Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli

Abstract

From two different specimens of a chronic prosthetic hip infection taken at an interval of 2 months a slow-growing gram-negative bacterium was isolated in pure culture. The strain grew with the typical features of a small-colony variant (SCV). 16S rRNA sequencing identified the bacterium as Escherichia coli. Biochemical characterization demonstrated multiple phenotypic alterations of a mutant carrying a defect in the heme biosynthetic pathway (Hem-): (i) catalase and nitrate reductase reactions were both negative, (ii) a negative benzidine reaction demonstrated the lack of heme-containing cytochromes, and (iii) growth stimulation under anaerobic conditions as well as gentamicin resistance indicated defective aerobic respiration. PCR and Southern hybridization demonstrated that the mutation of the SCV of E. coli was localized in the hemB gene and was most likely due to a deletion of the hemB gene. On blood agar plates revertants were recognized growing as normal-sized colonies between the dominant small colonies of the strain. Feeding experiments indicated that the revertants but not the small colonies were permeable for hemin. A strong antibody response against the infecting SCV of E. coli was found. To our knowledge, this is the first report of a Hem- E. coli strain as the etiological agent of a chronic bacterial infection.

FIG. 1

After 24 h of incubation at 37°C the large-colony type of E. coli Z-2376 showed hemin-dependent growth on McConkey agar only surrounding an X-factor disc (X DD; 5 μg of hemin).

FIG. 2

Southern hybridization. Two identical nylon membranes carrying immobilized, EcoRI-digested, genomic DNA were hybridized with the hemB probe (A) and the hemD probe (B). Lanes 1, DH5α; lanes 2: small-colony type of Z-2376; lanes 3, large-colony type of Z-2376; lanes M, marker (HindIII-digested λ DNA). The localization of the different fragments at 23.7, 9.46, 6.66, 4.2, 2.25, and 1.96 kb (top to bottom) are indicated by lines.

FIG. 3

Immunoblotting. Cell lysates of E. coli DH5α and the two colony types of Z-2376 grown under aerobic or anaerobic conditions on TSA-blood agar plates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and tested against antisera. (A) Antiserum from the patient diluted 1:500; (B) pooled antisera from 10 healthy volunteers diluted 1:100. Lanes 1, DH5α; lanes 2, large-colony type of Z-2376 grown aerobically; lanes 3, small-colony type of Z-2376 grown aerobically; lanes 4, large-colony type of Z-2376 grown anaerobically; lanes 5, small-colony type of Z-2376 grown anaerobically; lanes 6, prestained low-molecular-weight marker.