Human transferrin as a source of iron for Streptococcus intermedius

Streptococcus intermedius is well known to produce severe infections in various areas of the body. In this study, we evaluated the ability of S. intermedius to utilise human transferrin as a source of iron and investigated the mechanism by which iron can be obtained from this plasma protein. Adding either ferrous sulfate or holotransferrin to an iron-deficient culture medium allowed growth of S. intermedius. Cultivation of S. intermedius under an iron-poor condition was associated with the over expression of a 58 kDa cell surface protein. Neither siderophore activity nor reductase activity could be detected. Moreover, cells of S. intermedius did not show transferrin-binding activity or proteolytic activity toward transferrin. It was found that S. intermedius could rapidly decrease the pH of the medium during cell growth, resulting in a release of iron from holotransferrin. When the buffering capacity of the culture medium was significantly increased, the holotransferrin could not support growth of S. intermedius. It is suggested that under certain circumstances, S. intermedius may migrate from its normal niche (oral cavity), reach a particular site and create a localised environment where the pH can be lowered with the subsequent release of iron from transferrin. This would allow bacterial growth and initiation of the infectious process.     

Enterobacteria of emerging pathogenic significance from clinical cases in man and animals and detection of toads and wall lizards as their reservoirs

A total of 416 samples comprising faecal samples from diarrhoeic cases of man, calves, sheep and goats, and urine samples from patients with urinary tract infections, were examined for the presence of enterobacteria of emerging pathogenic significance.Citrobacter freundii from 20,C. intermedius biotype-a from four,Serratia marcescens (serotype 05:H13, bactericin type 16) from one andErwinia herbicola from two human stool samples were isolated. Only two urine samples yieldedC. freundii. Citrobacter freundii was isolated from 10 andC. intermedius biotype-a from two calves. From sheep and goats, two isolates ofC. freundii and three ofC. intermedius biotype-a were obtained. None of these samples yieldedEdwardsiella tarda orYersinia enterocolitica. The examination of 99 toads and 145 wall lizards revealed that toads were reservoirs forC. freundii, C. intermedius biotype-a andSalmonella brijbhumi, whereas wall lizards were reservoirs forC. freundii, C. intermedius biotype-a,E. herbicola, Enterobacter cloacae andSalmonella spp. These bacteria were present in the range of 2.0 × 10⁶ to 6.0 × 10¹¹ organisms per gram of intestinal contents. In addition, toads were carriers forEdwardsiella tarda (new serotypes 04167:H1 and 05159: nonmotile). None of the toads and wall lizards proved positive forC. intermedius biotype-b (C. koseri),S. marcescens andY. enterocolitica. C. freundii, C. intermedius biotype-a,E. herbicola andS. marcescens were resistant to penicillin and erythromycin whereasE. tarda isolates were also resistant to gentamycin, neomycin, colistin and sulfamethaxazole.     

Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the β-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-β-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.     

The effects of prolonged food deprivation on the covering behavior of the sea urchins Glyptocidaris crenularis and Strongylocentrotus intermedius

In this study, we investigated effects of 24 weeks of food deprivation on covering behavior of Glyptocidaris crenularis and Strongylocentrotus intermedius. Neither G. crenularis nor S. intermedius significantly reduced covering behavior during both short- and long-term food deprivation. However, G. crenularis and S. intermedius had significantly different behavioral patterns over the 24 weeks. Covering behavior of G. crenularis greatly increased from the first to the second week, with decreasing reaction time and increasing covering ability, while the covering behavior of S. intermedius generally remained unchanged during the experiment of 24 weeks. As prolonged food deprivation does not significantly affect covering behavior of G. crenularis and S. intermedius, the present study highlights the innate nature and biological significance of covering behavior in sea urchins, providing new insights into its behavioral mechanism.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

A comparative immunological study of catalases from coagulase-positive staphylococci

Protein homology studies with catalase as a reference point were carried out with coagulasepositive staphylococci belonging to Staphylococcus aureus, S. intermedius and S. hyicus. Antisera against catalases of S. aureus ATCC 12600 and S. aureus ATCC 12601 reacted very weakly employing double immunodiffusion and quantitative microcomplementfixation assay with cell-free extracts or catalase enriched preparations of S. intermedius or S. hyicus. The differences between coagulase-positive staphylococci could be confirmed by using the antiserum against S. intermedius H 11 catalase. Within the strains of the species S. intermedius immunological distances ranging up to 25 indicate a heterogeneity which justify the separation of the biotypes E and F on a subspecies level. Coagulase-positive strains of S. hyicus revealed neither a close relationship to S. aureus nor to S. intermedius.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

A simple assay to determine the nutritional suitability of cattle dung for coprophagous beetles

An assay using extracted dung fluid was developed to provide an objective method of assessing the suitability of cattle dung for beetle Euoniticellus intermedius. Fluid was extracted from whole dung by twisting gauze-wrapped samples by hand, or by use of an hydraulic press set sequentially at 2 MPa, 6 MPa and 10 MPa. Artificial dung was made by adding dung fluid to shredded fibre-board in a 15:1 weight ratio. The rate of brood-ball (= egg) production by female E. intermedius fed on the artificial dung made from fluid extracted by the hydraulic press at 2 MPa was the same as that on natural whole dung. Reproductive performance on all artificial dungs was significantly correlated with the percent dry matter content of the dung fluid used in the mixture. Thus the percent dry matter in the fluid extracted at 2 MPa pressure provides an objective measurement for comparing the quality of different dungs as food for adult E. intermedius, without the need for performing bioassays. The results confirm that the fluid component of dung is the major source of nutrition for adult dung beetles.Deceased February 1986.     

Age,growth and reproduction of Marcusenius pongolensis,Oreochromis mossambicus and Schilbe intermedius in an oligotrophic impoundment in Swaziland

The age, growth and reproductive biology of Marcusenius pongolensis, Oreochromis mossambicus and Schilbe intermedius were investigated in the Mnjoli Dam, Swaziland. Otolith annulus formation occurred in winter for M. pongolensis, and in spring/summer for O. mossambicus and S. intermedius. Maximum ages of 8, 6 and 8 years were recorded for M. pongolensis, O. mossambicus and S. intermedius, respectively. Growth was described by the von Bertalanffy growth model as L_t = 238.73(1 − exp^{−0.27(t+2.27}) mm fork length (FL) for M. pongolensis, L_t = 226.83(1 − exp^{−0.45(t+2.02)}) mm total length (TL) for O. mossambicus, and L_t = 214.59(1 − exp^{−0.60(t+1.20)}) mm FL for S. intermedius. Sexual maturity was estimated for male and female M. pongolensis at 134 mm FL and 119 mm FL, respectively. Marcusenius pongolensis matured within their first year. Female O. mossambicus and S. intermedius matured at 239 mm TL and 205 mm FL, corresponding to 2 and 4 years of age, respectively. Extended spawning periods, with two spawning peaks was observed for M. pongolensis, one in spring (September) and the second in autumn (March) and one peak over late‐summer for S. intermedius.     

Localization of alkaline phosphatase inBacillus intermedius cells

Alkaline phosphatase, an enzyme secreted byBacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth ofB. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na₂HPO₄ at a concentration of 0.01 % diminished the activity of membrane-bound and extracellular phosphatases. CoCl₂ at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

The reproductive and feeding biology of Schilbe intermedius Rüppell in the Okavango Delta,Botswana

The reproductive and feeding biology of 314 Schilbe intermedius Rüppell 1832 collected in the Okavango Delta, Botswana, between April 1985 and February 1986 during low, intermediate and high water level were investigated. S. intermedius matures at 130 mm standard length and is a monocyclic spawner. Females predominate in the population and have an average fecundity of 27107 eggs. In the northern riverine floodplain of the Delta fish spawn in mid-summer during the peak flood. In the southern drainage rivers fish spawn in spring, which is two to three months after the arrival of the winter flood waters. S. intermedius is an opportunistic predator with a diet consisting largely of fish 41%, aquatic larvae 25%, terrestrial insects 14%, aquatic insects 7% and crustacea 5%. Juvenile S. intermedius feed predominantly on insect larvae, while adults are largely piscivorous.     

Bactericidal activity and oral pathogen inactivation by electromagnetic wave irradiation

Aims: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens. Methods and Results: A Gram‐negative (Porphyromonas gingivalis) or Gram‐positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500–1000 kHz, 5–15 times, 1 s time^?1). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number‐dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5–10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro‐inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat‐killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis. Conclusions: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens. Significance and Impact of the Study: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.     

Molecular characterization of <Emphasis Type="Italic">Staphylococcus pseudintermedius</Emphasis> strains isolated from clinical samples of animal origin

The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable.     

Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus(1)

Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.     

Isozyme evidence on the genetic diversity, mating system and evolution of Bromus intermedius (Poaceae)

Genetic diversity and differentiation among the B. intermedius accessions of different geographic origin has been studied using isozyme analysis. The mating system was evaluated on the basis of allozyme polymorphism. Outcrossing rate (t) in B. intermedius was mostly 0, except one population with t = 0.16, indicating nearly complete autogamy in this species. Given that B. arvensis and B. intermedius had common allozymes of all isozymes studied, it is suggested that B. intermedius may be a direct autogamous derivative of the outcrosser B. arvensis. Contrary to expectations, the allozyme diversity in B. intermedius was higher than in B. arvensis, 23 and 16 allozymes, respectively. Geographic pattern was found among the accessions of B. intermedius.     

Preliminary study on polymorphism analysis of SpRunt-1 gene by PCR–SSCP in <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and its association with growth traits

In current study, the SpRunt-1 gene was screened for the polymorphisms using PCR-single strand conformation polymorphism (PCR–SSCP) and DNA sequencing methods in Strongylocentrotus intermedius (S. intermedius). Three polymorphisms were found with two site mutations, G833A and C1505T mutation, and an insertion mutation (GT) between 931 nt and 932 nt. These two site mutations are synonymous mutations and the insertion mutation is frame shift mutation. One way ANOVA analysis of correlation between the single nucleotide polymorphisms (SNPs) and growth traits showed that the gonad weight of AA genotype (G at 833 nt) was significantly higher than that of BB genotype (A at 833 nt) (P = 0.029). The body weight of the CC genotype (C at 1505 nt) was significantly higher than that of DD genotype (T at 1505 nt) (P < 0.01) and the shell height of CD genotype was significantly higher than that of DD genotype (P = 0.032). These results provided the evidence that S. intermedius SpRunt-1 gene could be selected as a candidate gene for the growth traits.     

Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria

In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Ga. europaeus exhibited the highest resistance to acetic acid (10%), whereas Ga. intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid.     

Reproductive behavior of the Lake Malawi cichlid fish, <Emphasis Type="Italic">Tramitichromis intermedius</Emphasis>

Synopsis We studied the reproductive behavior of wild caught and captive-born, first generation offspring of the Lake Malawi fish, Tramitichromis intermedius(Teleostei, Cichlidae), held in aquaria. Spawning behavior includes an exchange of actions with dominant males performing bower construction and courtship behaviors while females focus on oviposition and mouthbrooding. Egg counts per oviposition and brooding and interbrooding periods of wild caught T. intermedius follow records of other mouthbrooding cichlid fishes. Observation of circling behavior suggests this behavioral trait may be used in mate choice as longer circling, indicating a secure territory and thus male dominance, leads to more oviposition events and hence the potential for larger broods. Comparisons of clutch size and total length of young at release from full-sized females vs. first spawners reveal smaller clutches and young from the younger females, most likely stemming from differences in body size. Investigation of spawning photoperiodicity also noted distinctions between the two groups with wild caught T. intermedius spawning activity peaking in the middle of the light cycle and captive-born, first spawners exhibiting no significant peak in activity. The trend to spawn midday is most likely influenced by predation factors, while the lack of a spawning periodicity in the offspring may be explained by developmental processes, the absence of environmental cues or the tendency for smaller males to be opportunistic in spawning events. The details of spawning behavior recorded in this study provide a database to investigate species differences and to indicate changes due to chemical and physical disturbances.