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AEROBACTIN-MEDIATED IRON UPTAKE: A VIRULENCE DETERMINANT IN ENTEROPATHOGENIC ESCHERICHIA COLI?
763
NALIDIXIC-ACID-RESISTANT SHIGELLA
AEROBACTIN PRODUCTION BY ENTEROPATHOGENIC ISOLATES OF
DYSENTERIAE I
E COLI
SIR,-In our report of an epidemic of multiresistant Shigella dysenteriae we highlighted the indiscriminate use of nalidixic acid by village practitioners in rural areas. Resistance to nalidixic acid is chromosomally mediated and the drug’s use in shigellosis has been supported by some workers. McCormack2described nalidixic acid as first-line therapy for shigellosis, resulting in lower morbidity and case-fatality rates, especially among the very young and old. Malengreau3reported that during treatment with nalidixic acid only one strain out of 10 000 patients was resistant to this drug. In another epidemic of dysentery due to S dysenteriae in July to September, 1984, we isolated 52 strains from faecal samples of patients from two districts of the Kashmir Valley. These patients had a history of passing blood and mucus for 4-15 days. Antibiotic susceptibility was tested by disc diffusion with Difco discs. 38 (73%) strains were resistant to nalidixic acid (30 lAg) and ampicillin 10, chloramphenicol 30, tatracycline 30, streptomycin 10, sulphadiazine 300, and co-trimoxazole I-25+23’75 g discs. All except 1 strain were sensitive to furazolidone 100 pig and neomycin 30 g. In transfer studies by direct crosses and triparental crosses we observed that nalidixic acid resistance was the only resistance that
could be transferred to Escherichia coli C600 KI2 P- Lac-Rif by overnight crosses. The frequency of transfer was low (10-6 to 10’). Nevertheless, the observation is worrying. Another important observation was the high degree of nalidixic acid resistance transferred among transconjugants; while the donor strains had minimum inhibitory concentrations of 30 tg/ml, the transconjugants had MICs of 60 g/ml or more, by plate dilution method. This could be due to variable gene expression in different hosts. Studies of phage restriction and pilus production could not confirm the plasmid nature of the resistance. Transposon mediation is possible since nalidixic acid resistance was not reversed by ethidium bromide. These findings lead us to stress that nalidixic acid should not be used in cases of shigellosis until the nature of this resistance has been
thoroughly investigated. Department of Microbiology, Shar-i-Kashmir Institute of Medical Sciences,
Soura-190011, Srinagar, Kashmir, India
B. R. PANHOTRA BHAVANA DESAI P. L. SHARMA
1 Panhotra BR, Desai B Resistant Shigella dysenteriae. Lancet 1983; ii 1420 2. McCormack JG Nalidixic acid for shigellosis Lancet 1983, ii 1091. 3 Malengreau M Nalidixic acid in Shigella dysenreriae outbreaks. Lancet 1984; ii: 172. 4 Stokes EJ, Waterworth PM. Antibiotic sensitivity tests by diffusion method. Assoc Clin
*Serotypes represented were 026, 055, 0111, 0114, 0119, 0128, and 0142 for classical EPEC, 0124, 0143, 0152 for EIEC; and 06H16, 08:H9, 015:H1I, 025:H42, 027:H7, 078:HI 1, 0148:H28, and 0159:H34 for ETEC. tbioassay uses the E co" K-12 mutant stram LG 1522, which can grow m conditions of Iron limitation when supplied with aerobactin, but not enterochelin. Minimal agar containing the iron-chelating compound a,a’-dIPyndyl (160 pmol/1) was seeded with strain LGI522 (10’ cells/plate), and inoculated with the bacterial isolates to be tested (10/plate). Individual strains that secrete aerobactin were identified by a halo of growth of the lawn of LG 1522 around the point of inoculum after overnight incubation.
(transferrin in serum and lactoferrin in secretions). Several surveys suggest a high frequency of aerobactin production among E coli isolates from patients with bacteraemia,3,5,6 peritonitis,6and 7 urinary-tract infections.7 Less obvious, perhaps, is a role for aerobactin in diarrhoeal diseases, the gut lumen being rich in iron. The table shows, however, that among a small number of clinical isolates chosen at random aerobactin was produced by nine enteropathogenic E coli (EPEC) strains associated with neonatal diarrhoeal disease, two invasive isolates categorised as "facultatively enteropathogenic" from severe dysentery-like disease of infants, and one "classical" enteroinvasive E coli (EIEC) strain.
Conversely, of seventeen E coli
enterotoxigenic (ETEC) isolates, strains produced enterochelin.
none
secreted aerobactin. All
Our results suggest that iron assimilation mediated by aerobactin contribute to the virulence of some non-toxigenic enteropathogenic isolates of E coli. Such strains are usually associated with disease in infants, whose diet of breast milk is rich in the iron-binding protein lactoferrin. Moreover, lactoferrin is also present in mucosal secretions. Therefore the very close adherence to epithelial cells characteristic of some enteropathogenic and invasive strains would commit such bacteria to a microenvironment at the mucosal surface in which efficient iron sequestration might be selectively advantageous for effective colonisation. may
We thank Dr S. H. bacterial strains.
Department of Genetics, University of Leicester, Leicester LE1 7RH
Parry
and Dr M. A.
Lmggood
for
providing
some
P. H. WILLIAMS M. ROBERTS
Pathol Broadsheet 1972, 55: 1-2.
AEROBACTIN-MEDIATED IRON UPTAKE: A VIRULENCE DETERMINANT IN ENTEROPATHOGENIC ESCHERICHIA COLI?
1 Neilands JB. Microbial iron compounds. Ann Rev Biochem 1981, 50: 715-31 2 Warner PJ, Williams PH, Bindereif A, Neilands JB. ColV plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infect Immun 1981; 33: 540-45. 3 Williams PH Novel iron uptake system specified by ColV plasmids: an important
component in the virulence of invasive strains of Escherichia coli. Infect
Immun 1979;
26: 925-32
PH, Warner PJ. ColV plasmid-mediated, colicin V-independent iron uptake system of invasive strains of Escherichia coli. Infect Immun 1980; 29: 411-16 5 Stuart SJ, Greenwood KT, Luke RKJ. Iron-suppressible production of hydroxamate by Eschsrichia coli isolates. Infect Immun 1982; 36: 870-75. 6 Mootgomerie JZ, Bindereif A, Neilands JB, Kalmanson GM, Guze LB. Association of hydroxamate siderophore (aerobactin) with Escherichia coli isolated from patients with bacteremia Infect Immun 1984; 46: 835-38 7 Carbonetti NH, Williams PH Aerobactin and the pathogenesis of extraintestinal infections of man In: Plasmids in bacteria New York: Plenum, 1985 4 Williams
in nature but most of it would be virtually unavailable for bacterial metabolism were it not for efficient ironsequestering mechanisms. Many bacteria produce low-molecularEnteric weight iron-chelating compounds called bacteria, for example, secrete enterochelin; the ferric-enterochelin complex formed is actively transported back across the bacterial membranes, iron being then released by enzymatic degradation of the siderophore in the cell. «-e found that certain strains of Escherichia coli isolated from patients with systemic infections synthesise the hydroxamate siderophore aerobactin;this phenotype contributes strongly to virulence in experimental infections.3Ferric-aerobactin, despite an association constant many orders of magnitude lower than that of ferric-enterochelin (about 1023 as against 1052),1 seems to confer a selective advantage for bacterial growth in the tissues and body fluids of infected animals.3,4 Clearly, therefore, such a system of iron assimilation is likely to be crucial in the pathogenesis of extraintestinal diseases, where sites of infection will be low in available iron because of the presence of iron-binding glycoproteins
SIR,-Iron is abundant
siderophores.
FIFTY YEARS OF SULPHONAMIDES
SIR,-Your editorial celebration of the 50th anniversary of the use of sulphonamides (Feb 16, p 378) fails to mention the benefits of sulphasalazine in rheumatoid arthritis. When Nanna Svartz developed this combination of 5-aminosalicylic acid and in the late 1930s, it was for the treatment of rheumatoid arthritis, not ulcerative colitis. Although the drug fell into disrepute for many years, its efficacy was rediscovered in the 1970s by McConkey and his co-workers in Birmmgham. Later controlled trials have confirmed that the drug does have second-line
sulphapyridine
NALIDIXIC-ACID-RESISTANT SHIGELLA
AEROBACTIN PRODUCTION BY ENTEROPATHOGENIC ISOLATES OF
DYSENTERIAE I
E COLI
SIR,-In our report of an epidemic of multiresistant Shigella dysenteriae we highlighted the indiscriminate use of nalidixic acid by village practitioners in rural areas. Resistance to nalidixic acid is chromosomally mediated and the drug’s use in shigellosis has been supported by some workers. McCormack2described nalidixic acid as first-line therapy for shigellosis, resulting in lower morbidity and case-fatality rates, especially among the very young and old. Malengreau3reported that during treatment with nalidixic acid only one strain out of 10 000 patients was resistant to this drug. In another epidemic of dysentery due to S dysenteriae in July to September, 1984, we isolated 52 strains from faecal samples of patients from two districts of the Kashmir Valley. These patients had a history of passing blood and mucus for 4-15 days. Antibiotic susceptibility was tested by disc diffusion with Difco discs. 38 (73%) strains were resistant to nalidixic acid (30 lAg) and ampicillin 10, chloramphenicol 30, tatracycline 30, streptomycin 10, sulphadiazine 300, and co-trimoxazole I-25+23’75 g discs. All except 1 strain were sensitive to furazolidone 100 pig and neomycin 30 g. In transfer studies by direct crosses and triparental crosses we observed that nalidixic acid resistance was the only resistance that
could be transferred to Escherichia coli C600 KI2 P- Lac-Rif by overnight crosses. The frequency of transfer was low (10-6 to 10’). Nevertheless, the observation is worrying. Another important observation was the high degree of nalidixic acid resistance transferred among transconjugants; while the donor strains had minimum inhibitory concentrations of 30 tg/ml, the transconjugants had MICs of 60 g/ml or more, by plate dilution method. This could be due to variable gene expression in different hosts. Studies of phage restriction and pilus production could not confirm the plasmid nature of the resistance. Transposon mediation is possible since nalidixic acid resistance was not reversed by ethidium bromide. These findings lead us to stress that nalidixic acid should not be used in cases of shigellosis until the nature of this resistance has been
thoroughly investigated. Department of Microbiology, Shar-i-Kashmir Institute of Medical Sciences,
Soura-190011, Srinagar, Kashmir, India
B. R. PANHOTRA BHAVANA DESAI P. L. SHARMA
1 Panhotra BR, Desai B Resistant Shigella dysenteriae. Lancet 1983; ii 1420 2. McCormack JG Nalidixic acid for shigellosis Lancet 1983, ii 1091. 3 Malengreau M Nalidixic acid in Shigella dysenreriae outbreaks. Lancet 1984; ii: 172. 4 Stokes EJ, Waterworth PM. Antibiotic sensitivity tests by diffusion method. Assoc Clin
*Serotypes represented were 026, 055, 0111, 0114, 0119, 0128, and 0142 for classical EPEC, 0124, 0143, 0152 for EIEC; and 06H16, 08:H9, 015:H1I, 025:H42, 027:H7, 078:HI 1, 0148:H28, and 0159:H34 for ETEC. tbioassay uses the E co" K-12 mutant stram LG 1522, which can grow m conditions of Iron limitation when supplied with aerobactin, but not enterochelin. Minimal agar containing the iron-chelating compound a,a’-dIPyndyl (160 pmol/1) was seeded with strain LGI522 (10’ cells/plate), and inoculated with the bacterial isolates to be tested (10/plate). Individual strains that secrete aerobactin were identified by a halo of growth of the lawn of LG 1522 around the point of inoculum after overnight incubation.
(transferrin in serum and lactoferrin in secretions). Several surveys suggest a high frequency of aerobactin production among E coli isolates from patients with bacteraemia,3,5,6 peritonitis,6and 7 urinary-tract infections.7 Less obvious, perhaps, is a role for aerobactin in diarrhoeal diseases, the gut lumen being rich in iron. The table shows, however, that among a small number of clinical isolates chosen at random aerobactin was produced by nine enteropathogenic E coli (EPEC) strains associated with neonatal diarrhoeal disease, two invasive isolates categorised as "facultatively enteropathogenic" from severe dysentery-like disease of infants, and one "classical" enteroinvasive E coli (EIEC) strain.
Conversely, of seventeen E coli
enterotoxigenic (ETEC) isolates, strains produced enterochelin.
none
secreted aerobactin. All
Our results suggest that iron assimilation mediated by aerobactin contribute to the virulence of some non-toxigenic enteropathogenic isolates of E coli. Such strains are usually associated with disease in infants, whose diet of breast milk is rich in the iron-binding protein lactoferrin. Moreover, lactoferrin is also present in mucosal secretions. Therefore the very close adherence to epithelial cells characteristic of some enteropathogenic and invasive strains would commit such bacteria to a microenvironment at the mucosal surface in which efficient iron sequestration might be selectively advantageous for effective colonisation. may
We thank Dr S. H. bacterial strains.
Department of Genetics, University of Leicester, Leicester LE1 7RH
Parry
and Dr M. A.
Lmggood
for
providing
some
P. H. WILLIAMS M. ROBERTS
Pathol Broadsheet 1972, 55: 1-2.
AEROBACTIN-MEDIATED IRON UPTAKE: A VIRULENCE DETERMINANT IN ENTEROPATHOGENIC ESCHERICHIA COLI?
1 Neilands JB. Microbial iron compounds. Ann Rev Biochem 1981, 50: 715-31 2 Warner PJ, Williams PH, Bindereif A, Neilands JB. ColV plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infect Immun 1981; 33: 540-45. 3 Williams PH Novel iron uptake system specified by ColV plasmids: an important
component in the virulence of invasive strains of Escherichia coli. Infect
Immun 1979;
26: 925-32
PH, Warner PJ. ColV plasmid-mediated, colicin V-independent iron uptake system of invasive strains of Escherichia coli. Infect Immun 1980; 29: 411-16 5 Stuart SJ, Greenwood KT, Luke RKJ. Iron-suppressible production of hydroxamate by Eschsrichia coli isolates. Infect Immun 1982; 36: 870-75. 6 Mootgomerie JZ, Bindereif A, Neilands JB, Kalmanson GM, Guze LB. Association of hydroxamate siderophore (aerobactin) with Escherichia coli isolated from patients with bacteremia Infect Immun 1984; 46: 835-38 7 Carbonetti NH, Williams PH Aerobactin and the pathogenesis of extraintestinal infections of man In: Plasmids in bacteria New York: Plenum, 1985 4 Williams
in nature but most of it would be virtually unavailable for bacterial metabolism were it not for efficient ironsequestering mechanisms. Many bacteria produce low-molecularEnteric weight iron-chelating compounds called bacteria, for example, secrete enterochelin; the ferric-enterochelin complex formed is actively transported back across the bacterial membranes, iron being then released by enzymatic degradation of the siderophore in the cell. «-e found that certain strains of Escherichia coli isolated from patients with systemic infections synthesise the hydroxamate siderophore aerobactin;this phenotype contributes strongly to virulence in experimental infections.3Ferric-aerobactin, despite an association constant many orders of magnitude lower than that of ferric-enterochelin (about 1023 as against 1052),1 seems to confer a selective advantage for bacterial growth in the tissues and body fluids of infected animals.3,4 Clearly, therefore, such a system of iron assimilation is likely to be crucial in the pathogenesis of extraintestinal diseases, where sites of infection will be low in available iron because of the presence of iron-binding glycoproteins
SIR,-Iron is abundant
siderophores.
FIFTY YEARS OF SULPHONAMIDES
SIR,-Your editorial celebration of the 50th anniversary of the use of sulphonamides (Feb 16, p 378) fails to mention the benefits of sulphasalazine in rheumatoid arthritis. When Nanna Svartz developed this combination of 5-aminosalicylic acid and in the late 1930s, it was for the treatment of rheumatoid arthritis, not ulcerative colitis. Although the drug fell into disrepute for many years, its efficacy was rediscovered in the 1970s by McConkey and his co-workers in Birmmgham. Later controlled trials have confirmed that the drug does have second-line
sulphapyridine