Mortality in kittens is associated with a shift in ileum mucosa-associated enterococci from Enterococcus hirae to biofilm-forming Enterococcus faecalis and adherent Escherichia coli

Abstract

Approximately 15% of foster kittens die before 8 weeks of age, with most of these kittens demonstrating clinical signs or postmortem evidence of enteritis. While a specific cause of enteritis is not determined in most cases, these kittens are often empirically administered probiotics that contain enterococci. The enterococci are members of the commensal intestinal microbiota but also can function as opportunistic pathogens. Given the complicated role of enterococci in health and disease, it would be valuable to better understand what constitutes a "healthy" enterococcal community in these kittens and how this microbiota is impacted by severe illness. In this study, we characterized the ileum mucosa-associated enterococcal community of 50 apparently healthy and 50 terminally ill foster kittens. In healthy kittens, Enterococcus hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium. These E. hirae isolates generally lacked virulence traits. In contrast, non-E. hirae enterococci, notably Enterococcus faecalis, were more commonly isolated from the ileum mucosa of kittens with terminal illness. Isolates of E. faecalis had numerous virulence traits and multiple antimicrobial resistances. Moreover, the attachment of Escherichia coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent E. hirae. These findings identify a significant difference in the species of enterococci cultured from the ileum mucosa of kittens with terminal illness compared to the species cultured from healthy kittens. In contrast to prior case studies that associated enteroadherent E. hirae with diarrhea in young animals, these controlled studies identified E. hirae as more often isolated from healthy kittens and adherence of E. hirae as more common and extensive in healthy kittens than in sick kittens.

Fig 1

Demonstration of enterococci adhering to the small intestinal epithelium of an apparently healthy kitten. Extensive colonies of bacteria are shown along the apical epithelium by means of Gram stain and fluorescence in situ hybridization using an oligonucleotide probe specific for eubacteria (Eub-338-FAM) or Enterococcus spp. (Enc-221-Cy3). Sections were nuclear counterstained with DAPI (4′,6-diamidino-2-phenylindole). Bar = 20 μm.

Fig 2

Representative results of semiquantitative scoring of the number and extent of enteroadherent enterococci in 4 healthy group A kittens. (A) Scant, focal; (B) mild, focal; (C) moderate, diffuse; (D) severe, diffuse. Fluorescence in situ hybridization was performed using an oligonucleotide probe specific for eubacteria (Eub-338-FAM) or Enterococcus spp. (Enc-221-Cy3). Specimens were nuclear counterstained with DAPI.

Fig 3

Transmission electron micrographs of enterococci interacting directly with the intestinal epithelial microvilli. The micrographs in the left panel were taken of a specimen with light microscopic evidence of scant, focal adherence of enterococci. The micrograph in the right panel was taken of a specimen with light microscopic evidence of severe diffuse adherence of enterococci. These specimens share the same origins as those shown in Fig. 2A and D, respectively.

Fig 4

Population diversity of enterococcal species from 12 group A (n = 120 isolates) and 13 group B (n = 159 isolates) kittens, each of which had ≥8 individual isolates identified as to species. Four common species, E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum, were identified using multiplex PCR (30). PCR amplification and sequencing of the sodA gene were carried out for isolates not identified by multiplex PCR (35).

Fig 5

Correlations among biofilm formation, gelatinase phenotype, and presence of virulence genes (gelE, asa1, esp, and cylA) in enterococci isolated from 12 group A kittens (n = 120 isolates) (A) and 13 group B kittens (n = 133 isolates) (B). The dotted lines indicate biofilm formation activity levels (<0.2, no biofilm; 0.2 to 0.7, biofilm; >0.7, strong biofilm). Kitten numbers are presented on the x axis followed by the total numbers of characterized isolates in parentheses. Letters following the kitten numbers indicate kittens with adherent enterococci (X), no adherent bacteria (Y), or adherent E. coli (Z). E. faecalis V583 was used as a positive control. Bars correspond to the means ± standard errors of the means (SEM) of 5 replicate experiments. OD₅₅₀, optical density at 550 nm.

Fig 6

Pulsed-field gel electrophoresis of 48 E. hirae isolates cultured from the ileum mucosa of apparently healthy kittens (group A; n = 15) and kittens that died or were euthanized due to severe illness (group B; n = 12). Bar denotes light microscopic findings in the kitten from which the isolate was obtained (white, no enteroadherent enterococci or EPEC observed; black, enteroadherent enterococci present; gray, EPEC present). Numerical values indicate the identities of the kittens and isolates. The type strain is E. hirae ATCC 8043.

Fig 7

Pulsed-field gel electrophoresis of 19 E. faecalis isolates cultured from the ileum mucosa of apparently healthy kittens (group A; n = 2) and kittens that died or were euthanized due to severe illness (group B; n = 10). Numerical values indicate identities of the kittens and isolates. The type strain is E. faecalis ATCC 29212.

Fig 8

Antimicrobial susceptibility test results for ileal mucosa culture isolates of E. hirae (n = 27) from 15 group A and 11 group B kittens and E. faecalis (n = 18) from 2 group A and 10 group B kittens. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Fisher's exact test). TGC, tigecycline; TET, tetracycline; CHL, chloramphenicol; DAP, daptomycin; STR, streptomycin; TYLT, tylosin tartrate; SYN, quinupristin-dalfopristin; LZD, linezolid; NIT, nitrofurantoin; PEN, penicillin; KAN, kanamycin; ERY, erythromycin; CIP, ciprofloxacin; VAN, vancomycin; LIN, lincomycin; GEN, gentamicin.