The quantification of the neutralization of the action of antibiotics by cationic iron.

This paper describes the reversal of antibiotic action by iron. Crude measurements of the molar ratio of iron to various antibiotics at reversal indicated that ampicillin, carbenicillin and lincomycin had low ratios of 0.2 to 2.4. With the remainder the ratios lay between 13 and 105; they averaged 46 for the tetracyclines, and 70 for the aminoglycosides. Precise measurement with Fe+++ and tetracycline revealed that the molar ratio for neutralization increased with increasing concentrations of tetracycline; from 1 to 625 mg/l the ratio increased over three-fold from 35 to 118. At the tetracycline concentration of 5 mg/l--usually achieved in plasma during effective therapy--the ratio is of the order of 45.

The neutralization of antibiotic action by metallic cations and iron chelators.

The neutralization of the action of a variety of antibiotics on klebsiellae by moderate doses of di- and tri-valent metallic cations, was measured in vitro. Some beta-lactams tested were affected by Mg++ and by Cu++. Of six tetracyclines one was moderately neutralized by Ca++, two by Mg++, three by Cu++, and all, strongly, by Fe+++. Erythromycin was affected by Ca++, lincomycin by Ca++, Mg++ and Cu++. Aminoglycosides were affected by Ca++ and Mg++ and strongly by Fe+++. Five antibiotics (three beta-lactams and two macrolides) with high MICs for klebsiellae were tested against staphylococci: most of the reversing agents were ineffective. The microbial iron chelators, desferrioxamine and enterochelin were largely inactive, affecting only two aminoglycosides.

The antagonism of tetracycline and ferric iron in vivo.

To test the hypothesis that the in-vivo antibiotic action of tetracycline might be affected by ferric iron and the enhancement of infection by ferric iron by tetracycline, the actions of intraperitoneal antibiotic and local ferric ammonium citrate, given separately and together, were measured in the dorsal skin of guinea-pigs bearing lesions due to staphylococci, streptococci, a Proteus sp., an Erysipelothrix sp., Clostridium perfringens, Pseudomonas aeruginosa, Aeromonas hydrophila and Klebsiella pneumoniae. Tetracycline, given in two intraperitoneal doses of 25 mg/kg at 0 and 2 h after intracutaneous challenge, maintained plasma concentrations of 4-6 micrograms/ml for more than the first 4 h of infection, after which the local lesions had become largely insusceptible to the antibiotic. The intracutaneous injection of Fe 10 micrograms in a volume of 0.1 ml containing the bacteria was sufficient to enhance infection by those strains susceptible to this effect. The in-vivo efficacy of tetracycline was not always related to low MIC; a low MIC was sometimes associated with little action and a high MIC with moderate action. Sixteen organisms were tested. The iron diminished the tetracycline effect only feebly with one staphylococcal strain and the strain of E. rhusiopathiae. In only one case, with a strain of Proteus sp., was the tetracycline action grossly diminished. On the other hand, tetracycline diminished the enhancement effect of iron moderately with three strains of staphylococci and one strain each of K. pneumoniae, P. aeruginosa and C. perfringens, and strongly with two strains of staphylococci, a group-C streptococcus and one strain each of K. pneumoniae, E. rhusiopathiae and A. hydrophila. It is evident that the diminution of tetracycline action by moderate excess of readily available Fe , whether endogenous or administered, is an unlikely event (three instances among the 16 tested) whereas the diminution of the infection-enhancing effect of iron by tetracycline is much more likely (12 instances among the 16). Insofar as a decrease in iron available for enhancement of infection is valid evidence of a diminution of the iron available for necessary physiological processes of the subject treated, our results suggest that these processes might be affected by tetracycline.

The variable response of bacteria to free haemoglobin in the tissues.

The local enhancement of infection by exogenous ferric iron, as ferric ammonium citrate, and by ferrous iron as guinea-pig haemoglobin, was assessed in studies with 55 strains of bacteria injected into the skin of guinea-pigs. The test organisms included Staphylococcus aureus, Streptococcus spp., Klebsiella spp., Escherichia coli and Pseudomonas aeruginosa. Four strains of Bacteroides spp. were tested with haemoglobin only. As previously reported with other strains, enhancement of infection by members of a given species by ferric iron was variable; in this study infection with only 11 of 59 strains was enhanced. Haemoglobin either of equal or lesser iron content was a more potent enhancer, affecting 27 of the 59 strains. The enhancement ranged from two-fold to 80-fold, the higher figures on the whole being characteristic of haemoglobin enhancement. Some few instances of depression by both haemoglobin and ferric ammonium citrate were noted. A few tests were made with systemic haemoglobin but the concentrations attainable were largely ineffective. Enhancement of infection did not appear to be related to the capacity of a strain to lyse or digest host red blood cells. In so far as guinea-pigs, whose antibacterial defences are lowered by ferric or ferrous iron, represent human subjects at risk of infection because of clinical circumstances characterised by excess of available iron--either exogenous or as a result of haemolysis--our results with organisms of a kind commonly associated with infection in hospitals suggest that only a small proportion of environmental bacteria can take advantage of any decreased resistance associated with iron excess.

The variable response of bacteria to excess ferric iron in host tissues.

The enhancement by exogenous ferric iron, both systemic and local, of the infectivity of 120 strains of bacteria, representing 17 genera, was measured in the skin of guinea-pigs. Systemic iron enhanced only 23% of 115 strains, and local iron 49% of 71 strains. Systemic iron, by an apparently anti-inflammatory action, depressed the size of lesions produced by 27 of the non-enhanced strains from nine of the genera tested. For most strains, the degree of enhancement was small, ranging from 2- to 8-fold, and often evident only with the more effective local iron; among these were some near-saprophytes like Mycobacterium phlei, M. smegmatis, Bacillus cereus and Clostridium bifermentans. Substantial enhancement, from 14- to 50-fold, was observed with the more pathogenic among the strains tested: namely BCG, Corynebacterium ovis, C. murium, Listeria monocytogenes, Erysipelothrix rhusiopathiae, Cl. perfringens, Cl. septicum, Cl. oedematiens, and some strains of Klebsiella spp., Proteus spp. and Aeromonas hydrophila. The enhancement of BCG by a single dose of iron given locally with the inoculum was only feebly manifest after 7 days, but substantial after 14--19 days, indicating the decisive effect of interference with an early humoral defence on the establishment of chronic infection some time later. Insofar as guinea-pigs whose antibacterial defences are lowered by substantial amounts of exogenous iron in the circulation represent human subjects at risk of infection because of clinical states characterised by excess of available iron, the results of the survey suggest that only a minority among the environmental bacteria can take advantage of the decreased resistance associated with such states; but that this minority is likely to include the more virulent strains in the environment.

Microbial iron-chelators and their action on Klebsiella infections in the skin of guinea-pigs.

Preparations of catechols from ethyl acetate extracts of cultures of Klebsiellae in a low-iron medium contained iron-chelators whose potency was measured by the reversal of the bacteristasis of Escherichia coli and klebsiellae in unheated horse serum, and of the growth-inhibition of these two organisms by ethylene diamine di-orthohydroxyphenyl acetic acid (EDDA). As revealed by in situ tests of paper chromatograms, there was a multiplicity of biologically active chelators in the preparations. Catechols from strains both of high and low virulence for guinea-pigs enhanced the skin infectivity of most of the 10 Klebsiella strains tested. The enhancement was roughly proportional to iron-enhanceability with the 6 iron-enhanceable (E+) strains, though not as great as that by iron. But of the 4 (Eo) strains not enhanceable by iron, two were moderately enhanced by the catechols. The Streptomyces iron-chelator desferrioxamine B also enhanced infectivity, again roughly in proportion to the iron enhanceability of the strains; though one Eo strain was substantially enhanced. The synthetic iron-chelator EDDA did not enhance infection.

The action of iron on local Klebsiella infection of the skin of the guinea-pig and its relation to the decisive period in primary infective lesions.

The infectivity of 16 strains of Klebsiella spp. and its modification by systemic and local ferric iron were tested in the skin of the guinea-pig. The in vivo proliferation of 11 strains was enhanced in varying degrees by Fe+++ (E + strains); 5 strains (Eo) were not enhanceable even by large doses of Fe+++. Of 10 strains examined in detail, 6 were E + and 4 were E0. Guinea-pig and human sera were consistently bacteriostatic for E + strains and bactericidal for Eo strains. Both Fe+++ and microbial iron-chelators abolished the bacteriostasis of E + strains but did not affect the lethal effect on Eo strains. Both effects were diminished by heating the sera to 56 degrees for 30 min and by the anticomplementary substance Liquoid; neither appeared to be due to specific antibody. Virulence, as measured in the skin and by intravenous injection, was roughly associated with degree of enhanceability by iron, the EO strains being among the least virulent. The volume of plasma exudate entering the skin during the first 5 h was sufficient to kill a large proportion of the infecting doses of Eo strains and to inhibit the growth of infecting doses of E + strains. Enhancement of the latter by Fe+++ is predominantly the result of inhibition of the non-specific bacteriostasis exerted by the extravascular plasma. Lesions by E + strains aged 4 h or more are insusceptible to systemic Fe+++ and only moderately susceptible to large doses of local Fe+++. The insusceptibility appears to be due to segregation of the infecting bacilli within exudate leucocytes. Klebsiella infections accordingly provide another example of an initial decisive period of action of the antibacterial defences-in this case non-specific and humoral-which cease to be locally effective after the first few hours. Besides enhancing lesions due to E + strains, systemic Fe+++ has an opposite, apparently anti-inflammatory action on klebsiella lesions, slightly decreasing their size. It was evident with all the strains tested, whether dead or alive, but not in E + lesions in circumstances when they were susceptible to enhancement by the Fe+++.

Enterobacterial chelators of iron: their occurrence, detection, and relation to pathogenicity.

In or on agar media, low-density seedings of enterobacteria fail to grow in the presence of certain concentrations of ethylene diamine-di-orthohydroxyphenyl acetic acid (EDDA); on the other hand, high-density seedings not only grow but secrete iron chelators which release the iron bound by the EDDA in the medium and stimulate the growth of low-density seedings. Plates of media containing EDDA with low-density seedings of indicator organisms were used to survey iron-chelator production in seven enterobacterial genera, including a number of virulent smooth (S) forms from which rough (R) mutants had been obtained. An examination of over 80 strains of Aeromonas, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella and Shigella species indicated that the iron chelators from bacteria in all these genera were functionally interchangeable. Chelator production was equally good with randomly selected avirulent and virulent strains of Klebsiella spp. and E. coli; and with the S forms and their avirulent R mutants in one pair of escherichiae, six pairs of salmonellae (4 species) and six pairs of shigellae (3 species). As determinable in vitro, the capacity to synthesise iron chelators is clearly no index of the capacity of a strain to proliferate in vivo.