Virulence factors of Eschericha coli in urinary isolates

Escherichia coli strains isolated from 100 urine samples taken from patients with urinary tract infections (UTI) and from 20 normal fecal (NF) samples were examined for serum resistance, mannose-resistant hemagglutination of human erythrocytes (MRHA) and for production of aerobactin, hemolysis and colicin. Among the UTI E. coli strains, 79% produced aerobactin, 69% showed serum resistance, 44% produced MRHA, 32% were beta-hemolytic and 22% were colicinogenic. A greater proportion of UTI E. coli strains produced aerobactin, colicin V, beta-hemolysis and MRHA when compared to NF strains. Production of MR hemagglutins was significant correlated with that of aerobactin and hemolysin. These results suggest that the presence of aerobactin may be a significant etiological factor in UTI, and that the production of MR adhesins and of hemolysin also might contribute to the virulence of these strains

Analysis of ferrichrome biosynthesis in the phytopathogenic fungus Ustilago maydis: cloning of an ornithine-N5-oxygenase gene.

By using a non-enterobactin-producing enb-7 mutant of Salmonella typhimurium LT2 as a biological indicator, a novel screening method was developed for identifying mutants of Ustilago maydis defective in the biosynthesis of the siderophores ferrichrome and ferrichrome A. Two classes of siderophore mutations, both recessive, were isolated after mutagenesis of haploid cells of the corn smut fungus. Class I mutants no longer produced ferrichrome while retaining the ability to produce ferrichrome A; class II mutants were defective in the production of both ferrichrome and ferrichrome A. Genetic and biochemical data suggest that class II mutants are defective in the ability to hydroxylate L-ornithine to delta-N-hydroxyornithine, the first step in the biosynthesis of these siderophores. A genomic library of wild-type U. maydis DNA was constructed in the cosmid transformation vector pCU3, which contains a dominant selectable marker for hygromycin B resistance. Two cosmids, pSid1 and pSid2, were identified in this library by their ability to complement class II siderophore auxotrophs. The production of both siderophores was concomitantly restored in the majority of the resultant transformants. Transforming DNA could be recovered from the fungal, cosmid-containing transformants by in vitro packaging with lambda bacteriophage extracts. Alternatively, the clones could be identified by a sib selection procedure. Cotransformation was found to occur at a high frequency in the fungus and was used to determine that a 2.5-kilobase HindIII-NruI fragment in pSid1 was responsible for complementing the class II siderophore biosynthetic mutation.

Assessment of biological and colony hybridization assays for detection of the aerobactin system in Escherichia coli from urinary tract infections.

A total of 466 E. coli strains from urinary tract infections (UTI) were screened for the presence and expression of the aerobactin system by a colony hybridization test and a bioassay. A probe carrying part of the genes for aerobactin synthesis was used. A total of 43.1% (201) of the strains were positive in the probe test and undoubtedly positive in the bioassay. When doubtfully positive bioassays were included, this figure rose to 49.8% (232). An additional 4.9% (23) of the strains were positive in the colony hybridization test only while 44% (205) of the strains were negative in both tests. Doubtfully positive bioassays were probably due either to a false positive reaction or to a weak expression of the aerobactin system. 01:K1:H- strains were characteristically probe positive and doubtfully positive in the bioassay. The incidence of isolates positive by both methods or by only one of them was significantly higher among isolates from cases of pyelonephritis (Py) than among those from asymptomatic bacteriuria (ABU) and normal feces (FN) (P less than 0.01).

Incidence of aerobactin-positive Escherichia coli strains in patients with symptomatic urinary tract infection.

A total of 323 Escherichia coli strains from children with primary acute non-obstructive pyelonephritis (n = 144) or cystitis (n = 56) and from adults with acute non-obstructive pyelonephritis (n = 66) or cystitis (n = 57) were examined for presence of the aerobactin-mediated iron uptake system and expression of P-fimbriae. Overall, pyelonephritogenic Escherichia coli strains were significantly more often aerobactin-positive (72%) than cystitis strains (42%) (p less than 0.001). Seventy-three percent of the isolates from children with acute pyelonephritis were aerobactin-positive compared to 54% of the cystitis strains (p less than 0.05). Pyelonephritogenic Escherichia coli strains from adults were also significantly more often aerobactin-positive (70%) than cystitis strains (30%) (p less than 0.001). The cystitis strains from children were more often aerobactin-positive than cystitis strains from adult patients (p less than 0.05). There was a significant correlation between presence of the aerobactin-mediated iron uptake system and expression of P-fimbriae in all strains (p less than 0.001).

Aerobactin production as a virulence factor: a reevaluation.

Iron starvation is one of the major barriers that virulent bacteria must overcome in order to proliferate in the host. Virtually all microorganisms possess high affinity iron (III) transport systems mediated by low molecular weight iron specific chelators called siderophores, the synthesis of which is activated under iron-limiting conditions. Siderophore aerobactin is frequently produced by enterobacteria which cause various types of infections in humans and animals. The status of aerobactin production as a virulence factor is evaluated both from data derived from experimental infection systems and the actual presence of this siderophore in clinical isolates. Aerobactin appears to be an important contributor to extracellular pathogenesis (mostly, that of Escherichia coli strains causing septicaemia and urinary tract infections) and to the extracellular stages of growth of intracellular pathogens like Shigella. When invasive bacteria actually enter target cells, acquisition of iron seems to occur independently of siderophore production. The feasibility of an antimicrobial therapy aimed at interfering with siderophore functioning is discussed.

Nucleotide sequence of the iucD gene of the pColV-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions.

Gene iucD of the aerobactin operon of the Escherichia coli plasmid ColV-K30 encodes a membrane-bound enzyme synthesizing N6-hydroxylysine, the first product of the aerobactin biosynthesis pathway. The entire nucleotide sequence of the cloned iucD gene was determined, from which the primary and some aspects of the secondary structure of the encoded peptide were deduced. E. coli cells harboring multicopy plasmid pVLN12 (iucD+) hyperproduced an approximately 50-kilodalton peptide which was purified and identified as the product of the gene by examination of its amino-terminal sequence. Two iucD'-'lacZ gene fusions were constructed in vitro and four iucD'-'phoA gene fusions were generated in vivo by mutagenesis of iucD with transposon TnphoA (Tn5 IS50L::phoA). Analysis of the corresponding fusion proteins suggested at least two domains of attachment of the IucD protein to the inner side of the cytoplasmic membrane. The first apparent membrane-bound domain was found within the first 25 amino acids of the protein and showed a sequence which resembled that of the signal peptides.

Epidemiology of aerobactin production in Enterobacteriaceae.

Clinical isolates (496) of 10 different enterobacterial genera were studied for aerobactin excretion, colicin production and antibiotic resistance. In the case of Escherichia coli, the incidence of aerobactin-positive strains in 108 blood isolates (45%) was not significantly different from that corresponding to faecal sources (41%). Although colicin V production was much more frequently associated with aerobactin production than other colicins, colicin V was only produced by 26% of aerobactin excreters. Some strains were aerobactin-negative and colicin V producers. Aerobactin production seemed to be significantly associated with plasmidic antibiotic resistance. The production of the siderophore is described for the first time in Proteus, Serratia and Morganella strains.

Mapping of mutations in Pseudomonas aeruginosa defective in pyoverdin production.

Twelve mutants of Pseudomonas aeruginosa PAO defective in pyoverdin production were isolated (after chemical and transposon mutagenesis) that were nonfluorescent and unable to grow on medium containing 400 microM ethylenediaminedi(o-hydroxyphenylacetic acid). Four mutants were unable to produce hydroxamate, six were hydroxamate positive, one was temperature sensitive for pyoverdin production, and another was unable to synthesize pyoverdin on succinate minimal medium but was capable of synthesizing pyoverdin when grown on Casamino Acids medium (Difco Laboratories, Detroit, Mich.). The mutations were mapped on the PAO chromosome. All the mutations affecting pyoverdin production were located at 65 to 70 min, between catA1 and mtu-9002.

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.

Production of aerobactin by some species of the genus Yersinia.

Seventy-seven isolates of Yersinia species were examined for evidence of hydroxamate biosynthesis. A total of 7 of 12 Y. frederiksenii, 1 of 5 Y. kristensenii, and 5 of 5 Y. intermedia isolates synthesized an hydroxamate species which was chromatographically and electrophoretically identical to aerobactin. Proof that this material was indeed aerobactin was obtained for one strain of Y. frederiksenii by using mass spectrometry. None of 50 Y. enterocolitica nor any of 5 Y. pseudotuberculosis isolates produced hydroxamates.

[Excretion of 2,3-dihydroxybenzoic acid by an enterobactin-negative multiresistant wild strain of Salmonella typhimurium]. / DHBA (2,3-Dihydroxybenzoesäure)-Ausscheidung durch einen enterobactinnegativen multiresistenten Salmonella typhimurium-Wildstamm.

In investigations of iron siderophores produced by different Salmonella typhimurium strains we found a strain deficient in enterobactin production. Using microbiological and chemical methods, we detected the production and excretion of 2,3-dihydrobenzoic acid, a metabolite of the enterobactin biosynthesis pathway. The strain was able to produce aerobactin. We suggest that the iron uptake of pathogenic strains can be mediated by aerobactin alone. For a correct enterobactin bioassay it is necessary to use a mutant unable to grow on 2,3-dihydroxybenzoic acid.

Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli.

One of the chromosomal segments associated with the virulence of Shigella flexneri and transferred to Escherichia coli K-12 by conjugation has been shown to code for the production of aerobactin and for the synthesis of an iron-regulated 76,000-dalton (76K) outer membrane protein. Analysis of various E. coli K-12-S. flexneri transconjugants showed that the genes involved with the synthesis of aerobactin and with the production of the 76K protein were linked to the mtl region of the S. flexneri chromosome. S. flexneri itself synthesized a 76K protein in its outer membrane under iron restriction as well as traces of 81K and 74K proteins. An examination of four enteroinvasive strains of E. coli showed that each produced aerobactin and a 76K outer membrane protein during iron-restricted growth. The profile of the iron-regulated proteins expressed by the enteroinvasive strains of E. coli was virtually identical to that expressed by the laboratory-constructed E. coli K-12-S. flexneri hybrids under the same growth conditions.

Genetic and biochemical characterization of the aerobactin synthesis operon on pColV.

Certain ColV plasmids of Escherichia coli contain genes that specify functions for the acquisition of iron(III) via aerobactin. The locus for aerobactin synthesis of pColV-K311 was cloned into pBR322. Mutagenesis with the transposon Tn1000, and the generation of deletions with restriction enzymes resulted in multicopy plasmids which complemented pColV mutants impaired in various steps of aerobactin synthesis. The insertion and deletion mutants were mapped and assigned to three loci termed aerA, aerB, and aerC. It is proposed that these genes mediate the synthesis of aerobactin by specifying functions for hydroxylation (aerA), acetylation of the 6-amino group of 6-hydroxylysine (aerB), and the coupling of N-acetyl-N-hydroxy-lysine with citrate (aerC). The order and transcription polarity of the structural genes was found to be aerB, aerC, aerA.

Cloning of the aerobactin-mediated iron assimilation system of plasmid ColV.

The high-affinity iron assimilation system of plasmid ColV-K30 was cloned on the vector plasmid pPlac. Plasmid pABN1 was isolated by means of sensitivity to cloacin, a bacteriocin using the same outer membrane receptor as ferric aerobactin. Restriction maps were determined for this plasmid and for a subclone, pABN5. Plasmid pABN1 codes for the complete gene complex, whereas plasmid pABN5 encodes only the biosynthetic genes for aerobactin. Regulation of the uptake system by iron is retained in cloned sequences of pABN1.

Iron-suppressible production of hydroxamate by Escherichia coli isolates.

A total of 476 strains of Escherichia coli isolated from humans, pigs, cattle, poultry, potable water, or effluent were examined for iron-suppressible ability to produce hydroxamate. Isolates able to produce such material (Hyd+ isolates) are presumed to be able to carry out hydroxamate-dependent transport of iron. The percentages of Hyd+ isolates found among E. coli isolated from the feces of breast-fed babies (71%), adults (46%), milk-fed calves (32%), or poultry (28%) were significantly greater (P less than 0.01) than the percentages isolated from potable water and effluent (6%) or from the feces of suckling piglets (6%), weaned pigs (6%), or weaned cattle (4%). The percentages of Hyd+ isolates found among E. coli associated with diarrhea in humans (51%), weaned pigs (7%) or calves (25%) were not significantly different (P greater than 0.1) from those found among strains isolated from corresponding nondiarrheic hosts. Many of the E. coli isolated from cases of E. coli bacteremia in humans and poultry were Hyd+ (64% and 83%, respectively). We conclude that ability to carry out hydroxamate-mediated transport of iron is widely distributed among natural isolates of E. coli but that the distribution of Hyd+ E. coli is not random. E. coli isolated from sources where levels of available iron might be expected to be low tend to be Hyd+. It seems that a link may exist between prevalence of Hyd+ E. coli and active host-defense based on restricted availability of iron.