion fluids is therefore clinically significant. Bacteria present in the air and sink drains in wards at the Ife State Hospital were isolated, identified, and comprised fifteen Gram-positive and five Gram-negative organisms. Fourteen of the organisms obtained were introduced into sterile distilled water and five different infusion fluid preparations and were monitored over a 48 h period. It was found that, in general, Gram-positive organisms did not grow in the fluids used, while Gram-negative organisms were able to proliferate in the same fluids. Some Gram-positive organisms showed remarkable ability to remain viable in infusion fluids for more than 48 h.     

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

Abstract 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.     

Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity

A comparative study on the antimicrobial properties of extracts from medicinal plants obtained by two different methods was carried out. The screening of the antimicrobial activity of extracts from six plants was conducted by a disc diffusion test against Gram-positive, -negative and fungal organisms. The most active extracts (inhibition diameter >/=12 mm) were assayed for the minimum inhibitory concentration and submitted to phytochemical screening by thin-layer chromatography and bioautography. The results obtained indicate that the diethyl ether extracts were the most efficient antimicrobial compounds. The activity was more pronounced against Gram-positive and fungal organisms than against Gram-negative bacteria. Bioautography showed that the antimicrobial activity was probably due to flavonoids and terpenes.     

Human peptidoglycan recognition proteins require zinc to kill both gram-positive and gram-negative bacteria and are synergistic with antibacterial peptides

Mammals have four peptidoglycan recognition proteins (PGRPs or PGLYRPs), which are secreted innate immunity pattern recognition molecules with effector functions. In this study, we demonstrate that human PGLYRP-1, PGLYRP-3, PGLYRP-4, and PGLYRP-3:4 have Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiologic Zn(2+) concentrations found in serum, sweat, saliva, and other body fluids. The requirement for Zn(2+) can only be partially replaced by Ca(2+) for killing of Gram-positive bacteria but not for killing of Gram-negative bacteria. The bactericidal activity of PGLYRPs is salt insensitive and requires N-glycosylation of PGLYRPs. The LD(99) of PGLYRPs for Gram-positive and Gram-negative bacteria is 0.3-1.7 muM, and killing of bacteria by PGLYRPs, in contrast to killing by antibacterial peptides, does not involve permeabilization of cytoplasmic membrane. PGLYRPs and antibacterial peptides (phospholipase A(2), alpha- and beta-defensins, and bactericidal permeability-increasing protein), at subbactericidal concentrations, synergistically kill Gram-positive and Gram-negative bacteria. These results demonstrate that PGLYRPs are a novel class of recognition and effector molecules with broad Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria that are synergistic with antibacterial peptides.     

The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions.     

What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram-positive bacteria? A new proposal for the classification of prokaryotic organisms

The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria. A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/protein sequences. To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low-G + C Gram-positive bacteria are phylogenetically distinct from the high-G + C Gram-positive group and that the diderm prokaryotes (i.e. Gram-negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram-negative eubacterium. Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported. These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three-domain proposal.     

In vitro activity of ramoplanin and comparator drugs against anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Synthesis and antibacterial activity of new N-linked 5-triazolylmethyl oxazolidinones

A new series of N-linked 5-triazolylmethyl oxazolidinones with varying substitution at the piperazine nitrogen 4-position were synthesized and tested against a panel of Gram-positive and Gram-negative bacteria including clinical isolates. Most of the compounds showed excellent antibacterial activity against susceptible and resistant Gram-positive organisms. One of the compounds showed enhanced antibacterial activity against Moraxella catarrhalis.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies

Polyreactive antibodies bind to a variety of structurally unrelated antigens. The function of these antibodies, however, has remained an enigma, and because of their low binding affinity their biological relevance has been questioned. Using a panel of monoclonal polyreactive antibodies, we showed that these antibodies can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway can inhibit bacterial growth by lysis, generate anaphylatoxin C5a, enhance phagocytosis, and neutralize the functional activity of endotoxin. Polyreactive antibody-enriched, but not polyreactive antibody-reduced, IgM prepared from normal human serum displays antibacterial activity similar to that of monoclonal polyreactive IgM. We conclude that polyreactive antibodies are a major contributor to the broad antibacterial activity of the natural antibody repertoire.     

Antimicrobial activity and phytochemical analyses of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt

Polygonum aviculare (Polygonaceae) is an herb commonly distributed in Mediterranean coastal regions in Egypt and used in folkloric medicine. Organic and aqueous solvent extracts and fractions of P. aviculare were investigated for antimicrobial activities on several microorganisms including bacteria and fungi. Phytochemical constituents of air-dried powered plant parts were extracted using aqueous and organic solvents (acetone, ethanol, chloroform and water). Antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of inhibition zone against both Gram-negative and Gram-positive bacteria and fungi using paper disc diffusion method.Results of the phytochemical studies revealed the presence of tannins, saponins, flavonoids, alkaloids and sesquiterpenes and the extracts were active against both Gram-negative and Gram-positive bacteria. Chloroform extract gave very good and excellent antimicrobial activity against all tested bacteria and good activity against all tested fungi except Candida albicans. Structural spectroscopic analysis that was carried out on the active substances in the chloroform extract led to the identification of panicudine (6-hydroxy-11-deoxy-13 dehydrohetisane).Evaluation of the antimicrobial activity of panicudine indicated significant activity against all tested Gram-negative and Gram-positive organisms. Panicudine displayed considerable activity against the tested fungi with the exception of C. albicans. Antimicrobial activity of the extracts was unaffected after exposure to different heat treatments, but was reduced at alkaline pH. Studies of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of panicudine on the tested organisms showed that the lowest MIC and the MBC were demonstrated against Salmonella paratyphi, Bacillus subtilis and Salmonella typhi and the highest MIC and MBC were against Staphylococcus aureus.     

Antimicrobial activity of extracts of the lichens <Emphasis Type="Italic">Cladonia furcata,Parmelia caperata,Parmelia pertusa,Hypogymnia physodes</Emphasis> and <Emphasis Type="Italic">Umbilicaria polyphylla</Emphasis>

Antimicrobial features of acetone, methanol and aqueous extracts of lichens of Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla were investigated by two different methods at the same time. Testing of antimicrobial activities of extracts from five species of lichens was performed by disc diffusion test in relation to Gram-positive and Gram-negative bacteria and fungal organisms, and through determination of minimal inhibitory concentration (MIC) by Broth Tube Dilution method. The obtained results indicated that acetone and methanol extracts of all investigated lichens in different concentrations manifested selective antibacterial and antifungal activity. That activity was more evident in relation to Gram-positive, than Gram-negative bacteria and fungal organisms. Acetone and methanol extracts of lichens Parmelia pertusa, Hypogymnia physodes and Umbilicaria polyphylla inhibited the growth of all tested microorganisms, most of all of lichens Cladonia furcata and Parmelia caperata. Although, the methanol extracts were generally the most active against the test organisms, the lowest MIC value was measured for acetone extract of species Cladonia furcata 0.39 mg/mL in relation to bacterium Bacillus subtilis. Aqueous extracts of investigated lichens were inactive against all tested organisms.     

Antimicrobial spectrum of bacteriocin-like substances produced by rumen staphylococci

Five strains of rumen coagulase-negative adherent and ureolytic staphylococci were obtained as bacteria producting bacteriocin-like substances or, lantibiotics. All examined staphylococci produced inhibitory agents which showed a wide range of inhibition against Gram-positive and Gram-negative indicator organisms from different sources. Clear zones of inhibition (diameter 1–6 mm) dominated. Most bacteriocin-like substances produced by the strains were stable and sensitive to trypsin, susceptible to chloroform vapours and heat-sensitive.     

Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Antimicrobial properties of phenolic compounds from berries

AIMS: To investigate the antimicrobial properties of phenolic compounds present in Finnish berries against probiotic bacteria and other intestinal bacteria, including pathogenic species. METHODS AND RESULTS: Antimicrobial activity of pure phenolic compounds representing flavonoids and phenolic acids, and eight extracts from common Finnish berries, was measured against selected Gram-positive and Gram-negative bacterial species, including probiotic bacteria and the intestinal pathogen Salmonella. Antimicrobial activity was screened by an agar diffusion method and bacterial growth was measured in liquid culture as a more accurate assay. Myricetin inhibited the growth of all lactic acid bacteria derived from the human gastrointestinal tract flora but it did not affect the Salmonella strain. In general, berry extracts inhibited the growth of Gram-negative but not Gram-positive bacteria. These variations may reflect differences in cell surface structures between Gram-negative and Gram-positive bacteria. Cloudberry, raspberry and strawberry extracts were strong inhibitors of Salmonella. Sea buckthorn berry and blackcurrant showed the least activity against Gram-negative bacteria. CONCLUSION: Different bacterial species exhibit different sensitivities towards phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: These properties can be utilized in functional food development and in food preservative purposes.