Characterization of small-colony variants of Enterococcus faecalis isolated from chickens with amyloid arthropathy

Abstract

In this study we report the isolation and characterization of normal-sized and small-colony variants of Enterococcus faecalis from outbreaks of amyloid arthropathy in chickens. Postmortem examinations of 59 chickens revealed orange deposits in the knee joints, typical for amyloid arthropathy. Bacterial cultures from 102 joints and 43 spleens exhibited pure (n = 88) and mixed (n = 11) cultures of normal (n = 60) and pinpoint (n = 28) colonies of E. faecalis. Pulsed-field gel electrophoresis of 62 isolates demonstrated seven different band patterns with at most two band size variations, and multilocus sequence typing demonstrated two different sequence types, sharing six out of seven alleles, suggesting a close evolutionary relationship between isolates obtained from four outbreaks. In addition, all isolates were clonally related to an amyloid arthropathy reference strain from The Netherlands, previously shown to be globally dispersed. Initial investigation of the isolated small-colony variant phenotype revealed no difference in whole-cell protein profiling between normal and pinpoint colonies. However, the pinpoint colony isolates appeared to be more virulent in an in vivo challenge model in chickens than their normal-sized-colony counterparts. In addition, pinpoint morphology and associated slow growth were expressed without reversion after in vitro and in vivo passage, suggesting a genuine altered phenotype, and in some instances normal colonies converted to pinpoint morphology postinfection. In conclusion, small-colony variants of E. faecalis are described for the first time from veterinary clinical sources and in relation to amyloid arthropathy in chickens.

FIG. 1.

Primary plate with mixed culture of pinpoint (predominant) and normal-sized colonies of Enterococcus faecalis.

FIG. 2.

PFGE of Danish outbreak isolates of Enterococcus faecalis associated with amyloid arthropathy. Lanes 1 and 13, PFGE Low Range marker set (New England Biolabs); lanes 2 to 5 and 7 to 9, PFGE type A; lanes 6, 10, and 11, PFGE type B; lane 12, PFGE type C.