Peptide-like substances as antimicrobial barriers to Corynebacterium sp. adhesion to silicone catheters

AIMS: To show medical application of antimicrobial peptides such as Pep5 and epidermin in inhibiting adhesion of Corynebacterium spp. to silicone catheters. METHODS AND RESULTS: The inhibitory activity of crude preparations of Pep5 and epidermin was tested on Corynebacterium spp. isolated from catheter-related infections. The addition of these substances at 640 AU ml(-1) to a cell suspension of Corynebacterium sp. 633544 resulted in a decrease of 3 log cycles in the number of viable cells over a period of 12 h. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a decrease of 1 log unit (P < 0.01) in the cell number of Corynebacterium spp. adhered to silicone catheters. Scanning electron microscopy revealed that antimicrobial-treated catheters presented zones with absence of adhered cells, and some parts of the catheter presented aggregates suggesting damaged cells. CONCLUSIONS: The crude preparations of Pep5 and epidermin were able to inhibit Corynebacterium sp. 633544 isolated from catheter-related infection. The capability of Pep5 and epidermin to inhibit catheter colonization may indicate their usefulness as a barrier to block or to reduce the bacteremia by Corynebacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Peptide-like antimicrobial substances used to reduce bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.     

Role of first stimulating agents in the production of tumor necrosis factor

Summary The conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Microflora associated with the skin of the bowhead whale (Balaena mysticetus)

A study of the microbiological flora isolated from cultures of normal and lesional skin tissue samples collected from 19 bowhead whales (Balaena mysticetus) over a 4 yr period is presented. These cultures were obtained from 30 tissue samples (17 normal, 13 lesion) and 248 swab samples (157 normal, 91 lesion). Seven hundred-thirty bacterial and yeast isolations were made (285 normal, 445 lesion). Distribution revealed that 56% of the gram positive bacterial isolates, 75% of the gram negative bacterial isolates and 64% of the yeast isolates recovered were associated with lesional skin. It was found that 80% of one group of Corynebacterium sp. isolates, 90% of the Acinetobacter sp. isolates and 94% of the Moraxella sp. isolates were associated with lesional skin. Although the primary yeasts recovered were Candida spp., they were found on both normal and lesional skin. Enzymatic assays of isolates from normal and lesional skin demonstrated production of enzymes capable of causing necrosis. The majority of the microorganisms recovered were facultative anaerobes and many of them could be considered potential pathogens of mammalian hosts.     

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.     

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer. Reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.     

Elimination of Lactobacillus plantarum, Corynebacterium spp., Staphylococcus saprophyticus and Pseudomonas paucimobillis from micropropagated Hemerocallis, Choisya and Delphinium cultures using antibiotics

C. LEIFERT, H. CAMOTTA, S.M. WRIGHT, B. WAITES, V.A. CHEYNE & W.M. WAITES. 1991. The antibiotic sensitivity patterns of bacterial contaminants isolated from micropropagated plant cultures were determined. Lactobacillus plantarum, Corynebacterium spp., Staphylococcus saprophyticus and Pseudomonas paucimobilis were eliminated from plant tissue cultures of Hemerocallis, Choisya and Delphinium by incorporating combinations of antibiotics into the plant growth medium. Elimination of contaminants resulted in increased growth rates of all plant cultures, except Choisya infected with Staph. saprophyticus. Pseudomonas maltophilia was not eliminated from Delphinium by any of the antibiotic treatments tested.     

The Significance of Propionibacterium Species and Micrococcus lactilyticus to the Ensiling Process

The fermentation of lactic acid by two Propionibacterium spp. and by Micrococcus lactilyticus was examined in simulated silage. Fermentation occurred in silages which achieved a low pH (<4·0) but this resulted only in a small rise in pH and a secondary clostridial fermentation did not occur. Lactic acid-fermenting organisms resembling Propionibacterium spp. were isolated from authentic silages but not from fresh grass.     

Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses

In this study, a total of 29 Propionibacterium spp. were isolated from traditional home-made Turkish cheese samples. As a result of the identification, isolates were identified as Propionibacterium freudenreichii subsp. freudenreichii (15 strains), Propionibacterium jensenii (12), and Propionibacterium thoenii (2). All isolates and 5 reference strains were examined for their abilities to survive at pH 2.0, 3.0, 4.0, 5.0 and in the presence of 0.06, 0.15 and 0.30% bile salts, their influence on the growth of food-borne and spoilage bacteria, as well as their sensitivity against 11 selected antibiotics. Only seven propionibacteria strains survived in both the acidic and bile salt environments. Propionibacterium spp. strains strongly inhibited growth of the Escherichia coli ATCC 11229 and Shigella sonnei Mu:57 strains (91%). All propionibacteria strains were sensitive to a majority of the antibiotics used in the investigations. Overall, dairy propionibacteria showed high antibacterial activity, resistance to pH 4.0, 5.0, high resistance to bile salts and will provide an alternative source to Lactobacillus and Bifidobacterium as probiotic culture.     

Facultative alkalophilic bacteria from mangrove soil with varying buffering capacity and H+ conductance

Facultative alkalophilic bacteria Planococcus sp. (EMGA-26), Bacillus sp. (EMGA-29) and Corynebacterium spp. (EMGA-33 and 130) were isolated from mangrove soil samples. Neutrophiles were predominant than alkalophiles. Buffering capacity and membrane H+ conductance were investigated for the strains grown in PPYG medium at pH 10.5 using acid pulse technique. Bacillus sp. showed higher buffering capacity than Planococcus sp. and Corynebacterium spp. Buffering capacity was two-fold higher in Corynebacterium sp. EMGA-33 than in EMGA-130. The membrane H+ conductance was high in Bacillus sp. and was directly proportional to the buffering capacity values. The Bacillus sp. (EMGA-29) had higher cell membrane adaptability in high pH environment than the Planococcus sp. and Corynebacterium spp.     

Identification of the emerging skin pathogen Corynebacterium amycolatum using PCR-amplification of the essential divIVA gene as a target

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.     

Analysis of microorganisms isolated from febrile neutropenic children with neoplastic disease

The microbiological evaluation of the blood was carried out as a continued monitoring of the microorganism culture using the colourmetric system VITAL 200 (bio-Mérieux). There have been analysed 4660 bacteriological research results of the peripheral blood, blood from Broviac and from Broviac liquid of the children suffering from cancer treated in the years 1997-2000 in the Pediatric, Hematology and Oncology Clinique of the State Clinical Hospital in Bydgoszcz. There have been gained 1032 positive cultures from KZZ, KZB, PZB. There have been recognized 259 bacteremia and 22 fungemia by the children with fever in the neutropenia period. In the analysed four years in the blood dominated Gram-positive bacteria. Among Gram-positive bacteria there were mostly Staphylococcus spp. (42.5%), mostly CNS, fewer numerous were Streptococcus spp. (14.5%) and Corynebacterium spp. (5.9%). Among Gram-negative bacteria mostly were isolated Acinetobacter spp. (25.6%), Pseudomonas spp. (9.7%), E. coli (13.8%), Enterobacter spp. (13.9%), Klebsiella spp. (9.2%). There were observed few infections by strains resistant to many antibiotics, S. maltophilia, B. cepacia, E. faecium, S. haemolyticus. All strains Staphylococcus spp. were sensitive to vancomycin. There have not been found Enterococcus spp. resistant to glycopeptides. Most active against Gram-positive rods were carbapenems and aminoglycosides.     

Identification of Actinomyces, Arachnia, Bacterionema, Rothia, and Propionibacterium Species by Defined Immunofluorescence

Fractionated fluorescein-isothiocyanate (FITC)-conjugated immunoglobulin G (dye-to-protein ratio <10), produced against whole cells of Actinomyces spp., Arachnia, Bacterionena, Rothia, and Propionibacterium spp., give species-specific conjugates with controlled nonspecific staining reactions when appropriately diluted on the basis of their antibody content (10 mg/ml). Using this standardization in immunofluorescence, serotype-specific conjugates are also available after dilution for all serotypes of these organisms except for Actinomyces viscosus type 2, and Propionibacterium acnes type 1. Adequately adsorbed conjugates could be used to differentiate these serotypes from A. viscosus type 1 and P. acnes type 2, respectively. A serological classification in defined immunofluorescence corresponded to species and serotype designation proposed on the basis of other serological analysis and biochemical characteristics. This includes a separation in immunofluorescence of two serotypes of Propionibacterium acnes. The detection of certain actinomycetes of the family Actinomycetaceae and Propionibacterium species by the defined immunofluorescence in direct smears prepared from clinical specimens agreed to 88% with parallel culturing when including a prereduced (PRAS) medium technique for isolation. Qualitative studies revealed that single cells of these organisms could be specifically identified by immunofluorescence when admixed with morphologically similar bacteria and a large number of other contaminants.     

In vitro evaluation of antibacterial substances produced by bacteria isolated from different marine organisms

Bacteria from several groups of marine organisms were isolated and, using direct antibiograms, identified those that produce antibacterial substances, using a human pathogenic strain of Staphylococcus aureus ATCC6538 as revealing microorganism. Bacteria which produce substances that inhibited S. aureus growth were identified through morphological, physiological and biochemical tests. Out of 290 bacteria, 54 (18.6%) inhibited the growth of S. aureus, but only 27 survived for identification. Bivalves, sponges and corals were the most represented from which 41.2, 33.3 and 29.7%, respectively, produced antibacterial substances of the isolated bacteria in each group. The marine species with highest proportions of these bacteria were the hard coral Madracis decactis (62.5%), the sponges Cliona sp. (57.1%) and the octocoral Plexaura flexuosa (50.0%). Out of the 27 strains that produced antibacterial substances, 51.8% were Aeromonas spp. and 14.8% Vibrio spp. Marine bacteria that produce antibacterial substances are abundant, most belong in the Vibrionacea group and were isolated mainly from corals and bivalve mollusks.     

Mitogenic Activity of the Cell Walls of Mycobacteria,Nocardia, Corynebacteria and Anaerobic Coryneforms

The mitogenic activity of the cell walls prepared from Mycobacterium bovis BCG, Nocardia rubra, Corynebacterium diphtheriae PW8, and four species of Propionibacterium, Corynebacterium parvum ATCC 11829, Propionibacterium acnes C7, Propionibacterium granulosum ATCC 25564 and Propionibacterium avidum ATCC 25577, were investigated. These cell walls were active as mitogens on normal spleen cells, anti-θ sera-treated spleen cells, macrophage-depleted spleen cells of C57BL/6J mice and cortisone-treated thymocytes of C57BL/6J mice. It was also shown that these cell walls were mitogenic on spleen cells and macrophage-depleted spleen cells of congenitally athymic (nude) mice. The above results suggest that the cell walls investigated in this study act as mitogens on both thymus-derived lymphocytes (T-cells) and bone marrow-derived lymphocytes (B-cells).     

Microbial pathways leading to steroidal malodour in the axilla

Odorous steroids, specifically the 16-androstenes, 5alpha-androstenol and 5alpha-androstenone, are widely accepted as being contributors to underarm odour, but the precursors and pathways to these odorous steroids were unclear. This study demonstrated that the axillary microflora could only generate odorous 16-androstenes from precursors that already contain the C16 double bond, such as 5,16-androstadien-3-ol and 4,16-androstadien-3-one. In incubations containing 5,16-androstadien-3-ol, mixed populations of Corynebacterium spp., isolated from the axilla, could generate many different 16-androstene metabolites, several of which were odorous. Isolation of individual Corynebacterium strains, followed by pure culture incubations with 5,16-androstadien-3-ol, revealed organisms capable of efficient, rapid reactions. However, no single isolate could carry out a full complement of the observed biotransformations. 16-Androstene metabolites were identified by gas chromatography-mass spectrometry (GC-MS), either by comparison with known standards, or by prediction from molecular ion and fragmentation patterns. Based on detection of these metabolites, a metabolic map for axillary corynebacterial 16-androstene biotransformations was proposed, detailing potential enzyme activities. In summary, the formerly implicated 4,16-androstadien-3-one, 5alpha-androstenone and 5alpha-androstenol were detected, along with previously unreported hydroxy- and keto-substituted 16-androstenes, 16-androstatrienones and 16-androstatrienols. Additionally, many other metabolites with steroidal fragmentation patterns were present, but have remained unidentified.A key observation was that very low prevalences of microorganisms capable of biotransforming 16-androstenes were present on skin. For example, from a panel of 21 individuals, only 4 of 18 mixed populations of corynebacteria, and only 4 of 45 Corynebacterium isolates, could biotransform 5,16-androstadien-3-ol.This study has increased understanding of the metabolic pathways involved in steroidal malodour formation, and has demonstrated that the biotransformations are more complex than previously anticipated. However, it is clear that further research is required, both to assess the level of contribution of 16-androstenes to underarm odour, and to further elucidate the pathways and odour molecules formed by corynebacteria.     

Are zoonotic <Emphasis Type="Italic">Staphylococcus pseudintermedius</Emphasis> strains a growing threat for humans?

Staphylococcus pseudintermedius is a species often isolated from animals, as a common element of their microbiota or an agent of infection, and from people associated with an animal habitat, including owners of home pets—dogs and cats. As with many other species, adaptation of these bacteria to the human body can occur, and they become important human pathogens. 59 S. pseudintermedius strains were investigated in this study to determine the factors contributing to human body colonization: inhibition growth of human skin residents isolated from human skin (Staphylococcus epidermidis, Corynebacterium spp., Cutibacterium acnes (formerly Propionibacterium acnes)), biofilm formation, and the presence of ten genes encoding infection-promoting features (including ebpS, spsE, lukS, lukF, pvl, lip, hlgA, hlgB). The ability of human skin to be colonized and the presence of genes that promote the development of skin infections showed the significant potential of the studied strains in their adaptation to the host. However, while a comparison of the characteristics of animal strains and those isolated from human infections does not allow us to claim that we are the witnesses of the speciation of a new human pathogen, it does indicate their gradual adaptation to the human organism.     

Bacterial competition for human nasal cavity colonization: role of Staphylococcal agr alleles

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal agr alleles. The Staphylococcus aureus colonization rate correlated negatively with the rate of colonization by Corynebacterium spp. and non-aureus staphylococci, especially S. epidermidis, suggesting that both Corynebacterium spp. and S. epidermidis antagonize S. aureus colonization. Most of the S. aureus and S. epidermidis isolates were agr typed by a PCR method. Only one S. aureus agr (agr(Sa)) allele was detected in each carrier. Multiple logistic regression of the two most prevalent agr(Sa) alleles (agr-1(Sa) and agr-2(Sa)) and the three S. epidermidis agr (agr(Se)) alleles showed a specific influence of the agr system. The results of this model did not support conclusions drawn from previous in vitro agr-specific cross-inhibition experiments. Our findings suggest that the agr alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties--including mediators of bacterial interference--in the strains that bear them.