Is the iron an essential nutrient for enterococci?

Enterococci were considered as not requiring iron. The aim of study was evaluation of relationship between enterococci and iron. This study examined these relationships in a 71 strains belonging to two species--Enterococcus faecalis and Enterococcus faecium, which are often isolated from human infections. The iron is an essential nutrient for enterococci. Demonstrated that iron--regardless of the concentration in the medium--is collected during growth. Iron deficiency in the nutrient medium resulted in changes in the kinetics of growth of enterococci. Inhibiting the growth of enterococci by iron chelators and lack of inhibition are further proof of this demand for iron bacteria. Enterococci have the ability to acquire this important element of its connections with natural and synthetic chetators with different strength of chemical bonding and structure. Bacteria of the genus Enterococcus have a natural resistance to many antimicrobial agents. In the hospital environment can easily acquire resistance genes to many other classes of antimicrobial compounds. For these reasons, treatment of enterococal infections poses more difficulties. Inhibition of iron uptake in enterococci can be helpful in reducing and combating enterococal infections.     

Assimilatory ferric reductases in enterococci

Enterococci produced assimilatory ferric reductases which are surface-associated enzymes. This is the first report of the intracellular enzymic reduction of iron by enterococci. A correlation between ferric reductases activity and species affiliation and origin of strains was found. The expression of ferric reductases has not affected by the presence or absence of iron, hemin and hemoglobin in the growth medium. Enterococcal ferric reductases exhibit a very broad specificity. A number of different ferric organic and inorganic compounds, natural and synthetic iron chelators and iron body sources including lactoferrin, transferin, ferritin, haemoglobin, could be reduced. A surface-associated ferric reductases may be one component of a general iron scavenging mechanism which can be used by enterococci growing in a variety of environments.     

The interchangeability of siderophores in the Enterococcus genus

The functional interchangeability of siderophores was tested among 62 strais belonging to 12 species of genus Enterococcus. Most investigated strains were from E. faecalis and E. faecium species. The majority of examined enterococcal strains appeared highly resistant to EDDA (ethylene-di-amine-di-ortho-hydroxyphenylacetic acid), therefore the group of sensitive strains involved only 11 used as indicator strains. The determination of interchangeability of siderophores within enterococcal strains was performed using EDDA-agar media into which the indicator strains were included. Test colonies (donor strains) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. Only two strains: E. solitarius DSM 5634 and E. pseudoavium DSM 5632 did not demonstrate the ability to utilize siderophores synthesized by all investigated strains. The other tested indicator strains appeared to be recipients of siderophores from 20-52 donor enterococcal strains. The ability to exchange siderophores in enterococci was found as the feature characterizing individual strains.     

The effect of synthetic iron chelators on bacterial growth in human serum

The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms (Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis, but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa. Desferrioxamine enhanced the growth of all except E. coli. These results suggest that the chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergoing chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

The effect of synthetic iron chelators on bacterial growth in human serum

Abstract The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms ( Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa ); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis , but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa . Desferrioxamine enhanced the growth chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergiong chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.     

Utilization of exogenous siderophores by enterococci]

The study was undertaken to investigate the ability of enterococci to assimilate iron via siderophores of bacteria living in the same habitats in the human organism. The potential recipients of exogenous siderophores were six Enterococcus faecalis and six Enterococcus faecium strains, isolated from clinical materials of human origin. The donors of siderophores were Gram-negative rods (various species of the Enterobacteriaceae, Pseudomonas and Acinetobacter) and Gram-positive cocci (various species of Staphylococcus and Streptococcus). All of the investigated E. faecium and only two E. faecalis strains demonstrated the ability to utilize the siderophores of the aforementioned bacterial groups, predominantly the chelators of Gram-negative rods, those of Gram-positive cocci were utilized to a smaller extent. Four recipient strains from E. faecalis species did not demonstrate the ability to utilize siderophores synthesized by all of 40 investigated donor strains.     

The functional interchangeability of enterobacterial and staphylococcal iron chelators

The functional interchangeability of staphylococcal and enterobacterial iron chelators was investigated with an indicator system in which minimally effective concentrations of ethylene diamine di-ortho-hydroxyphenyl acetic acid (EDDA) were used to inhibit the growth of indicator strains in the depth of simple agar media by making the iron unavailable. Test colonies were then applied to the surface of the media to determine whether the indicator organisms, by utilising chelators from the test colony could obtain the required iron for growth, in its vicinity.Approximately 50% of staphylococcal strains, both S. aureus and S. epidermidis, reversed the inhibition of enterobacterial indicators, whereas almost all enterobacterial test strains, representing five genera, reversed the inhibition of the staphylococcal indicators. A purified preparation of the enterobacterial iron chelator enterochelin also reversed the inhibition of four out of the five staphylococcal indicator strains.     

Iron Chelators as antimalarials: the biochemical basis of selective cytotoxicity

Like all living organisms, malania parasites need iron for vital cell functions and must handle their cellular contents in a highly regulated fashion. However, in the asexual stage of growth, parasites present a special case of iron metabolism. Despite dwelling in a sea of hemoglobin and carrying inside the remnants of its degradation products, intracellular parasites have no obvious means for mobilizing bioavailable iron from the host or from the medium. In that sense they differ from most mammalian cells which are exposed to body fluids and acquire the metal from circulating iron carriers. The uniqueness of iron handling by parasites is manifested in their susceptibility to drug-induced deprivation of the metal. Both natural and synthetic iron(III) chelators of the hydroxamate family have been shown to abolish cell growth in vitro, and to reduce malaria infection in vivo as well as in the clinic. Z. loav Cabantchik here explores the molecular basis for the selective cytotoxicity of iron(III) hydroxamate chelators and the potential of iron chelation therapy in the management of malaria.     

B群脑膜炎奈瑟氏球菌铁调蛋白的诱导表达及其性质研究

对２株Ｂ群脑膜炎奈瑟氏球菌的铁调蛋白的表达及其性质作了研究。在流脑半综合培养基中加人铁螯合剂,诱导了铁调蛋白的表达,并比较了三种铁螯合剂的效果。结果表明,ＥＤＤＨＡ是最令人满意的螯合剂。ＥＬＩＳＡ实验证实含有铁调蛋白的外膜蛋白可以增强对小白鼠的免疫原性。Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验证明有两种铁调蛋白是有效的抗原成分,分子量分别在７５ｋＤａ和８５ｋＤａ附近。由分子量来推算,分别应为ＦｅＲＰ－７０及ＴＢＰ２。     

Acquisition of iron by Aeromonas salmonicida.

The ability of six typical and three atypical strains of Aeromonas salmonicida to sequester Fe3+ from the high-affinity iron chelators ethylenediaminedihydroxy-phenylacetic acid, lactoferrin, and transferrin was determined. Typical strains were readily able to sequester Fe3+ and used two different mechanisms. One mechanism was inducible and appeared to involve production of a low-molecular-weight soluble siderophore(s). Iron uptake by this mechanism was strongly inhibited by ferricyanide. One virulent strain displayed a second mechanism which was constitutive and required cell contact with Fe3+-lactoferrin or -transferrin. This strain did not produce a soluble siderophore(s) but could utilize the siderophore(s) produced by the other strain. Fe3+ uptake by this stripping mechanism was strongly inhibited by dinitrophenol. Atypical strains displayed a markedly reduced ability to sequester iron from high-affinity chelators, although one of them was able to utilize the siderophores produced by the typical strain. In all strains examined, Fe3+ limitation resulted in the increased synthesis of several high-molecular-weight outer membrane proteins.     

Iron requirement and chelator production of staphylococci,Streptococcus faecalis and enterobacteriaceae

The effect of iron deprivation on growth of 101 aerobic strains of gram-positive and gram-negative bacteria was studied on agar media in the presence of various concentrations of the synthetic iron chelator ethylene diamine diorthohydroxyphenyl acetic acid (EDDA) and the iron binding protein transferrin.Growth of Staphylococcus epidermidis was inhibited by 15mm EDDA and 1.5mm transferrin. Staphylococcus aureus was only inhibited by 44mm EDDA and not by transferrin. None of the strains of S. faecalis was inhibited. The majority of the enterobacteriaceae (E. coli, Salmonella spp, Klebsiella spp) was inhibited by 44mm EDDA and 1.5mm transferrin. The relation between susceptibility and concentration of EDDA and transferrin was expressed as S-value for each species. Iron supply with various iron compounds could restore the effects of inhibition.In all species except in S. faecalis iron chelator production could be demonstrated, using indicator plates of media containing EDDA and flooded with 10⁴–10⁵ colony forming units of indicator organisms.The iron chelator of both S. epidermidis and S. aureus could stimulate growth of S. epidermidis, but not that of enterobacteriaceae. Iron chelators from all gram-negative bacteria were functionally interchangeable, but did not stimulate growth of gram-positive bacteria.     

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe³⁺ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe³⁺] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe³⁺ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.     

Studies on siderophore exchange properties between staphylococci and various species of gram-positive and gram-negative bacteria

The ability of iron utilizing by means of staphylococcal siderophores by bacteria belonging to genera: Acinetobacter, Corynebacterium, Curtobacterium, Clavibacter, Bacillus and Mycobacterium was investigated. The staphylococcal donor strains (18 species) used in these experiments were characterized by the ability to utilize siderophores produced by various strains belonging to aforenamed genera. The utilization of staphylococcal siderophores was studied on agar media in which minimally effective concentrations of ethylenediaminedi-ortho-hydroxyphenylacetic acid (EDDA) were used to inhibit indicator strains. Test colonies (staphylococcal) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. The growth inhibition by EDDA of most strains from the Acinetobacter rods and from the coryneform-organisms (plant pathogen) genera, and strains from the species: B. subtilis, M. phlei, M. smegmatis, M. fortuitum was reversed by staphylococcal siderophores. None of the staphylococcal strains investigated, had the ability to exchange siderophores with strains from the species: C. pseudodiphtheriticum, Corynebacterium ANF group, B. megaterium, M. vaccae, M. chitae and M. parafortuitum.     

Chelated iron sources are inhibitors of Pseudomonas aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the human lung

Aims:  To determine whether chelated sources of ferric iron were efficient inhibitors of biofilm formation in Pseudomonas aeruginosa and might be suitable for drug delivery to the lungs of cystic fibrosis (CF) patients via nebulization.
Methods and Results:  The response of P. aeruginosa biofilms to elevated iron concentrations in the form of eight structurally varied iron chelators in a microtitre plate assay for biofilm production was examined in the lab. Among these iron chelates, picolinic acid and acetohydroxamic acid-chelated iron were able to effectively thwart biofilm production in P. aeruginosa PA14 and in 20 clinical isolates of P. aeruginosa from a local hospital. The chelated iron sources showed excellent distribution in an Anderson cascade impactor model of particle size distribution in the human lung.
Conclusions:  Ferric picolinate and ferric acetohydroxamate are effective anti-biofilm compounds against both lab and clinical strains of P. aeruginosa and are readily nebulized into particles of suitable size for lung delivery.
Significance and Impact of the Study:  The data herein serve both to solidify the growing base of literature correlating high iron levels with biofilm inhibition in P. aeruginosa and to highlight the potential of these chelators as nebulized agents to combat biofilms of P. aeruginosa in CF patients.     

Modulation of cell proliferation in rat liver cell cultures by new calix[4]arenes

Cell cycle progression is dependent on intracellular iron level and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing two aspartic/glutamic acid, ornithine groups or hydrazide function at the lower rim, designed as potential iron chelators. The synthesis only afforded calix[4]arenes in the cone conformation. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the rat hepatoma cell line Fao by measuring mitochondrial succinate dehydrogenase activity. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicated that among all tested compounds, monohydrazidocalix[4]arene 2 which is not cytotoxic in Fao cells exhibits interesting antiproliferative activity. This effect, independent on iron depletion, remains to be further explored. Moreover, it also shows that new substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.     

In vitro characteristics of 1-phenyl-3-methyl-4-acylpyrazol-5-ones iron chelators

Iron chelators represent a group of structurally different compounds sharing the ability of iron binding. The group has been evolving in recent years mainly due to novel experimental indications associated with variable requirements for iron chelators. A group of synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones has been known for many years but data on their potential biological activity are rather limited.In this study, we analysed a series of these compounds for their iron-chelating properties as well as for their effects on iron based Fenton chemistry. For the former ferrozine spectrophotometric method and for the latter HPLC method with salicylic acid were used.All of the tested compounds were very efficient ferric chelators but their ferrous-chelating effects differed according to the acyl substitution. Notwithstanding various ferrous chelation activities, the individual Fe²⁺-affinities were not significantly different through pathophysiologically relevant pH conditions and some of the tested substances were more potent ferrous chelators at pH 4.5 than clinically used standard deferoxamine. Of particular interest is H₂QpyQ /2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine/ which iron-chelating affinity increased when pH was decreasing. In spite of ferrous chelation differences, most of the tested acylpyrazolones were similarly active powerful inhibitors of Fenton chemistry as deferoxamine.Conclusively, acylpyrazolones are efficient iron chelators and H₂QpyQ may represent a prototype of novel specific chelators designated particularly for chelation at acidic conditions.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes

Cell cycle progression is dependent on the intracellular iron level, and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing alkyl acid and alkyl ester groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670 (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in human hepatocarcinoma HepaRG cell cultures using the MTT assay. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicate that their antiproliferative effect is due to their cytotoxicity. The efficiency of these compounds, comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that novel substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.     

Evaluation of Chromocult enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

AIMS: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. METHODS AND RESULTS: The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. CONCLUSIONS: A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.     

Differentiation of Some Enterococci by Gas Chromatography

Relative fatty acid compositions of 37 enterococci were examined by gas chromatography. Streptococcus faecalis, S. faecium, and S. faecium var. durans yielded similar fatty acid patterns. Strains of S. faecium var. casseliflavus, and a motile yellow-pigmented streptococcus, contained very low levels of C(19:0) cyclopropane fatty acid and four unidentified components, compared to the other strains of enterococci examined. There were no significant differences in the fatty acid patterns of enterococci grouped according to plant, animal, or human source.